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VOLUME 126 SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander 1949-1988 1949-1984 ...

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VOLUME 126

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander

1949-1988 1949-1984 19671984-

ADVl SORY EDlTORS H. W. Beams Howard A. Bern Dean Bok Gary G. Borisy Bharat B. Chattoo Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham M. Nelly Golarz De Bourne Elizabeth D. Hay Mark Hogarth Keith E. Mostov Audrey Muggleton-Harris

Andreas Oksche Muriel J. Ord Valdimir R. Pantic M. V. Parthasarathy Lionel 1. Rebhun Jean-Paul Revel L. Evans Roth Jozef St. Schell Hiroh Shibaoka Joan Smith-Sonneborn Wilfred Stein Ralph M. Steinman Hewson Swift Masatoshi Takeichi M. Tazawa Alexander L. Yudin

K.W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

M. Friedlander Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME 126

Academic Press, Inc. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London

Sydney Tokyo Toronto

This book is printed on acid-free paper. (3 Copyright 0 1991 BY ACADEMIC PRESS, INC All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including photocopy, recording. or any information storage and retrieval system without permission in writing from the publisher

Academic Press, Inc. San Diego, California 92101

United Kingdom €dt/on published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NW1 7DX

Library of Congress Catalog Card Number 52-5203

ISBN 0-12-364526-3

(alk paper)

PRINTED IN THE UNITED STATES Of AMERICA

91329394

9 8 7 6 5 4 3 2 1

CONTENTS

Contributors

............................................................................................................

vii

Melanin-Concentrating Hormone: A General Vertebrate Neuropeptide Bridget I . Baker I. 11. 111 . IV. V. VI . VII .

Introduction ................................................................................................. Anatomical Distribution of MCH in Vertebrates and Invertebrates ............... Structure of MCH and Its cDNA Sequence ......................... ................. Biosynthesis of MCH .................................................................................... Structure-Activity and Molecular Modeling Studies ..................................... Physiology of MCH ...................................................................................... Conclusion: MCH, Past and Future ............................................................... References ...................................................................................................

1 3 14 20 21 30 40 44

Regulation of Synthesis and Transport of Secreted Proteins in Cereal Aleurone Russell L. Jones and John V . Jacobsen I. It. Ill. IV. V. VI . VII .

Introduction ....................................................... ...... Aleurone Tissue ........................................................................................... a-Amylase Genes and Control of Their Expression ...................................... Mechanism of Hormone Action .................................................................... lntracellular Transport and Exocytosis of Secretory Proteins ....................... The Role of Calcium ..................................................................................... Perspective ...................... ............................... References ...................................................................................................

49 49 54 63 70 79 83 84 V

CONTENTS

vi

Multiphasic Uptake Mechanisms in Plants Per Nissen ................... 1. .......................... .. ......................... II. ...................................... Ill. IV. Other Models ................................................... V. VI. .........................

89 91 93 122 126 128 131

Glycosylation in Intestinal Epithelium Douglas J. Taatjes and Jurgen Roth ................................... .... I. .......................................... ~. II. Ill. Methods Employed to Investigate Cellular Glycosylation Reactions .......................................... in Intestine .......................................... IV.

V. VI. VII.

nal Glycosyltransferases and Their Saccharide .......... ............,....................................... Agents on Intestinal Glycosyltran and Glycosylation .................................. ..................................... .. Differentiation and Glycosylation in Intestinal Cell Culture Systems ............. Concluding Remarks ........................... ................................. ................................................... References .,.,,, ..,......,

135 139 147 151 183 185 187 188

Physiological and Pharmacological Regulation of Biological Calcification Daniel C. Williams and Charles A. Frolik .............................................. I Introduction . ............. I I The Vertebrate Skeleton ............................................ Ill Physiological Regulation of Calcification in the Verteb IV Pharmacological Regulators of Calcification ............. ............................................ V. Summary and Conclusions ................ .......................................................... References ._................. Index .........................................................................................................................

195 199 217 246 262 263 293

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Bridget I. Baker (l), School of Biological Sciences, University of Bath, Cleverton Down, Bath BA2 7AY, England Charles A. Frolik (195),Department of Biochemistv Research, Lily Research LaboratoriesJEli Lil& and Company, Indianapolisl Indiana 46285 John V. Jacobsen (49),Division of Plant Industry, CSIRO, Canberra,Australia Russell L. Jones (49),Department of Plant Biology, University of California, Berkelev, Berkeley, California 94720 Per Nissen (89),Department of Biology and Nature Conservation,Agricultural University of Norway, N-I432&NLH, Norway Jurgen Roth (135),lnterdepartrnental Electron MicroscopyJBiocenter, Universily of Basel, CH-4056 Basel, Switzerland Douglas J. Taatjes (135),lnterdepartmntal Electron Microscopy, Biocenter, University of Basel, CH-4056 Basel, Switzerland Daniel C. Williams (195),Bone Biology Research Group, Department of Connective Tissue and Monoclonal Antibody Research, Lily Research Laboratories, Eli Lily and Company, Indianapolis, Indiana 46285

vii

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INTERNATIONAL REVIEW OFCYTOLOGY. VOL. 126

Melanin-Concentrating Hormone: A General Vertebrate Neuropeptide BRIDGETI. BAKER School of Biological Sciences, Universiv of Bath, Cleverton Down, Bath BA2 7AY. England

I. Introduction We owe much of our current appreciation of the endocrine repertoire to comparative studies on lower vertebrates, which have shown that while identical or highly homologous hormones occur in all vertebrate classes, their physiological roles may differ. A hormone may be involved with several diverse functions even within one animal, but the emphasis on a particular function can shift during the course of evolution, so that homologous hormones in fishes and mammals may come to serve different roles in the two classes. It follows that a hormone or neuropeptide exerting an obscure (although not necessarily unimportant) effect in mammals might elicit a strikingly obvious response in lower vertebrates, which is the cause of its discovery. This possibility alone would justify the research of lower vertebrates, since such studies can draw attention to previously unsuspected messenger molecules in mammals. In no tissue is this possibility greater than in the brain and the pituitary gland. The neuropeptide discussed in the present chapter - the melanin-concentrating hormone (MCH) - was discovered because of its striking effect in teleost fish, causing them to become pale in color. Although this pigmentary response appeared to be restricted to bony fishes, it soon became clear that a similar molecule existed in the brains of several vertebrate classes. More recent determination of the structure of MCH from fish, rat, and human shows that the peptide has been highly conserved. It seems to be abundant in the mammalian brain but we still know very little about its physiological role there. On the other hand, several physiological effects of MCH have now been studied in fish. It is the aim of this chapter to review these data and to consider whether this knowledge can assist our search for the potential roles of MCH in mammals. A. DISCOVERY OF MCH There are many examples of physiological events that are regulated by two opposing hormones, an arrangement which gives more rapid and finer control than changing titers of a single hormone. The idea that rapid changes in skin color, seen when lower vertebrates such as amphibians and fishes move between 1

CopyrightQ I991 by Academic Press. Inc. All rights of reproduction in any form reserved.

2

BRIDGET I. BAKER

dark and pale-colored backgrounds, are controlled by two antagonistic hormones seemed uncontentious when it was first proposed (Hogben and Slome, 1931, 1936). it turned out, however, that the evidence from amphibians on which the proposal was based had been misinterpreted. Hogben and Slome ( 1936) observed that both frogs and the clawed toad Xenopus became extremely dark after cautery of the pituitary lobe known as the pars tuberalis. They concluded that this lobe secreted a paling hormone which opposed the darkening effects of the melanocyte-stimulating hormone (MSH) secreted by the intermediate lobe of the pituitary. The pars tuberalis lies closely applied to the floor of the hypothalamus and its destruction can cause damage to the hypothalamo-pituitary tract which restrains the activity of the pars intermedia. Etkin (1962) showed some years later, that such damage results in the hypersecretion of MSH and this is now believed to be the only pituitary hormone regulating color change in amphibia. There is no doubt that rejection of the dual hormone hypothesis for amphibians discouraged credence in a paling or melanin-concentrating hormone when the concept was subsequently applied to bony fish. In this group of animals, however, the evidence was quite different. It was realized early on that some teleosts responded to injections of teleost pituitary gland extract by exhibiting pallor, even though the extract contained MSH (Fries, 1943; Hewer, 1926; Weisel, 1948). The evidence for the existence of a melanin-concentrating hormone was fully documented by Pickford and Atz (1957) but at that time the few attempts to purify the hormone and separate it from MSH were unconvincing (addendum in Pickford and Atz, 1957). Its site of production was another enigma. Kent (1959) showed that MCH bioactivity was most abundant in the neurointennediate lobe of the minnow pituitary gland but the report by Healey (1948) that removal of this lobe did not prevent pallor when minnows were transferred to a pale-colored background suggested that the hormone was associated with the remaining anterior pituitary lobe. Based on his observation that MCH bioactivity could be detected in the catfish hypothalamus, Enami (1955) proposed that it was a neurohypophysial hormone. We know now that this is the correct conclusion and it would, of course, have explained the findings of other workers, but his observations could not be repeated by Kent and were generally ignored, except by Pickford and Atz (1957). interest in the control of color change waned in the 1960s and the existence of a melanin-concentrating hormone did not attract attention again until two decades later, when observations by Baker and Ball (1975) on color changes in the teleost Poeciliu seemed to be explicable only in terms of a dual hormonal control. This reawakened interest in the hormones involved. Subsequent studies by Rance and Baker (1979) and Baker and Rance (1983) showed that MCH bioactivity did indeed exist in the trout hypothalamus but that it was rapidly destroyed when the brain was extracted at pH 5.0; that its concentration in hypothalamic and pituitary tissue changed when fish were switched from white to

MELANIN-CONCENTRATING HORMONE

3

black tanks; and that similar bioactivity existed in the hypothalami of other vertebrates, including lampreys, amphibians, and mammals, suggesting that the peptide might have a widespread distribution. Encouraged by these observations, Kawauchi and co-workers (1983) purified and sequenced the melaninconcentrating hormone from the salmon pituitary gland and showed that it was a heptadecapeptide. Antibodies raised against this sequence were then used by Naito et al. (1985) to reveal the MCH-secreting neurons in the basal hypothalamus of salmonids, from which axons projected not only to the pituitary neural lobe but also up into the brain. Subsequent immunocytochemical studies and radioimmunoassays have confirmed the existence of MCH-like neurons in the hypothalamus of other groups of vertebrates (Section 11).

11. Anatomical Distribution of MCH in Vertebrates and Invertebrates

The distribution of MCH perikarya and fibers has been examined in rather few species but these studies suggest that the organization found in bony fish may differ slightly from that in other vertebrates. The teleost pattern will be discussed first. OF MCH IN TELEOST FISH A. DISTRBUTION

In teleost fish, most of the MCH activity is found in the neurohypophysial lobe where the peptide is stored, ready for release into the blood (Naito et al., 1985; Bird et al., 1989). The relative abundance of MCH in the brain and pituitary depends on a number of factors including the method used to measure the hormone. Bioassays for MCH, which depend on the ability of the peptide to concentrate the melanin granules in isolated scale melanophores, respond poorly to the MCH precursor molecule (Kawazoe et al., 1987a). They also give low values in the presence of MSH, the functional antagonist of MCH on melanophores (Baker, 1988a). This accounts for the apparently low pituitary : hypothalamic ratio of MCH reported both by Enami (1955) and by Rance and Baker (1979) in early studies using scale bioassays. Antibodies against the active peptide also appear to recognize the precursor, so that radioimmunoassays (RIAs) of hypothalamic extracts indicate a higher content of MCH than do bioassays (Kawazoe et al., 1987a; B. I. Baker, unpublished observations). The abundance of immunoreactive MCH (ir-MCH) in the brain and pituitary gland of trout depends on the age of the fish and the color of the tank in which it is kept (Table I), since this affects the rate of hormone synthesis and its release. Changes in the pituitary content of MCH in response to changes of background color have been observed in other species of fish, including the eel Anguilla anguilla (Powell and Baker, 1988), and the grass carp Ctenopharyngodon idellus (Bird and Baker, 1989).

4

BRIDGET I. BAKER TABLE I EFFECTOF REARING TROUTIN BLACKOR WHITE TANKSON MCH CONTENT OF HYPOTHALAMUS AND PITUITARY GLAND"

MCH Contenth

wt

n

tg)

Hypothalamus (Pg)

Pituitary (Pg)

Pituitary/ hypothalamic ratio

4 Months

Black stock B --f B 6 days B + W 6 days White stock W -+ W 6 days W 4 B 6 days

0.8 f 0.06 1.3 f 0.30 0.3 f 0.03 8.1 f 1.9 4.1 f 0.60 8.2 f 0.60

2.3 2.4 2.6 7.6 2.3 2.8

4.7 k 0.6 6.5 + 1.5 0.6 f 0.1' 15.9 f 5.0 2.5 f 0.2"Y 12.0 k 1.4

5.6 5.3 1.8 2.0 0.6 1.6

15 months

Black stock White stock

200

12

200

12

9.2 k 1.4 249.5 ?r 29

231.2 f 25.0 1088.4 f 93.0

25.0 4.0

Trout were reared from the egg stage onwards in off-white- or black-colored tanks (black or white stock). At 4 months, some fish were transferred to clean black or white test tanks and killed after 6 days ( B + B, B -+ W, W -+ W and W + B). Other stock fish were killed at 15 months. "Valuesare means f SEMS. All MCH concentrations in white-reared fish are significantly higher than in their black-reared counterparts. ' p < .01 compared with stock fish.

An early attempt to map the distribution of bioactive MCH within the brain (Baker and Rance, 1983) suggested that the peptide was concentrated in two sites; the majority was located in the ventral hypothalamus near the pituitary stalk, while about 30% was found in the dorsal thalamus, near the posterior commissure. This distribution was clarified by an immunocytochemical study of salmon and trout brains by Naito and co-workers (1985), using antiserum raised against synthetic salmonid MCH (sMCH). They showed that MCH is produced in magnocellular neurons located on the floor of the hypothalamus near the pituitary stalk, in the lateral region of the nucleus lateralis tuberis (NLTpI) (Fig. 1). While most axons are directed to the pituitary, others project dorsally into the brain and are found in the preoptic area and even more abundantly within the pretectal region, either side of the posterior commissure and habenular nuclei (Fig. 2), thus explaining the bioactivity detected in this region. The authors drew attention to the fact that neuroanatomical studies, using tracers such as horseradish peroxidase or [3H]leucineinjected into the eye, have shown this pretectal region receives fibers from the retina and pineal gland, and they proposed that the MCH neurones might receive photosensory information at this site. The


5

FIG.1. Sagittal section through the basal hypothalamus and pituitary gland of a trout, immunostained with anti-MCH and counterstained with light green. (A) Parasagittal section at the level of the nucleus lateralis tuberis, pars lateralis (NLTpl), showing numerous MCH perikarya with axons extending ventrally to the pituitary gland and dorsally towards the thalamus. (B) Near mid-sagittal section, showing axons ramifying through the neural tissue (NT) surrounded by the pars intermedia (PI) and extending also into the pars distalis (PD). (LR) Lateral recess of ventricle; (IR) infundibular recess; *, third ventricle. [From Naito et al. (1985). Immunocytochemical identification of melaninconcentrating hormone in the brain and pituitary gland of the teleost fishes Oncorhynchus keta and Salmo gairdneri.]

projection of MCH fibers into the brain implies that the peptide plays a neuromodulatory or neurotransmitter role, apart from its hormonal effect on color change. One may speculate that the release of MCH in the pretectal region could influence the transmission of photic information from the retinal or pineal nerves. The location of MCH immunoreactivity in the trout brain was confirmed by Bird er al. (1989) who showed using fluorescence irnmunostaining on cryostat sections, that MCH fibers also penetrated specific layers of the optic tectum. A comparable distribution of magnocellular MCH perikarya and fibers has been described in the Chinese grass carp, C . ideffus (Bird er af., 1989) and in the molly Poecifia faripinnu (Batten and Baker, 1988). Again, the MCH perikarya are concentrated in the lateral region of the NLT; the majority of fibers supply the pituitary while others project to various regions of the brain - the preoptic area and telencephalon, the pretectal area, lateral hypothalamus, and optic tectum. In all species, MCH perikarya are not totally restricted to the NLT; isolated

6

BRIDGET 1. BAKER

I amprey

trout

dogfish

frog

rat

\*-..

..

... a :

;.: .

,.,.:la:


7

cell bodies can sometimes be found near the lateral ventricular recess but their axonal projections have not been traced. The MCH neurons appear to be bi- or multipolar cells, sometimes with branching fibers. It has not been established whether the fibers projecting to the pituitary and the brain arise from the same perikaryon, although this seems likely, judging from the histological picture and from data showing that conditions influencing MCH release from the pituitary can also affect the concentration of MCH in the central regions of the brain (Section VI,B). The data suggest ;hat the availability of MCH for neuromodulatory functions in the brain can be influenced by conditions such as background color, although it does not show whether the release of MCH from the different terminals occurs simultaneously. Most of the axons entering the pituitary gland terminate in the neural lobe but a few penetrate the pars distalis (Naito et al., 1985; Batten and Baker, 1988), coursing near the corticotropes and other cells (Powell and Baker, 1987). No pituitary portal system exists in teleosts but pituitary cells are controlled by hypothalamic neurons which terminate on or near the secretory cells. The presence of MCH fibers in the pars distalis could indicate a potential regulatory effect on one or more pituitary cell types. MCH fibers in the neurohypophysial lobe concentrate near major blood vessels (Naito ef al., 1985) and also, in the case of the eel (Powell and Baker, 1988), terminate on the vascular basement membrane which separates the neural and intermediate lobes. This location permits MCH access to the general circulation and also to cells of the pars intermedia, which accords with the observation that MCH can exert an inhibitory effect on the melanotrope cells (Section VI,A). In some species such as the carp and Poeciliu, in which a basement membrane is less developed, or in the trout in which the fibers can penetrate the basement membrane, the MCH nerve terminals make direct contact with the pars intermedia cells (Batten and Baker, 1988; Powell and Baker, 1987; Naito et al., 1986b). Ultrastructural studies show that MCH is contained within membrane-bound, electron-opaque secretory granules which have a modal width and length of 90 x 120 nm in the carp and eel (Powell and Baker, 1987, 1988); they appear of similar size in Poecilia (Batten and Baker, 1988). In the eel, an increase in the secretory activity of the MCH neurones, caused by adapting the fish to a palecolored background, is accompanied by a decrease in the abundance and pack-

FIG.2. Diagrams of transverse and sagittal sections through the brain of different vertebrates showing the position of the MCH cell bodies (large dots) and their major axonal projections (small dots). Arrows on the sagittal sections indicate the level of the transverse section. Cells and axons are shown on one side only. AVA, area ventralis anterior thalamus; CH, cerebral hemisphere. CP, choroid plexus; LH, lateral hypothalamus; NID,nucleus infundibularis dorsalis; NIL,neurointermediate lobe; NLT, nucleus lateralis tuberis; NPO, preoptic nucleus; NTP, nucleus tuberculi posterioris; NSV, nucleus saccus vasculosus; OT, optic tectum; P, pituitary gland; pc, posterior commissure.

X

BRIDGET 1. BAKER

ing density of the MCH secretory granules at the nerve terminals, together with an increase in the number of synaptoid thickenings on the nerve terminal membrane (Powell and Baker, 1988). In the carp, about 40% of the MCH neurons are also immunostained by antiserum against a-melanocyte stimulating hormone (aMSH) and ultrastructural studies show that both immunoreactivites are located in the same neurosecretory granule (Powell and Baker, 1987). Immunocytochemical localization of other pro-opiomelanocortic (POMC) fragments in the carp brain suggests this immunoreactive a melanocyte-stimulating hormone (iraMSH) cannot be attributed to the coexpression of the POMC precursor in the MCH neurons, and thus does not signal the presence of conventional aMSH (Bird er al., 1989). More recent studies (Section 111) show that the immunostaining is probably attributable to the presence within the MCH precursor sequence of an epitope with minimal resemblance to the C-terminal region of crMSH. Colocalization of these two immunoactivities has not been observed in other teleosts, but occurs in several other vertebrates. Terlou and co-workers (1985) showed that the MCH neurons of trout were immunostained with antiserum raised against methyl-S-glucagon. It seems likely that this antiserum, too, recognizes some epitope within the MCH precursor. Computer sequence comparison (Clustral program) shows that the 15 C-terminal residues of glucagon share three identities and eight similarities with the N-terminal region of prohormone MCH (proMCH) (B. I. Baker, unpublished observations). B. DISTRIBUTION OF MCH IN NONTELEOSTEAN VERTEBRATES

In all nonteleostean vertebrates that have been studied, the MCH perikarya are located more centrally within the hypothalamus and it seems likely that the ventral situation of these neurons in teleosts is associated with their strong axonal projection to the pituitary gland. The hypothalamo-neurohypophysialcomponent is usually much less well developed in nonteleostean vertebrates, while the central projections may be very extensive. In our experience, the MCH neurons are best preserved in tissues that have been fixed by perfusion, and may be poorly preserved in whole brains that have been fixed only by immersion. Thus, even in the small brains of lampreys, good preservation of the cell bodies and their axonal tracts requires that the fixative be injected into the brain ventricles (unpublished observations). Additionally, i.c.v. injection of colchicine enhances the visibility of MCH fibers in mammals. Perfusion and colchicine have not always been used in the studies described below, and it is possible that the distribution of MCH fibers may be more widespread than the current descriptions suggest. I. Cyclostomes

Ln lampreys, MCH neurons can be detected even in young ammocetes (larvae) and become more apparent with increasing age. The cell bodies are lo-

MELANIN-CONCENTRATINGHORMONE

9

cated in a single group near the third ventricle of the hypothalamus, level with the pituitary neurointermediate lobe (Fig. 2). Many neurons are strikingly bipolar, one axon making contact with the ventricular cavity while the other is directed laterally into the brain (Fig. 3). The most prominent axonal tracts are seen in the lateral hypothalamus, projecting backward down the spinal cord and forward toward the optic chiasma and olfactory lobes, and some apparently crossing from side to side above the optic chiasma. Less prominent axonal tracts are directed dorsally toward the region of the habenular nuclei. Immunoreactive MCH fibers are evident in the neurohypophysial lobe of the ammocoete but are less obvious in this location after metamorphosis (unpublished observations). 2 . Elasrnobranchs

The distribution of MCH immunoreactivity has been studied in the dogfish Scyliorhinus canicula by Vallarino and co-workers ( 1989). Immunoreactive MCH cell bodies are found in three hypothalamic sites; the most prominent group is found in the nucleus saccus vasculosus (NSV), where they also react with crMSH antiserum. Fewer irMCH neurons, which apparently do not react with aMSH antiserum, are scattered through the nucleus tuberculus posterioris (NTF’) and the nucleus lateralis tuberis (NLT) (Fig. 2). In addition, some of the secretory cells situated around ventricular spaces within the pituitary gland also reacted positively with the MCH antiserum. No MCH fibers have been detected in the pituitary neural lobe, but they project throughout the posterior hypothalamus, and are found also in the dorsal thalamus and central grey of the mesencephalon.

3. Amphibia In this class of vertebrates, irMCH perikarya may again be located in more than one well defined hypothalamic nucleus (Fig. 2). In Rana ridibunda, the greatest number are found in the preoptic nucleus (NPO),where some make contact with the third ventricle (Andersen el al., 1986). Melanin-concentrating hormone neurons in this area are also immunostained by aMSH antiserum (Andersen el al., 1987). A few MCH perikarya are located in the area ventralis anterior thalami (AVA), lying dorsal to the preoptic nucleus while others, which are not immunostained with aMSH antiserum, occur in the dorsal infundibular nucleus (NID) of the posterior hypothalamus. Scattered nerve fibers are seen in the brain, and a well developed tract of fibers, probably arising from the preoptic region and dorsal infundibular nucleus, supplies the median eminence and the neurohypophysis where numerous fibers terminate around blood sinuses. These two destinations suggest that in R . ridibunda, MCH might serve both as a hormone and as a pituitary regulator. Within the neural lobe, ultrastructural studies show that the hormone is contained within electron-dense granules of about 80-90 nm diameter.

10

BRIDGET 1. BAKER


11

A different distribution of MCH cell bodies and fibers has been reported for R. temporaria and Xenopus laevis, in which MCH perikarya have been detected only in the postero-lateral hypothalamus. Fibers from this site project to the midbrain and forebrain bundle, but none were evident in the median eminence or neurohypophysis (Baker, 1988b; Batten et al., 1986). 4. Mammals

Following an early report of MCH-like bioactivity in the rat brain (Baker and Rance, 1983), Naito and co-workers (1988) confirmed that MCH bioactivity and immunoreactivity co-eluted during high-pressure liquid chromatography (HPLC) and thus were probably attributable to the same molecule. They and earlier workers noted, however, that rat MCH (rMCH) behaves as though structurally different from sMCH since it elutes later from HPLC columns, suggesting a more hydrophobic nature (Zamir et al., 1986a; Sekiya et al., 1988). The location of irMCH neurons in the rat brain has been mapped by immunocytochemistry using antiserum against sMCH (Skofitsch et al., 1985; Naito et al., 1988). The perikarya have an extensive distribution through the mid and caudal region of the dorso-lateral hypothalamus (LH) (Figs. 2 and 4A). At their anterior limit they are found dorsal to the paraventricular nuclei, and surround the fornix and the medial forebrain bundle. More caudally, they occur in the subzona incerta region, located above the ventromedial nuclei and medial and dorsal to the optic tract. From these perikarya arises an extensive network of fibers which project widely to most regions of the brain except the cerebellum, and into the spinal cord; a distribution that is confirmed by radioimmunoassays for MCH in dissected regions of the brain and spinal cord (Zamir et al., 1986b; Sekiya et al., 1988). A few MCH fibers project to the median eminence and posterior pituitary. When rat brains are examined by immunocytochemistryat intervals after birth, it is seen that the MCH neurons are poorly developed on postpartum day 1, with sparse granulation apparent in only a few cell bodies. The size of the MCH perikarya and the granulation they contain increases markedly during the first week, so that a greater number become evident. During the next three weeks the neurons increase only slightly in size but become more densely granulated and assume their adult appearance (Fig. 4B). Cell counts in serial sections through the hypothalamus of four rats revealed approximately 8,460 immunostained neurons/hypothalamus on day 4, 19,900 neurons on day 7, 23,960 neurons by day 24 and 22,080 neurons in an adult rat (M. Coles and B. I. Baker, unpublished observations). FIG.3. (A) Transverse section through brain of the lamprey Pefrumyzon mnrinus showing MCH cells with projections both to the ventricle and to the lateral hypothalamus (x 23). (B) Detail of neuronal cell bodies (x 122). LH, lateral hypothalamus; NIL, neurointermediate lobe; V, ventricle of brain.

12

BRIDGET I. BAKER


13

In the human, the only other mammal in which the MCH neurons have been located by immunostaining (Pelletier et al., 1987; Bresson et al., 1989), the cell bodies are discernible already in fetuses of 14 weeks (Bresson et al., 1989). In older individuals, the MCH system is said by some workers to be similar in terms of number, morphology, and topography of neurons to that found in the rat (Bresson et al., 1989). But other workers have reported that MCH cells are restricted to the periventricular region (Pelletier er al., 1987). The difference could perhaps be a problem of fixation, as mentioned earlier. As in the rat, axons extend to various regions of the brain, with a few MCH fibers projecting to the median eminence and pituitary stalk. Naito et al. (1988) reported that the MCH cells in rat are of two morphological types. One, located mainly around the zona incerta, is of medium size (15-25 pm diameter) and fusiform in shape; the other type, situated in the lateral hypothalamic and perifornical areas, is described as larger (20-25 pm) and multipolar. Ultrastructural studies show that, as in fish and amphibia, the MCH is contained within golgi-derived elyptical, dense-cored secretory granules whose diameter ranges from 80-110 nm to 90-250 nm, depending on the study (Pelletier et al., 1987; Naito et al., 1988). Nerve terminals of unknown type make synaptic contact with the MCH cell bodies; it is possible that some of these fibers are cholinergic since some MCH neurons in the rat contain acetylcholine esterase (Risold et al., 1989). In addition, MCH fibers make synaptic contact with dendrites of other MCH cells in both the rat (Naito ef al., 1988) and human (Bresson et al., 1989). In the human they are described as forming basketlike endings around both MCH-positive and MCH-negative perikarya. As in several lower vertebrates, the MCH neurons in the rat brain also react with antiserum against aMSH (Naito et al., 1986a; Fellmann et al., 1986). They correspond to the system of hypothalamic aMSH neurons that attracted attention previously because, in contrast to conventional MSH-secreting nerves in the arcuate nucleus, they fail to react with antisera against other POMC-derived peptides such as adrenocorticotropin (ACTH) or P-endorphin (Watson and Akil, 1980; Guy et al., 1980; Kohler et al., 1984; Khachaturian et al., 1986). Other workers subsequently found that the MCH neurons in rat also react with some antisera against human growth hormone-releasing factor (hGRF-37) (Fellmann et al., 1985, 1987) and corticotropin-releasing factor (CRF) (Antoni and Linton,

FIG.4. Immunoreactive MCH cells in the rat brain. (A). Vibrotome section (50 pm) through the hypothalamus of a colchicine-treated rat, showing MCH immunoreactive cells predominantly in the lateral hypothalamus above the fornix (F) and below the medial forebrain bundle (M). At this section thickness, the multipolar nature of the cells is apparent. (*), ventricle (courtesy of N. Naito). (B) Immunoreactive MCH cells (arrowed) in the developing rat. (a) 4 days; (b) 7 days; (c) 24 days; (d) adult, showing axon fibers. Rats were not treated with colchicine and at this section thickness (8 pm) the multipolar nature of the cells is not visible.

14

BRIDGET 1. BAKER

1979: Kawano er al., 1988). The epitopes responsible for this surprising wealth of co-existing immunoreactive molecules have recently been identified as sequences within the MCH precursor, bearing a lesser or greater resemblance to portions of aMSH. hGRF and CRF (Section 111). The observation that MCH and hGRF-37 antisera present a slightly different intracellular immunostaining pattern (Fellmann et al., 1987) might reflect the ability of the antisera to detect their epitopes at different stages of precursor processing. In the human, the MCH neurons are said not to immunoreact with aMSH antiserum but they do stain with antiserum against hGRF-37 (Pelletier et al., 1987; Bresson et al., 1986, 1989). C. INVERTEBRATES MCH-immunoreactive neurons have been described in the locust where they are found in the distal region of the optic lobe, bordering the junction with the ommatidia. and in the cerebral ganglion (Schoofs et al., 1988). The neurons in the optic lobes can also be immunostained by aMSH antiserum.

111. Structure of MCH and Its cDNA Sequence

A. STRUCTURE OF MCH The primary structure of MCH was first determined from purified pituitary extracts of the chum salmon Onchorhynchus keta (Kawauchi et al., 1983) and has since been determined for three further teleost species, the bonito Katsunionus pelamis and the eel Anguilla japonica by amino acid sequencing (Kawauchi, 1989), and the chinook salmon from its cDNA structure (Minth et al., 1989). The hormone is highly conserved in these fish. In all, it is a cyclic heptadecapeptide (Fig. 5) with an identical structure for the two salmonids and bonito and a conservative substitution to Asn' in the eel. More recently, the primary structure of rat MCH has been determined by traditional purification methods (Vaughan et al.. 1989), and that of the human was deduced from its cDNA sequence (Presse er al., 1990). Rat and human MCH are identical (Fig. 5 ) and, in spite of the fact that fish and mammals are separated by approximately 800 million years of evolutionary time, their MCHs are very similar. The central (ring) sequence, between the two cysteines, contains only a conservative mutation, that is, Leu in place of sMCH Val'. The C-terminal sequence also contains a single mutation of glutamine for sMCH GluI6, but the greatest changes involve the N-terminal region where there are two substitutions and two additional terminal residues (Fig. 5 ) . The biological significance of these changes is still uncertain. In teleosts, the ring structure is the most important for melanin concentration and the exocyclic sequences apparently potentiate this bioactivity,

15


Primary structure of MCH in different species 5

1

17

14

Eel’

H-~Thr*Met.Arg.Cys.Met.Val*Gly.Arg.Val.Tyr.Arg.Pro.Cys~Trp.Glu.Val*OH I

Salmonids

H-Asp.Thr*Met*Arg.Cys.Met.Val.Gly.Arg.Val.Tyr.Arg.Pro.Cys~Trp.Glu.Val.OH

and Bonito’.* Rat and human3

I

I

H-BSp.~Asp.~L8Y’Arg.Cys.Met.~Gly.Arg.Val.Tyr.Arg.Pro.Cys*Trp.~Val.OH

1

I

FIG. 5. Primary structure of MCH from three fish, the rat and human. Variant amino acids are underlined. I Kawauchi, 1989; * Kawauchi et al., 1983; Minth er al., 1989; Vaughan er al., 1989; Nahon er a/., 1989b; Presse er al., 1990.

perhaps by altering ring conformation and/or enhancing binding to the receptor site (Section V). Rat MCH has about 60% bioactivity compared with sMCH when tested on carp melanophores (unpublished observations) but no bioassay based on mammalian target tissue is yet available. OF TELEOST MCH B. PRECURSOR MOLECULE

Investigation of the molecular size of MCH in the fish hypothalamus initially suggested that, as for other peptides, it must come from a larger precursor molecule (Kawazoe et al., 1987b). This is confirmed by the MCH cDNA structure of two salmonid species, the chum salmon 0. keta (Ono et al., 1988) and the chinook salmon 0. tshawytcha (Minth el al., 1989). The gene for MCH mRNA in 0. keta contains just over 600 base pairs and lacks introns (Takayama et al., 1989). Salmonid fish are tetraploid and have two genes for MCH which are 86% identical. The MCH mRNA, whose cDNA sequences are shown in Fig. 6, each code for a pre-proMCH of 132 amino acids. An initiator MET is followed by a hydrophobic signal sequence and, assuming that signal peptide cleavage occurs after AlaZ4,this is followed by a prohormone of 108 amino acid residues in which MCH occurs at the C terminus (residues 116-132), separated from the rest of the precursor by a classical dibasic amino acid (Arg-Arg) cleavage site. The presence of other dibasic and monobasic amino acids within the precursor suggests that posttranslational processing might liberate additional molecules with potential biological activity. For instance, cleavage at Arglo0 could liberate a 13-amino acid peptide (101-113) which has been termed MCH-gene-related peptide (Mgrp) (Bird et al., 1990). In contrast to the mature MCH, which is identical in the two precursors, the remaining salmonid prohormone is less well conserved. Thus, depending on the species, Mgrp shows one or two amino acid variants (Asp or Gly at position 103 in both salmonids; Asn or Ser at position 107 in 0. keta). The two pre-proMCHs from the same species share 80% (chum) or 81% (chinook) amino acid

CHINOOK ppMCH CHUM p p M C H CHUM cDNA 1

1 10 20 M e t Arg H I S Ser V a l L e u Ser I L E S E R Phe A l a VAL A l a L e u Phe L e u G l u C y s T y r Thr ATG AGA CAC T C T G T C C T C T C C A T C T C C T'lT GCC GTG GCA CTT ? T C T T G GAG T G C T A C ACA

CHUM c D N A 2 CHUM p p M C H CHINOOK ppMCH

ATG AGA GAC T C G G T C C T C T C C G T C A T C TIT G C C T T G GCA CTT TTC T T G GAG T G C T A C ACA M e t A r g A S P S e r V a l L e u S e r V A L ILE Phe A l a L E U A l a L e u Phe L e u G l u C y s T y r Thr

C H I N O O K pp . MCH CHUM p C H CHUM c g N A 1

PRO 30 40 21 P r o S e r THR A l a I l e S E R ILE G l y Lys M e t ASP A s p VAL A l a Leu G l u G l n A s p Thr L e u

CHUM c D N A 2 CHUM p p M C H CHINOOK ppMCH

CCG T C C ATG GCG A T C CCG ATG GGC AAG ATG GAG GAC ACA GCC l T G GAG CAA G A T ACC C T A P r o Ser MET A l a I l e PRO MET G l y Lye M e t G L U A s p THR A l a Leu G l u G l n A s p Thr L e u

CHINOOK ppMCH CHUM p p M C H CHUM c D N A 1

50 ARG 60 A s p Ser L e u L e u SER VAL G l u V a l SER Q L U A S N SER P r o A s p S e r V a l A r g G L Y ARG Ser GAC T C C C T A C T G A G T GTA GAG G T G T C T GAA AAC AGC C C T G A T T C A G T C AGA GGC AGG AGC

CHUM c D N A 2 CHUM p p M C H C H I N O O K ppMCH

GAC T C T C T A C T G AAC GAA GAG GTG G C C G A T AAA AAC C C T G A T T C A G T C AGA AGC GGG AGC A s p Ser Leu L e u A S N Q L U G l u V a l A L A ASP LYS A S N P r o A s p Ser V a l A r g SER G L Y Ser

CHINOOK ppMCH CHUM p p M C H CHUM c D N A 1

61 70 80 Ser Lys I l e V A L L E U L e u A l a A s p Ser G l y L E U T r p MET A s n L e u A s n A r g G l y L e u P r o T C C AAG A T T G T C 7 1 G CTG GCA GAC T C T GGC CTG TGG ATG AAC C T G AAC AGA GGA C T T C C T

CHUM c D N A 2 CHUM p p M C H C H I N O O K ppMCH

T C C AAG A T C A T C GTG T T G GCA GAC T C A GGC ATG TGG AAG AAC C T G AAC AGA GGA ClT C C T Ser Lys I l e ILE V A L L e u A l a A s p Ser G l y MET T r p L Y S A s n Leu A s n A r g G l y L e u P r o

*

t.

L

CCG T C C ACG GCG A T C T C C A T T GGC AAG ATG GAC G A T G T C G C C T T G GAA CAA G A T A C T C T C

* *

*

* ***

*

*

t

*

t

41

**

* *

*

*

* *

*

*

t

*

*

*

*

CHINOOK ppMCH CHUM ppMCH CHUM cDNA 1

81 90 GLN P H E Q r Lys Leu ARQ Ala Ala Ala Ala Gly PRO Asp Arg Ala Leu Thr Leu Asp Arg ARG TTC TAC AAG CTG AGA GCT GCA GCC GCC GGG CCT GAC AGA GCC CTG ACT CTG GAC CGC AGA

CHUM cDNA 2 CHUM ppMCH CHINOOK ppMCH

CTC TAC AAG CTG AAA GCT GCA GCT GCA GGG CTT GAC AGA GCC CTG ACC CTG GAC CGC AGA LEU Tyr Lys Leu LYS Ala Ala Ala Ala Gly LEU Asp Arg Ala Leu Thr Leu Asp Arg ARG

*

h

*

*

*

L A


Glu Ala GLY Gln Asp Leu SER Pro Ser Ile Ser Ile Val Arq Arq Asp Thr Met Arg Cys GAG GCT GGC CAG GAC CTA AGC CCC AGC ATC TCC ATC GTC AGG AGG GAC ACC ATG AGG TGT


GAG GCT GAC CAG GAC CTG AAC CCC AGT ATC TCC ATT GTC AGG AGG GAC ACC ATG AGG TGC Gly Ala ASP Gln Asp Leu ASN Pro Ser Ile Ser Ile Val Arg Arg ASP Thr Met Ara Cys SER L A


Met Val Gly Arg Val Tyr Arg Pro Cys Trp Glu Val end ATG GTG GGA AGG GTG TAC CGA CCC TGC TGG GAG GTG TAC


101

110

*

*

120

*

*

121

*

130

*

*

ATG GTG GGA AGG GTG TAC CGG CCT TGC TGG GAA GTG TAC Met Val Glv Ara Val Tyr Ara Pro CYS Trp Glu Val end

FIG. 6. The nucleotide and amino acid sequence of the two chum salmon (Onochorhynchus keral pre-proMCHs (ppMCH; Ono et al., 1988). Residues which differ in chinook salmon MCH (Minth et al., 1989) are indicated above or below the rows. The predicted signal cleavage is indicated by an arrow. Dibasic, putative cleavage points are indicated by arrowheads. Asterisks show nucleotide mutations. The MCH sequence is underlined.

18

BRIDGET I. BAKER

identities, with 21 of the 26 residue changes in chum salmon and 20 of the 25 changes in chinook salmon involving single nucleotide mutations. Comparing chum with chinook pre-proMCHs reveals a much more striking level of homology (97-99%) between the species than between the two precursors from the same species, indicating that tetraploidy developed before the genus underwent speciation.

C. PRECURSOR MOLECULEOF MAMMALIAN MCH Mammalian MCH shows 76% amino acid identity with the salmonid peptide (excluding the two N-terminal residues of mammalian MCH) but the remaining prohormones (excluding signal) show only 20% identity, the homologous residues tending to be concentrated nearer the C terminus (Fig. 7) (Nahon et al., 1989b; Fellmann et al., 1989). As with fish, several potential proteolytic cleavage sites occur in the precursor so that a number of small peptides could be released. Following the system suggested by Tatemoto and Mutt (198 1 ) for naming neuropeptides, two of these fragments have been named NEI and NGE, viz. pre-proMCH ( I 3 1-144) is neuropeptide-glutamic acid-isoleucine (NEI), and pre-proMCH ( 1 10-128) amide is neuropeptide-glycine-glutamic acid (NGE) (Fig. 7). Fragment NEI is the more highly conserved, showing 30% identity (64% similarity) with salmonid Mgrp. Human pre-proMCH shares 81% identity with the rat precursor (Fig. 7). Both precursors contain 165 residues with the variant amino acids located mainly in the middle region of the molecule. The sequences from residue 114 onwards, which include MCH, NEI and most of the NGE peptide, are identical for the two species (Presse et al., 1990). The structure of the rat proMCH sequence was of considerable interest when it was elucidated because, quite apart from comparison with the teleost MCH precursor. it revealed why the MCH neurons are immunostained by antisera against several other neuropeptides; aMSH, hGRF-37, and CRF (Section 11,4). Workers from the Salk Institute (Nahon et ul., 1989b) showed that rat proMCH has epitopes in common with these other peptides which are recognized by antisera even though the sequence similarities may be very slight. By preabsorbing the antisera with synthetic fragments of rat proMCH, they showed that immunostaining with aMSH antiserum is attributable to the sequence Pro-Ile-NH2 derived from Pro-lle-Gly at the C terminus of peptide NEI. No other sequence similarity between proMCH and aMSH is apparent, and this very slight resemblance between the two molecules emphasizes the inherent pitfalls when interpreting immunostaining results. CRF (24-41) shares six out of a stretch of 18 residues spanning NGE + NEI but immunoabsorption tests suggested that the staining with their CRF antiserum was not attributable to these residues but to

19


I

SALMON RAT

1

HUMAN

1

-

Sianal Peptide

I

MRDSVLSVIFALALFLECYTPSMAI * : * ** * * :* MAKMSLSSYMLMLAFSLFSHGILLSASKSIRNVEDDIVFNTFRMGKAF . . .... . . ....* : * ; . . . . : * : : : : : : : : : : : : * * . * : * : : : : : : * : : * : MAKMNLSSYILILTFSLFSQGILLSASKSIRNLDDDMVFNTFRLGKGF

25 48 48

Signal Peptide

SALMON

26

PMGKMEDTALEQDTLDSLLNEEVA----DKNPDSVRSGSSKIIVLADS 69

RAT

49

QKEDTAERSVVAPSLEGYKNDESGFMKDDDDDK'ITKNTGSKQNLVTHGL 96

HUMAN

49

QKEDTAEKSVI APSLEQYKNDESSFMNEEENKVSKNTGSKHNFLNHG~ 96

.* * :*: * . * .. .. .. .. .. .. . * . . * . . . . . . . . . . .* . . * * * * : *

*

*

.. ..

*:

;:::::*:**

MarD

SALMON

70

RAT

97

HUMAN

*

...

v'

G~LNRGLPL---YKLKAAAAFLDRALTLD~E-ADQDLsPs * : * * : * * : : * : * * : * I sI: 1 13 * * :*: PLSLAVKPYLALKGPAVFPAENGVQNTESTQEKREIGDEENSAKFPIG 144 : : : : * : .. . .. ..... .*. . . ....... .. .. .. .. ....... .. .. .. .. ....... .. .. .. .. ......... .. .. 144

NGE

SALMON

114

RAT

145

HUMAN

145

RR--DTMRCMVGRVYRPCWEV

.. .. .. * : : : * : : : : : : : : * . RRDFDMLRCMLGRVYRPCWQV ..................... RRDFDMLRCMLGRVYRPCWQV

NE I

I

132 165 165

MCH FIG.7 Comparison between the pre-proMCHs from chinook salmon (Minth ef al., 1989). rat (Nahon er al., 1989b), and human (Presse et al., 1990).Gaps (-) are introduced to maximize homologies between sequences. (:), Identical amino acids of different species; (*), conservative substitutions; (A),putative proteolytic sites. Putative neuropeptides, NGE and NEI, which might be released from the mammal precursor, and Mgrp (MCHgene-related-peptide) from salmon, are also indicated. ~~

the same epitope recognized by aMSH antiserum, viz. the C-terminal bulky aliphatic amide of NEI. Lastly, the sequence apparently responsible for immunostaining with hGRF antiserum seems to be an 8-residue stretch in NGE, which shares five identical residues with hGRF (30-37). Within hGRF (30-37), residue Ser34corresponds to pre-proMCH SerIz5;it is probably significant that this single residue is not conserved between the human and rat GRF sequences and that antiserum against rGRF does not recognize the MCH precursor.

20

BRIDGET 1. BAKER

D. GENERAL COMMENTS ON MCH PRECURSOR The fact that the MCH neurons of salmonids are not immunostained by aMSH antiserum (Bird ef al., 1989) is probably explained by the sequence difference between the C-terminal region of NEI in rat (Pro-lle-Gly) and its homologue in the salmonid proMCH (residues 1 11-1 13, Ser-He-Val, Fig. 7). The Cterminal amide group, which is derived from the neighboring glycine residue and is a structural requirement for most antisera against aMSH, cannot be formed in salmonids. In the carp, elasmobranchs, and R. ridibundu at least some MCH cell bodies are immunostained with aMSH antiserum and it is possible that posttranslational processing of their proMCH liberates an appropriately amidated C-terminal region. It remains to be explained, nevertheless, why not all of the MCH perikarya in these species react with the aMSH antiserum. It could be that the unreactive MCH neurons produce a slightly different molecular form of proMCH or that posttranslational processing is different in these neurons. For instance, they may lack the relevant amidating enzymes. In the carp, not only the MCH perikarya, but also their nerve terminals in the neurohypophysis differ in whether or not they react with aMSH antiserum (Powell and Baker, 1987). so the distinction between the two neuronal types is unlikely to be simply a question of the rate of posttranslational processing. Many neuropeptides are expressed in nonneuronal tissues such as the gut or gonads, as well as in the central nervous system. The list includes such peptides as vasopressin, oxytocin, POMC. angiotensin 11, cholecystokinin (CCK) and many others. it is therefore interesting that Northern blot analysis, using both polynucleotide and oligonucleotide probes on mRNA extracted from a wide variety of tissues (pituitary gland, adrenal medulla, eye, testis, pyloric caeca, intestine, liver, kidney, and heart), has failed to provide evidence that MCH is expressed outside the hypothalamus (Ono et al., 1988; Minth et al., 1989).

IV. Biosynthesis of MCH The biosynthesis of MCH from its precursor has been studied only in the rainbow trout Uncorhynchus niykiss (formerly Salmo guirdneri; Bird ef al., 1990). In this species, the MCH perikarya are located as a group on the floor of the hypothalamus and radioactively labeled precursors, such as [3sS]cysteine,can easily be injected into their vicinity. If fish are killed at intervals after the injection, the labeled MCH precursor and its products can be extracted from the hypothalamus and pituitary gland, immunoprecipitated and separated by electrophoresis on sodium dodecyl sulfate (SDS) gels. In practice, the injected pool of labeled precursor becomes depleted quite rapidly, presumably due to diffusion or incorporation into other proteins, so that the MCH neurons behave as though they had

MELANIN-CONCENTRATINGHORMONE

21

received a long pulse of labeled precursor. Such studies show that one hr after injection, the label is associated largely with two high-molecular weight proteins (Fig. 8); these then decline in abundance, presumably as a result of conversion to the smaller molecular weight labeled-products which increase progressively in abundance. Melanin-concentrating hormone itself is barely detectable 1 hr after injection but forms a significant peak at 2 hr and is the predominant labeled product within the hypothalamus by 4 hr. The molecular identity of the intermediate-sized precursors has not yet been confirmed. More products are formed than can be accounted for by cleavage at only the dibasic residues in the precursor, and it is possible that some of the single Arg or Lys residues or other amino acids are used as cleavage sites, Separation of the MCH-immunoreactive products on Sephadex G50 reveals a labeled peak in the predicted position of preproMCH (101-132), i.e., MCH plus the preceding fragments (Mgrp, equivalent to rat NEI). This suggests that cleavage may occur first at Arg-Arg (99-100) and subsequently at Arg-Arg (1 14-1 15) but the low abundance of this immediate precursor of MCH implies that it is short-lived and processed rapidly to release mature MCH and the tridecapeptide (Mgrp). Most of the posttranslational cleavage presumably occurs within the secretory granules as they are translocated down the axons to the neurointermediate lobe. Judging from labeling studies, this passage takes 4 hr or more. Separation of MCH from its precursor by gel-permeation on Sephadex G75 shows that proMCH forms less than 1% of the total MCH-like material stored in the neurointermediate lobe. The change in rate of MCH synthesis following transfer of fish from a pale to a dark background and vice versa has been examined in preliminary experiments, making use of the observation that isolated hypothalamic fragments continue to incorporate ['sS]methionine into MCH precursors and process them to mature MCH in v i m (B. I. Baker and D. J. Bird, unpublished observations). Incorporation is doubled within about 7 days after fish are transferred from black- to white-colored tanks, and is enhanced further if fish are also given a daily stress. Incorporation is reduced at a similar rate when white-adapted fish are moved to black tanks. Processing of precursor to mature MCH also continues in vitro but the rate at which this occurs appears constant and unaffected by the rate of precursor synthesis.

V. Structure-Activity and Molecular Modeling Studies A peptide hormone interacts with its receptor in such a way that receptor conformation is changed and a chain of intracellular events, often referred to as signal transduction, elicits the target cell's response. Not all regions of the hormonal molecule interact with the receptor, but they may be important for other

P


23

reasons; they may assist interaction with the receptor by enhancing binding to the receptor site, influence the quaternary structure of the hormone, or protect it against degradation in the blood. It is useful to understand the role of the various amino acid residues in the hormone should one wish to modify molecular structure - e.g., by the addition of 1251 or during synthesis of tritiated analogs for binding studies - and still retain biological potency. Some insight into the importance of specific residues can be gained from structure-activity studies in which the bioactive potencies of various hormonal fragments and analogs are compared with the native molecule. Several studies have compared the in vitro effect of fragments and analogs of MCH on melanophores from different teleosts and tetrapods. They have been concerned mainly with the importance of the cyclic configuration of MCH, the effect of the N- and C-terminal exocyclic sequences, and the influence of a few specific residues. ON MELANOPHORES A. MCH HASaMSH-LIKEACTIVITY

The groups headed by Hadley and Hruby in Arizona and by Castrucci in Brazil have contributed much to our understanding in this field. They were the first to report that MCH has no melanin-concentrating effect when tested on tetrapod melanophores, but instead exhibits weak melanin-dispersing activity, being about 600-fold less potent than the melanocyte-stimulating hormone, aMSH, on frog Rana pipiens skin and 300 times less potent when tested on the skin of Anolis carolinensis (Wilkes et al., 1984a). The melanin-dispersing activity of MCH has been confirmed by other workers, although the reported potency of MCH relative to aMSH varies in different studies (Baker et al., 1985a; Ide et al., 1985). When tested on mammalian melanoma cells, sMCH similarly shows aMSH-like effects, stimulating tyrosinase activity and melanin synthesis (Baker et al., 1985a). Several observations suggest that the melanin-concentrating and dispersing activities are elicited by different sequences within the MCH molecule. Thus, when MCH is heated with 0.1 M NaOH (a treatment which racemizes aMSH and potentiates its activity) its aggregating activity is destroyed, while its dispersing activity is either enhanced (Baker et al., 1985a) or not affected (Matsunaga et al., 1989b). Analogs of MCH with truncated N- or C-terminal exocyclic sequences have diminished aMSH-like activity when tested on amphibian melanophores, but their melanin-aggregating effect on Synbranchus skin is not necessarily affected FIG.8. I n vivo incorporation of radioactive methionine and leucine into proMCH by the trout. Labeled amino acids were injected into the NLT, around the MCH perikarya. Trout were killed 1 hr, 2 hr, and 4 hr later, the basal hypothalamus was extracted, and immunoprecipitated MCH-related products were separated by SDS gel electrophoresis. [From Bird et al. (1990)l

24

BRIDGET 1. BAKER

(Hadley et al., 1987; Matsunaga et al., 1989b; Castrucci et al., 1989). Conversely, other MCH analogs in which the size of the ring structure, MCH (5-14), is diminished by shifting Cys5 to positions nearer the C terminus, show a markedly diminished MCH-like activity on Synbranchus skin which is dissociated from their melanin-dispersing activity (Lebl et al., 1988). In the case of the analog [Ala5,Cys'o]-MCH,for example, melanin-dispersing activity on frog skin is the same as for native MCH while its melanin-aggregating activity on eel skin is undetectable; another MCH analog, [AlaS,Cys'O]-MCH (5-17) which also has undetectable aggregating activity on eel skin, has diminished dispersing activity compared with sMCH when tested on Rana skin but is 7-fold more potent than MCH on Anolis skin (Castrucci et al., 1989), emphasizing the different requirements of MSH receptors in different species. This point might be important when interpreting the effects of MCH on teleosts. The tests for melanin dispersion described above used tetrapod melanophores but there is evidence that MCH may also cause melanin dispersion in teleosts. When tested on melanophores of Synhranchus, increasing doses of MCH cause progressive melanin aggregation (lo-" M ) followed by progressive melanin dispersion ( M), producing a bell-shaped dose-response curve (Castrucci et al.. 1987: Hadley et al., 1987). In other systems, a curve of this shape is often attributed to loss of receptor sensitivity at the higher hormone concentrations, but this seems not to be the case here. The melanin-dispersing effect of high peptide concentrations is not seen if calcium is absent from the incubation medium (Oshima et al., 1985; Castrucci et al., 1987) and since calcium is necessary for aMSH to bind to its receptors and cause melanin dispersion (Fujii and Miyashita, 1982; Eberle, 1988), it has been proposed that the melanindispersing effect of MCH results from its interaction with the aMSH receptor. The MCH analog [AlaS,Cys'"]-MCHsimilarly causes darkening of MCH-paled eel skin and requires extracellular calcium to do so (Lebl et al., 1989). The fragment MCH (5-17), which is equipotent with MCH in causing melanin aggregation in Synhranchus skin, does not show "autoantagonism" at high concentrations, suggesting that the MCH ( 1 4 ) sequence is of particular importance for aMSH receptor stimulation in teleost skin (Hadley et al., 1987; Castrucci ef al., 1987, 1989). So far, "autoantagonism"has been reported only for the eel Synbranchus, and only with high concentrations of MCH, but it is possible that MCH exerts some melanin-dispersing action at physiological concentrations in this and other species, since its melanin aggregating potency and that of MCH (1-14) can be enhanced up to 10-fold if tested in medium lacking calcium (Hadley etal., 1988; Visconti ef al., 1989; Castrucci et al., 1989). How MCH interacts with aMSH receptors is so far unexplained; the two hormones show no obvious similarities in their primary structure.

25


B. MELANIN-CONCENTRATING ACTIVITY OF MCH The data on the melanin-dispersing effect of MCH and the influence of calcium have been discussed because they have a bearing on the interpretation of work described below. The structure-activity tests with different MCH fragments, using melanophores from different teleost species, have been done in medium containing calcium. They appear to show that the molar potencies of sMCH, MCH (5-17), MCH (1-14), and MCH (5-14) vary in different species (Hadley et af., 1987) (Table 11), which could be interpreted to suggest a difference in structural requirements of their MCH receptors. However, should the melanin-dispersing effect of MCH vary depending on the species, this might contribute to the different aggregating potencies of MCH and some of its fragments. For this and other reasons, interpretation of the various structure-activity studies, done mainly on the eel Synbranchus by Hadley and Castrucci's groups, the Chinese grass carp Ctenopharyngodon by Baker and co-workers, and tilapia by Kawazoe and Kawauchi, is not easy, and only a few general points will be made here. The ring sequence MCH (5-14) is probably the most important for signal transduction. Although removal of the exocyclic sequences (arm structures) causes approximately 100- or 1000-fold loss of potency on eel and carp melanophores, respectively (Table 11), the combined exocyclic sequences, MCH (1-4, 15-17) or MCH (1-4, Aha, 15-17) (where Aha is a spacer molecule) have negligible bioactivity when tested alone (Brown et af., 1990; Baker el al., 1990). Both N - and C-terminal regions apparently serve to enhance the potency of the

TABLE I1 MELANIN-CONCENTRATING POTENCYOF CYCLICsMCH FRAGMENTS ON MELANOPHORES OF DIFFERENTTELEOST SPECIES MCH MCH (5-17) MCH (1-15) MCH (1-14) MCH(1-14) MCH (2-14) MCH (5-14)

Svnbranchus

CaID

Poecilia

TilaDia

100 10 0 h

100

100

looh 10 0 R

-

100 I8od -

-

4d

10 0 h

1O h

-

7R

-

-

Ih

0.lC

-

~

"Valuesare % potency, where MCH is 100%. "Hadley er al. (1987), Castrucci et al. (1987). 'Baker er al. (1990). Wscontin et al. (1989). 'Kawauchi and Kawazoe (1988). Watsunaga et a/.((1989a). PCastrucci et al. (1989). "Kawazoe et al. (1987a).

1'

loo' 100'

1 ow

26

BRIDGET I. BAKER

ring structure, possibly by changing its conformation or by helping to bind the peptide to its receptor site. The individual exocyclic residues vary in importance. Thus, Matsunaga and co-workers (1989a) found that TrpI5 was crucial for interaction with Synbranchus melanophores, MCH (5-1 5) being equipotent with sMCH but MCH (5-14) having only 1% bioactivity. MCH (1-14) also had full potency in some studies on Synbranchus melanophores (Castrucci e f al., 1989) (only 10% potency in other studies; Hadley et af., 1987; Matsunaga et af., 1989a.b) but removal of Asp' reduced potency to 7% that of sMCH, while removal of all four N-terminal residues, to give MCH (5-14), further reduced potency to 1.4%. Rat MCH which has, among other differences, four residue changes in the N-terminal portion (Fig. 5 ) has approximately 60%potency when tested on grass carp (B. I. Baker, unpublished observations). Molecular modeling studies suggest that the exocyclic sequences of sMCH affect the shape of the ring structure; they increase the local conformational flexibility of the two Cys residues and influence interactions between side groups of residues located near the Cys bridge within the ring (Paul et al., 1990). The conformation they induce may improve the interaction of the ring structure with the receptor molecule. It is probable, too, that the side arms enhance potency by helping to bind the peptide to its receptor site. Thus, the bioactivity of [C~S(AC~)'~]-M (9-14) C H is enhanced from aGIc > GlcNAc aMan > aGlc > GlcNAc aMan > aGlc = GlcNAc GlcNAc ( ~ I P G l c N A c ) ,>~ , PGlcNAc > NeuSAc GalNAc al.3GalNAc >> aGalNAc GalNAc al.3GalNAc > aGalNAc aGalNAc = PGalNAc aGal>> aGalNAc CalPl,3GalNAc > a and PGal pGal> aGal>> GalNAc P and a G a l > GalNAc GalPl,4GlcNAc = GlcNAc (PI,4GlcNAc),., a-L-Fuc a-L-Fuc aNeuSAc > aNeuSGc NeuSAc a2,6Gal/GalNAc NeuSAc a2,3Cal

uAbbreviations: Man, mannose; Glc, glucose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Gal, galactose; Fuc, fucose; NeuSAc, sialic acid.

GLYCOSYLATION IN INTESTINAL EPITHELIUM

151

with the publication of the first immunocytochemical localization of a glycosyltransferase at the electron microscopic level (Roth and Berger, 1982). This paper provided a key piece of evidence which led to the subcompartmentation model of the Golgi apparatus, which will be discussed in detail in Section IV,B,lb. At the same time, the opportunity had now arisen to explore the subcellular distribution of glycosyltransferases at the electron microscopic level in intestinal epithelial cells.

IV. Distribution of Intestinal Glycosyltransferases and Their Saccharide Products A.

STUDIES ON

WHOLE TISSUE

1 . Measurement of Glyco.$yltransferase Activities in Adult Animals As mentioned earlier, many of the studies concerning glycosyltransferases in intestinal cells sought to compare activities in the crypt with those in the villus, or to compare glycosyltransferases among various segments of the intestinal tract. Weiser (1 973a,b) compared the glycosyltransferase activity of mature cells in the villus with immature cells of the crypt by measuring the incorporation of radiolabeled monosaccharides into surface membrane glycoproteins, and using this as a measure of the corresponding glycosyltransferase activity. He employed a separation method based on citrate and EDTA to dissociate cells, resulting in epithelial cell fractions which defined a gradient of cells from villus tips to crypts. His results demonstrated that the levels of N-acetylgalactosaminyltransferase, galactosyltransferase, and fucosyltransferase were approximately 10-fold greater on crypt as compared to villus cells, whereas sialyltransferase activity was higher on villus cells. In a subsequent study, however, Weiser and co-workers (1978) not only separated crypt from villus cells, but also prepared membrane fractions, and reported that the basolateral plasma membrane of villus cells was rich in galactosyltransferase activity. This discrepancy with their previous results (Weiser, 1973a,b) was explained as resulting from the presence of glycosidases on the microvilli of intact villus cells in the earlier study which had interfered with the detection of glycosyltransferases on the lateral plasma membrane. However, an alternative explanation was proposed by Lau and Carlson (1981). They found that nucleotide pyrophosphatase, an enzyme that interferes with glycosyltransferase assays, is particularly enriched in intestinal mucosa (especially at the villus tips). By assuring inactivation of this enzyme, they determined that the activity of two galactosyltransferases (one acting on asparagine-linked and the other acting on 0-linked oligosaccharides) displayed essentially identical activities on both crypt and villus cells. They

152

DOUGLAS J. TAATJES AND JURGEN ROTH

advocated exercising caution when interpreting the measurement of intestinal glycosyltransferase activities without recognizing the potential influence of nucleotide pyrophosphatase. In subsequent studies, Weiser’s group (Weiser el a/., 1987; Wilson el al., 1987) took precautions against nucleotide pyrophosphatase activity and analyzed the rat intestinal distribution of two different galactosyltransferases; one acting on N-linked oligosaccharides and the other acting on 0-linked (mucintype) oligosaccharides (this enzyme may be identical to that investigated by Lau and Carlson mentioned above). They were still able to detect both crypt : villus differences as well as differences among intestinal segments for both enzymes. The galactosyltransferase acting on 0-linked oligosaccharides showed increased activity in the duodenum and distal ileum of the small intestine, and the cecum and proximal colon of the large intestine (Wilson eta/. , 1987). These areas of increased activity corresponded to areas of increased mucus production. Moreover, within the duodenum this galactosyltransferase showed a moderately increased activity in cells from the crypt region as compared to those of the villus; however, no such difference was detectable in the jejunum or ileum. The galactosyltransferase acting on N-linked oligosaccharides displayed highest activities in the terminal ileum, cecum, and proximal colon, with lesser amounts detected in the jejunum and duodenum (Weiser et af., 1987). Although they could not demonstrate a difference in total homogenate galactosyltransferase activity between crypt and villus cells, they found that assays for cell surface galactosyltransferase revealed an elevation in the crypts (Section IV,C). Kim and co-workers (1975) devised a planar sectioning technique utilizing a mounted razor blade to cut frozen sections for the separation of crypt from villus cells. Upon homogenization the sections were assayed for glycosyltransferase activity. The results showed that sialyltransferase activity was enriched in crypt cells. whereas galactosyltransferase activity was approximately equal in both regions (Kim ef al.. 1975). These results are in contrast to those from Weiser’s group mentioned above. The discrepancy may have arisen from the different methodologies employed by the two groups for the separation of cell populations or from glycosyltransferase activity variation among intestinal sections. Nevertheless, both separation techniques suffer from the questionable purity of the fractions. Indeed, the importance of the purity of intestinal fractions cannot be overstated, since elements other than intestinal epithelial cells have recently been shown to be the major, if not the only source for sialyltransferases in rat small intestine (Paulson et af., 1989). Glycosyltransferase activities were also shown to vary from the proximal to the distal regions of the rat small intestine (Morita et al., 1986). Specifically, activities for two galactosyltransferases (acting on N- and 0-linked oligosaccharides), two sialyltransferases (acting on N- and 0-linked oligosaccharides), fucosyltransferase and N-acetylgalactosaminyltransferasewere consistently found

GLYCOSYLATIONIN INTESTINAL EPITHELIUM

153

to be higher in distal regions of the small intestine compared with proximal regions. These results were corroborated by the carbohydrate analysis of brush border membranes in proximal and distal small intestine. Interestingly, both sialyltransferase enzymes displayed the lowest activity of all the glycosyltransferases assayed. In an immunocytochemical investigation (Section IV,C) we found that the distribution of the a2,6-sialyltransferase was regionalized within the rat intestine (Taatjes et al., 1988a). Although abundant staining was detectable in all regions of the large intestine, no labeling was detectable in any portion of the small intestine from the same animals. These results were corroborated by direct measurement of enzymatic activity for the a2,6- and a2,3-sialyltransferases. In this case, the activity of these two sialyltransferases (both acting on N-linked oligosaccharides) was undetectable in rat small intestine. These results would appear to conflict with those of Morita et al. (1986) mentioned above, as well as those of Van Halbeek er al. (1983) who reported the presence of sialic acid a2,3 linked to galactose in much glycoproteins from rat small intestine. The apparent discrepancy may be explained in part by the fact that we examined sialyltransferase activity only for the mucosal surface of the intestine scraped from the intestinal wall. Indeed, in a subsequent investigation, Paulson et al. (1989) found that homogenates of the intestinal wall itself contain substantial levels of this sialyltransferase in the small intestine. Moreover, in contrast to the results of Morita er al. (1986) they found that the activity for a sialyltransferase (adding sialic acid in an a2,3 position to galactose in 0-linked oligosaccharides) actually decreased in the mucosa from proximal to distal small intestine. Ironically, no activity was detectable in the ileal mucosa, whereas substantial activity was measurable in the ileum wall. Thus, results obtained from intestinal homogenates or segments are not directly comparable. This also indicates that these sialyltransferase enzymes are differentially expressed within different regions of the small intestine, each having specialized functions, yet identical cell types. These results have recently been further supported by in siru lectin-binding studies (Section IV,A,6). The situation is further complicated by the acceptor substrates used for the measurement of glycosyltransferase activity. For instance, according to the “one-enzyme one-linkage’’ hypothesis (Hagopian and Eylar, 1968) at least a dozen different sialyltransferases must exist in order to form the known linkages of sialic acid to penultimate sugars. Thus, fetuin, which is quite often employed as an acceptor substrate for sialyltransferase activity, contains both N- and 0linked carbohydrate groups which are acceptors for at least four different sialyltransferases (Kim er al., 1975; Weinstein et af., 1982; Green et al., 1988). This may explain in part the sometimes variable results reported using different techniques with respect to glycosyltransferase distribution within the intestine. Earlier studies demonstrating glycosyltransferase activity based upon known acceptor substrates may have actually been measuring the activity of several

154


different enzymes. However, due to antibody specificity, immunological-based techniques are assaying for the presence of one highly linkage-specific glycosyltransferase. Therefore, these limitations should be borne in mind when interpreting results from different investigators employing different techniques. Much less information is available concerning the glycosyltransferase activity in the large intestine. Freeman et al. (1978) have demonstrated that in adult rat large intestine both gaiactosyltransferase and sialyltransferase activities were significantly greater in proximal than in distal colonic mucosa. We have reported (Taatjes e f al.. 1988a) enzymatic activity for two sialyltransferases acting on asparagine-linked oligosaccharides in mucosal scrapings from rat large intestine (Section 1V.C; Table IV).

2 , Measurement of Glvcosyltransferase Activity during Development The activity of several glycosyltransferases has been found to vary during postnatal development in both rat small and large intestine. Sialyltransferase activity was present in increased levels during the suckling period, and decreased 5-fold during the subsequent weaning and adult periods (Chu and Walker, 1986). On the other hand, fucosyltransferase activity was very low during suckling phase, rapidly increased during weaning, and reached adult levels by 5 weeks of age (Chu and Walker, 1986). The activities of two galactosyltransferases, the UDP-Gal : GlcNAc(P1-4)galactosyltransferase, and UDP-Gal : GalNAc(p 1 -3)-galactosyltransferase have also been found to be under developmental regulation (Ozaki et al., 1989). Both glycosyltransferases demonstrated a marked elevation in activity after the weaning period and into adulthood in all regions of the rat small intestine. All of these results considered together demonstrate that activities for galactosyltransferase, N-acetylgalactosaminyltransferase and fucosyltransferase all increase during postnatal development of rat small intestine. On the other hand, sialyltransferase activity declines during the same developmental period. These data correlate well with known changes in terminal glycosylation of microvillar proteins during postnatal development of rat small intestine (Sections IV,A,4 and 6). The activity of galactosyltransferase has been measured in fetal and postnatal rat large intestine (LaMont and Ventola, 1978). The activity in fetal homogenates increased 4-to 7-fold between 18 and 22 days, the last 4 days of gestation. The enzyme activity then gradually increased postnatally, reaching adult levels by day 15. Determination of the autoradiographic incorporation of [3H]galactose into fetal large intestine glycoconjugates correlated well with the increase in galactosyltransferase activity during this period (Rampal et al., 1978). Interestingly, these autoradiographic studies revealed the selective incorporation of [3H]galactoseinto goblet cells but not into absorptive cells. These results therefore suggest that the maturation of fetal rat large intestine during the


155

last 4 days of gestation is accompanied by the appearance of goblet cells and enhanced mucus synthesis.

3. Analysis of Sugar Content of Membrane Glycoproteins in Adult Animals Two basic biochemical methods have traditionally been applied in order to determine the carbohydrate composition of intestinal membrane glycoproteins. The first method requires the administration of radiolabeled sugar precursors into the lumen of the intestine, followed by purification and analysis of the incorporation of the sugars into membrane glycoproteins. The second method employs the binding of labeled lectins to intestinal plasma membrane fractions. In an early study, Kim and Perdomo (1974) traced the incorporation of [14C]glucosamineinto the membranes of intestinal cells. They prepared three membrane fractions, consisting of smooth, rough, and brush border membranes. They observed incorporation first into smooth membranes, followed after a lag period by entrance into rough and brush border fractions. Aside from the peculiar late entrance into a rough membrane fraction, these results trace the transit of ['4C]glucosamine-containingglycoconjugates from the Golgi apparatus to the brush border. A further purification to distinguish apical (brush border) membranes from basolateral was not attempted. Quaroni and co-workers (Quaroni et al., 1980; Herscovics et al., 1980) took these studies further by separating Golgi apparatus, apical, and basolateral membranes, both in crypt and villus cells. They measured the incorporation of ~ - [ 5 , 6 3H]fucose and ~-[2-~H]mannose into intestinal membrane glycoproteins following an intraperitoneal injection of these radiolabeled sugars. The incorporation of mannose was roughly equal in crypt and villus cells, whereas fucose incorporation was elevated in the differentiated villus cells (Quaroni et al., 1980). Fucosylated glycoproteins were originally detected in the Golgi apparatus and basolateral membranes, followed by redistribution into villus membranes after 3 4 hr. In contrast, most mannose-labeled glycoproteins remained in the Golgi and basolateral membrane fractions. They interpreted their results to indicate that fucosylated glycoproteins represent a special class of membrane components that appear with differentiation (absent in undifferentiated crypt cells) and are specifically localized to the luminal portion of the intestinal cell plasma membrane. In an accompanying paper, Herscovics et al. (1980) used similar techniques to demonstrate that high-mannose oligosaccharides were the precursors of complex oligosaccharides. Moreover, they provided evidence that luminal membranes of both crypt and villus cells were greatly enriched in complex oligosaccharides as compared with basolateral plasma membranes, but no qualitative changes were found to occur during cellular differentiation. Thus, their results suggested that intestinal epithelial cells were polarized with respect to plasma membrane glycoconjugate oligosaccharide composition (Section IV,A,5).

156


The second biochemical method, utilizing lectin binding to isolated cell plasma membranes, is a more recent innovation, and has found widespread use for the comparison of membrane glycoproteins among intestinal segments as well as during maturation. Kim and co-workers (Morita et a [ . , 1986) examined the reactivity of brush border membrane components with lectins, and the binding of brush border membrane-associated enzymes to Ricinus communis lectin I (RCL I) and wheat germ agglutinin (WGA) in segments from the proximal and distal small intestine. In addition they analyzed the carbohydrate composition of the brush border membranes. Their results indicated that although brush border membrane glycoproteins from distal portions of the small intestine contained more complete oligosaccharide side chains, the glycoprotein profile on SDS gels was less complex than in proximal small intestine. Specifically, more WGA and succinylated-WGA-binding glycoproteins were present on brush border membranes from proximal compared to distal segments. However, the binding of RCL I to brush border membranes was two times higher in the distal as compared to proximal small intestine. Overall sugar content was higher in distal small intestine brush border membranes, reflected mainly by elevated galactose and sialic acid content. The content of N-acetylglucosamine appeared equal in the two intestinal segments. These results suggest that the carbohydrate content of brush border membranes changes with the progression of the gastrointestinal tract, with more distal regions of the small intestine containing more completed oligosaccharide chains. 4 . Analysis of Sugar Content of Membrane Glycoproteins During Development

Similar biochemical methods to those just mentioned above have also been used extensively to investigate changes in the glycosylation pattern of microvillar proteins in postnatal intestine. Mahmood and Torres-Pinedo ( 1 983) incubated microvillar membrane preparations from postnatal rats with radiolabeled lectins to determine the carbohydrate profile of membrane glycoconjugates. They found that the microvillus membrane of suckling rats (from birth to about 2 weeks of age) was rich in glycopeptides containing binding sites for peanut lectin (PNL) in sialyl-substituted form. During the weaning phase (14-21 days postnatal), the membranes lost about half of these binding sites, accompanied by decreased sialic acid content and increased content of glycopeptides containing unsubstituted binding sites for soybean lectin (SBL) and RCL I. Perhaps the most important result from this study was the finding that the sialic acid content of microvillar plasma membrane drastically decreases from the suckling to the weaning period. Indeed, in a subsequent paper (Torres-Pinedo and Mahmood, 1984) they found that this decrease in microvillar plasma membrane sialic acid content was accompanied by a dramatic rise in fucose content. They observed that the binding of '2sl-labeled WGA to neuraminidase-sensitive sites in the microvillar membrane decreased markedly from early suckling to weaning ages.


157

At the same time, the binding of Iz5I-labeledUlex europaeus lectin I (UEL I) to microvillar membranes showed an opposite increase from suckling to weaning periods. This developmentally related shift from sialylation to fucosylation was found for both glycoproteins and glycolipids of the microvillar membrane, suggesting that it is a general phenomenon for membrane constituents. They postulated that such a dramatic shift from a strongly acidic to a more neutral microvillar glycocalyx could relate to the physiological changes occumng in the intestine concomitant with development. These studies were followed up by examining the carbohydrate profile of individual microvillar membrane proteins during postnatal development of the rat small intestine. Srivastava et al. (1987) found that the terminal glycosylation of several microvillar glycoproteins of >90,000 Da (most likely hydrolases) does not reach complete maturation until after weaning, although their content within the membrane has reached adult levels by this time. Moreover, several of these glycoproteins were fully sialylated during the suckling period, whereas addition of N-acetylgalactosamine and fucose continued well into the weaning period. Buller el al. (1990) took such investigations one step further by examining the glycosylation of a known glycoprotein, lactase-phlorizin hydrolase, during development of the rat small intestine. Lectin binding to the enzyme immunoprecipitated from microvillus membranes revealed the presence of both N- and 0linked oligosaccharide chains containing mannose and galactose, which did not vary throughout development. In contrast, the content of fucose and sialic acid was developmentally regulated; sialic acid was present at weaning and declined through adulthood, whereas fucose was not detectable until rats were 20 days of age. Thus, by examining a single enzyme, it was established that the core N- and 0-linked oligosaccharide structures of this microvillar hydrolase remain constant during development, whereas alteration in terminal glycosylation occurs with a shift from sialic acid at suckling to fucose in adulthood. The above studies taken together show that a definite change in glycosylation occurs on specific microvillar membrane glycoproteins during the postnatal developmental period. In a related study (Jaswal et al., 1988), the content of sialic acid and fucose in enterocytes was measured in crypt and villus cells from suckling and adult animals. In suckling animals, no change was found in the sialic acid content of enterocytes during progression from crypt to villus. In contrast, the sialic acid content decreased precipitously from crypt to villus in adult animals. The fucose content of enterocytes from suckling animals was greater in the crypts than in the villus, whereas in adult animals fucose content was much greater in the villus.

5 . Cytochemical Detection of Lectin Binding to Intestinal Cells in Situ Much cytochemical data based upon lectin binding studies have contributed to the understanding of intestinal glycosylation patterns. In the absence of

158

DOUGLAS J . TAATES AND JURGEN ROTH

glycosyltransferase enzymatic measurements, lectin-binding sites can be taken as indicative of specific glycosyltransferase activity. The earliest studies used lectins conjugated to fluorescent dyes in a direct labeling technique, usually on frozen or paraffin sections. Such investigations demonstrated differences in lectin-binding patterns to intestinal epithelial cells in the various segments of the intestinal tract. For instance, Etzler and Branstrator (1974) examined the binding of FITC-conjugated lectins from Dolichos biflorus (DBL), Lotus terragonolobus (LTL), Ricinus communis I, and Triticum vulgare (WGA) to the various regions from rat small intestine. They observed differences in the binding of the lectins to both the epithelial cell plasma membranes, as well as to the goblet cell mucus. With respect to plasma membrane staining, LTL, RCL I, and WGA bound to the microvillar portion of the epithelial cells lining the crypts and villi in the proximal regions of the small intestine. This pattern of staining was altered along the first 15 cm of the small intestine, such that distal to this point the apical surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with LTL and RCL I, while WGA stained the apical surfaces of cells lining the villi. In the distal small intestine, LTL, RCL I, and WGA stained the cell surfaces of only those epithelial cells at the base of the villi and in the crypts. DBL did not stain the epithelial cell surface in any portion of the small intestine. With respect to staining of the mucus content of goblet cells, WGA and DBL stained the goblet cells in proximal portions of the intestine, whereas in middle and distal regions all four lectins were found to stain goblet cell mucus. These results suggested that the content of complex carbohydrates in goblet cell mucus increases from proximal to distal regions of the small intestine. In a preembedding peroxidase study, Ovtscharoff and Ichev (1984) showed that in rat small intestine (middle regions), the pea and soybean lectins stained the microvillar membrane from epithelial cells in the crypts and lower villus more intensely than those in the upper villus and lumen. Essner et al. (1978) also used several lectins conjugated to FITC to investigate the lectin-binding pattern to cryostat sections from portions of the descending colon of the rat. Besides reactivity in goblet cell mucus and plasma membrane, they identified cytoplasmic staining which they attributed to the Golgi apparatus. Binding sites for the lectins from Glycine max (SBL) and Dolichos hiflorus were observed in goblet cell mucus, apical and basolateral plasma membranes, and in the apical cytoplasm, indicating the presence of terminal nonreducing N-acetylgalactosaminyl residues at these sites. WGA, RCL I, UEL I, and concanavalin A (Con A) all stained the cytoplasm of epithelial cells, but did not, or only weakly, stain mucus droplets and plasma membranes. Gorelick et al. (1982) examined lectin-binding patterns in the plasma membrane and goblet cell mucus of epithelial cells in the various regions of guinea pig large intestine. Staining of the brush border with the various rhodamine-labeled lectins tended to be heterogeneous across the regions of the large intestine.

GLYCOSYLATION IN INTESTINALEPITHELIUM

159

In general, though, Con A, WGA, RCL 11, and PNL stained the brush border of the right colon, whereas RCL I and SBL stained the transverse colon intensely. Limulin reacted with the brush border only in the left colon. Staining of the goblet cell mucus was even more variable. Gorelick et al. (1982) not only examined the lectin binding to the goblet cell mucus in the various regions of the large intestine, they also separated the intestinal segments according to crypt regions: basal, middle and apical. Again, the staining patterns varied not only among the large intestinal segments, but also within crypt region of individual segments. Noted exceptions were LTL and PNL which did not stain the goblet cell mucus in any area examined. Moreover, goblet cells in the transverse and left portions of the large intestine tended to react more intensely with the various lectins employed, suggesting a maturation of goblet cell mucus along the large intestine. The paper by Gorelick et al. (1982) also served to usher in the modem approach to investigating intestinal lectin-binding sites; namely, the postembedding application of colloidal gold-labeled lectins to ultrathin sections. They prepared a complex of colloidal gold particles with RCL I1 and applied this to sections from intestine embedded in Epon-Araldite. They were able to demonstrate binding of this complex to goblet cell mucus, apical plasma membrane, apical cytoplasmic vesicles, and Golgi apparatus. Since this time, numerous papers have been published utilizing both colloidal gold-labeled lectins and peroxidase-labeled lectins at the light and electron microscopic level to investigate intestinal glycosylation patterns. The main,benefit of such studies has been the increase in resolution over fluorescence studies obtainable with these methods. Many of these studies were interested in investigating the role of the Golgi apparatus in glycosylation, and their results will be described in Section IV,B,3. In the current section we will detail the results of these studies as they relate to plasma membrane and mucous glycoconjugates. Helixpomatiu lectin (HPL) binding has been observed (Fig. 10) in the mucus and apical plasma membrane of chick duodenum (Roth, 1984), rat duodenum (Ellinger and Pavelka, 1985), and rat jejunum (Murata er al., 1986). RCL I binding has been reported in the mucus and apical and basolateral plasma membranes of chick (Roth, 1983b) and rat duodenum (Ellinger and Pavelka, 1985), and the basolateral plasma membrane of rat proximal colon epithelial cells (Roth et al., 1988a). Pavelka and Ellinger (1989b) have shown binding of Erythrina cristagalli lectin (ECL) to apical and basolateral plasma membranes and goblet cell mucus in rat duodenum, while Egea et al. (1989) have reported identical results by using Daturu srramonium lectin (DSL). Lotus terragonolobus lectin (LTL) binding to the apical plasma membrane and goblet cell mucus has been described in chick duodenum (Roth, 1983b), while UEL I binding to apical plasma membrane and goblet cell mucus has been reported for rat duodenum (Ellinger and Pavelka, 1988a). Binding of sialic acid-specific lectins has also been documented in intestinal cells. Roth et ul. (1984) found binding of

160



161

the Limaxflavus lectin (LFL) to the apical plasma membrane and goblet cell mucus in rat distal colon. Similar results were also found in rat proximal colon (Taatjes and Roth, 1988). Recently, staining with Maackia amurensis lectin (MAL) has been reported in the apical and basolateral plasma membranes and goblet cell mucus in pig colon (Sata el al., 1989). Furthermore, we have recently observed staining with Sambucus nigra L. lectin (SNL I) complexed with colloidal gold particles (Taatjes et al., 1988b) in mucus droplets, but not in the plasma membrane of rat jejunal epithelial cells (Taatjes and Roth, 1990; Section IV,A,6). The main message resulting from all of these studies is that lectins recognizing complex carbohydrate structures bind to plasma membranes and goblet cell mucus in both small and large intestine from various species.

6 . CyfochemicalDetection of Lectin Binding to Intestinal Cells in Situ During Development and Diferentiation As described in Section IV,A,2, the activities of several glycosyltransferases are altered during intestinal cell development. Such alterations are also reflected in the modification of lectin binding to epithelial cells that occurs during postnatal development. In the rat small intestine, Etzler and Branstrator (1979) found developmental changes in the binding of RCL I, LTL, and WGA. RCL I stained the brush border of epithelial cells as early as 1 hr after birth. The staining became patchy at the cell surface over the next few days, reacting uniformly with the surface 5-14 days after birth. By 19-24 days postnatal, the epithelial cell surface began to lose its ability to react with RCL I, and by 30 days postnatal, the cell surfaces were no longer stained with RCL I. The onset of LTL staining was a much later event, commencing between 11 and 19 days after birth. By 28 days after birth, regional differences were apparent with respect to LTL binding to intestinal cell surfaces; brush borders of cells lining the villi in the distal portion of the small intestine were no longer bound by LTL. Wheat germ agglutinin (WGA) stained the brush border of epithelial cells from 1 hr after birth, until about postnatal day 19 when cells lining the villi were no longer stained with this lectin. We have recently investigated the binding of sialic acid-specific and fucosespecific lectins to developing rat small intestinal cells (Taatjes and Roth, 1990). In line with the results detailed in Sections IV,A,2 and 3 concerning developmental-related changes in sialyltransferase and fucosyltransferase activities, as FIG. 10. Low-power electron micrograph demonstrating H . pomatiu lectin-gold binding sites in chick duodenum. In the center of the micrograph a prominent goblet cell is displayed, with intense gold particle labeling present in the goblet cell mucus (asterisk), in the Golgi apparatus, and in the apical plasma membrane (arrowheads). Label is also observable in the apical plasma membrane of adjacent absorptive cells (arrowheads). X 1,710. Bar = 5 p n . (Reproduced from the J . Cell B i d . 1984.98, 399406 by permission of the Rockefeller University Press.)

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DOUGLAS J. TAATES AND JURGEN ROTH


163

well as the lectin-binding results of Etzler and Branstrator (1974), we found that binding of SNL I, LFL, and UEL I to intestinal cells changed with postnatal development. SNL I (Fig. lla,b) and LFL stained the brush border and mucus droplets in animals during the suckling phase. During weaning (day 23) we found that individual epithelial cells were no longer stained with SNL I (Fig. llc,d and Fig. 12) and LFL. By adulthood, staining with these two sialic acidspecific lectins was restricted to goblet cell mucus and cells in the lamina propria and submucosa (Fig. 1 le,f). In contrast, binding of fucose-specific UEL I was restricted to goblet cell mucus during the suckling phase, but by day 23 postnatal appeared in the brush border of some epithelial cells. In adults, intense staining with UEL I was found in goblet cell mucus and in the brush border of epithelial cells. All of these results taken together support the premise that during postnatal development of rat small intestine, a progressive change from sialylation to fucosylation of brush border glycoconjugates occurs. Caldero et al. (1988) have performed a detailed investigation of changes in glycoconjugate composition of the rat colonic mucosa during development. They used a battery of eight fluorescein-conjugated lectins recognizing a variety of sugar residues. Their results demonstrated that each lectin showed a unique developmental staining pattern, including differences between the various regions of the colon. In all cases, the adult pattern of staining was achieved 25-30 days after birth. Differentiation-related changes in intestinal cell glycosylation patterns have been described in adult animals during cell migration from crypts to the villus or lumen. Some of these were already described above (Section IV,A,S; Etzler and Branstrator, 1974). We have investigated the localization of LFL binding sites in the plasma membrane of rat colonic epithelial cells during differentiation (Taatjes and Roth, 1988). We found that in the crypt regions, goblet and absorptive cell precursors were stained along their entire plasma membrane (Fig. 13); that is, both apical and basolateral plasma membranes were stained. However, when cells reached the zone of migration (Eastwood, 1977) the staining with LFL became restricted to the apical plasma membrane (Fig. 13). This polarized staining remained a feature of fully mature epithelial cells (both absorptive and goblet) located at the intestinal lumen. These results suggest that a feature of

FIG.1 1 . Light micrographs illustrating the detection of SNL I-gold binding sites in epithelial cells during postnatal development of rat jejunum. At postnatal day 1 (a,b), staining is present in the epithelium along the apical plasma membrane (arrowheads)and in the goblet cell mucus (asterisk). By postnatal day 23 (c,d), individual cells in the epithelium are not stained by the SNL I-gold complex (arrows). In adult animals (e,9, the apical plasma membrane (arrowheads)of all epithelial cells is not stained by SNL I-gold complex, whereas goblet cell mucus (asterisks) and the plasma membrane of cells in the lamina propria (Ip) are intensely stained. a,c,e, Bright-field micrographs; b,d,f, correspondingphase-contrast images. lp, lamina propria. X 368 (a-9. Bar = 5 pm.

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DOUGLAS J . TAATJES AND JURGEN ROTH


165

fully differentiated colonic epithelial cells is the polarization of the plasma membrane with respect to the distribution of sialic acid residues on membrane glycoconjugates. A similar phenomenon was apparent in the small intestine, although fully differentiated small intestinal epithelial cells display very sparse LFL binding sites. B. GOLGIAPPARATUS In an early study, Kim and co-workers (1 97 1) investigated the subcellular distribution of the then called “multienzyme system” of glycosyltransferases in rat small intestinal mucosal scrapings. They determined that the polypeptide : Nacetylgalactosaminyltransferase, galactosyltransferase, N-acetylglucosaminyltransferase, and N-acetylgalactosaminyltransferase were enriched in a smooth microsome fraction. This was the first detailed report of the localization of glycosyltransferases in intestinal tissue, and quite accurately determined them to be located in a fraction most likely representing Golgi apparatus membranes. In a subsequent investigation, Kim and Perdomo (1974) investigated the intestinal membrane distribution of five glycosyltransferases: two galactosyltransferases (acting on N- and 0-linked oligosaccharides), sialyltransferase, fucosyltransferase, and N-acetylgalactosaminyltransferase.They found that all five enzymes were enriched in a smooth membrane fraction (Golgi apparatus), with only background amounts detected in a rough membrane fraction and a brush border membrane fraction. More recently the techniques of immuno- and lectin cytochemistry have helped to unravel the pattern of glycosylation reactions in the intestinal cell Golgi apparatus. However, before we begin to detail these intestinal studies, it will be helpful to briefly review the concept of general Golgi apparatus glycosylation as formulated by the assimilation of data from several different techniques.

1. Subcompartmentation Model of the Golgi Apparatus Several recent reviews have considered this topic in detail and the interested reader should refer to them for more information (Dunphy and Rothman, 1985; Farquhar, 1985; Kornfeld and Kornfeld, 1985; Roth, 1987a; Roth and Taatjes, 1989). Briefly, this model proposes that the Golgi apparatus cisternal stack is FIG. 12. Demonstration of SNL I-gold binding sites in the jejunum of sections from postnatal day 23 rat. The apical plasma membrane (brush border) of adjacent epithelial cells is shown. Large gold particles (14-nm diameter) indicative of SNL I binding sites are restricted to the cell on the right. To rule out possible processing artifacts, this section was also stained with RCL I/asialofetuin-gold (small gold particles; 10-nm diameter). As can be seen, both cells are RCL I positive, indicating that loss of binding sites is specific for SNL I. The lateral plasma membrane (arrowheads) separating the two cells contains binding sites for both lectins. X 66,000. Bar = 0.15 pm.

166


FIG. 13. Detection of sialic acid residues with the Limo.rf7avus lectin/fetuin-gold technique at the plasma membrane of rat colonic absorptive cells. in differentiated cells at the surface epithelium (a). gold particle label is restricted to the apical plasma membrane (arrowheads). Note the lack of staining in the basolateral plasma membrane (asterisks). In contrast, the basolateral plasma membrane of undifferentiated absorptive cells from the crypts region (b) is intensely stained for sialic acid residues (arrowheads). n, nuclei of absorptive cells. X 18,000 (a); X 55,000 (b). Bars = 0.6 p n ( a ) and 0.2 p n (b). (Reproduced with permission from Taatjes and Roth, 1988.)


I67

functionally subcompartmentalized with respect to the steps involved in the processing of oligosaccharide side chains of glycoconjugates. Although based upon the maturation of N-linked oligosaccharides of glycoproteins, support for this model has also been presented for the case of 0-linked oligosaccharides of glycoproteins. Inherent in this concept is the premise that glycosyltransferases which act early in the pathway are preferentially located in cis-cistemae of the Golgi apparatus, whereas those acting at intermediate steps are located in middle cisternae, and those acting at terminal steps are housed in trans-cisternae. This compartmentation, or spatial separation, would allow the enzymes to act upon an oligosaccharide chain in an “assembly line” progression, without risk of interfering with the action of one another. This model is thus very attractive biochemically, and indeed has received much experimental support. For instance, in cell fractionation studies utilizing analytical sucrose gradients, activities for earlier and later acting oligosaccharide-processing enzymes were detected in distinct Golgi apparatus fractions (Dunphy et al., 1981; Dunphy and Rothman, 1983; Goldberg and Komfeld, 1983). More direct evidence, however, has been provided by numerous investigations analyzing the in situ cytochemical detection of various sugar residues with lectins (Pavelka, 1987; Roth er al., 1988b) and by the immunolocalization of a few glycosyltransferases (Roth and Berger, 1982; Dunphy er al., 1985; Roth et al., 1985a). Indeed, the first direct demonstration of Golgi apparatus subcompartments was provided by the immunocytochemical localization of galactosyltransferase by Roth and Berger (1982). They found that galactosyltransferase immunoreactivity colocalized with thiamine pyrophosphatase activity in one or two trans-Golgi apparatus cisternae in HeLa cells. This result indicated that the Golgi apparatus contained at least two compartments with respect to glycosylation reactions: cis (defined as galactosyltransferase negative) and trans (defined as galactosyltransferase positive). The number of identifiable subcompartments increased to three with the localization of N-acetylglucosaminyltransferase I to middle cisternae of the Golgi apparatus stack (Dunphy et al., 1985). Finally, the most distally acting glycosyltransferase, sialyltransferase, was detected in two trans-cisternae and a complex trans-tubular network continuous with these cisternae in rat hepatocytes (Roth er al., 1985a). Interestingly, sialyltransferase immunoreactivity was found in portions of the Golgi apparatus stack which also contained cytochemically demonstrable thiamine pyrophosphatase activity, a classical trans-Golgi marker, or acid phosphatase (CMPase) activity, a classical marker for the GERL element of the Golgi apparatus. These results suggested that in hepatocytes the Golgi apparatus is composed of three subcompartments with respect to glycosylation reactions: cis, so far delineated by what it does not contain; middle, containing N-acetylglucosaminyltransferase I; and trans, containing sialyltransferase. We include the trans-tubular network (Rambourg and Clermont, 1990), or trans-Golgi network (Griffiths and Simons, 1986) as part of the trans-Golgi apparatus since

168


functionally it is involved in sialylation as are trans-cisternae, and structurally it is continuous with trans-Golgi cisternae (Roth et al., 1985a; Taatjes and Roth, 1986). This view differs from that of Griffiths and Simons (1986), who regard the trans-Golgi network as perhaps a fourth Golgi subcompartment, separate from trans-cistemae. Moreover, other investigators have proposed that galactosyltransferase is housed in trans-Golgi apparatus cisternae, whereas sialyltransferase is separated and housed in the more distally located trans-Golgi network (Berger and Hesford, 1985; Thorens and Vassalli, 1986; Berger et al., 1987). Several pieces of evidence contest this view. First, we have shown that sialyltransferase is not only localized in the trans-Golgi network of hepatocytes, but also quite clearly in two trans-cistemae of the Golgi apparatus stack (e.g., Fig. 3 in Roth ef al., 1985a). Second, by double-labeling immunofluorescence we found an identical codistribution of galactosyltransferase and sialyltransferase irnmunoreactivity in cultured rat hepatocytes (Taatjes el al., 1987). Third, Geuze and co-workers ( 1985) found that galactosyltransferase was detectable in the trans-Golgi network, in addition to trans-cisternae in hepatoma cells and liver hepatocytes. Fourth, galactose residues detected with RCL I, were found in the trans-cisternae and trans-Golgi network of hepatocytes (Lucocq et al., 1987). Clearly, the ability to resolve more and more Golgi apparatus subcompartments will come with the introduction of more Golgi apparatus-specific antibodies. Of certain importance for the previous discussion will be the simultaneous immunocytochemical demonstration of galactosyltransferase and sialyltransferase in the same Golgi apparatus at the electron microscopic level. Due to the constraints placed upon imrnunocytochemical investigations by antibody cross-reactivity with other animal species, this experiment has not proven possible. Moreover, cell-specific variability with respect to the organization of Goigi apparatus subcompartments may have been a factor in the above described discrepancies.

2 . Immunoc:\.tochemicalLocalization of Glycosyltransferases in Golgi Apparatus of Intestinal Epithelial Cells To this date, only two glycosyltransferases have been immunocytochemically detected in the Golgi apparatus of intestinal cells; yet, their localization has yielded most interesting results. After having reported on the localization of sialyltransferase in hepatocytes, we sought to expand on these findings by performing similar localizations on intestinal cells. When we examined the Golgi apparatus distribution of sialyltransferase in goblet cells from the rat colon, we observed the expected result (Fig. 14). Namely, immunoreactivity was restricted to transGolgi apparatus cisternae (Roth et al., 1986). Likewise, sialic acid residues, as detected with LFL, were localized to trans-cisternae. However, quite surprisingly when we examined neighboring absorptive cells a quite different pattern of labeling emerged: the entire Golgi apparatus cisternal stack (with the exception of the fenestrated first cis-cistema) was labeled (Figs. 14 and 15a). In a fashion similar


169

FIG.14. Immunocytochemical localization of sialyltransferasein surface epithelial cells from rat proximal colon. Gold particle label is restricted to trans-Golgi apparatus cistemae in a goblet cell (gc), whereas in a neighboring absorptive cell (ac) label is detectable throughout the Golgi apparatus cistemal stack (with the exception of the fenestrated first cis-cistema). Label is also present in the goblet cell mucus droplets (md) and in the lateral plasma membrane separating the two cells (arrowheads). X 25,500. Bar = 0.4 pm. (Reproduced with permission from Roth er al., 1986.)

to that observed in goblet cells, the distribution of sialic acid residues in absorptive cells was found to mirror that of the sialyltransferase enzyme (Fig. 15b). Quantitative evaluation of the distribution of sialyltransferaseimmunolabel in the Golgi apparatus of absorptive versus goblet cells confirmed the differential label

I70


FIG. 15. Gold particle label for sialyltransferase (a) and sialic acid residues (b) is distributed throughout the absorptive cell Golgi apparatus cistemal stack (with the exception of the fenestrated first cis-cistema) from rat proximal colon. X 63,000 (a): X 66,500 (b). Bar = 0.16 pm (a) and 0.15 pm (b). (Reproduced with permission from Roth PI of., 1986.)

171

GLYCOSYLATION IN INTESTINAL EPITHELIUM TABLE I1 QUANTIFICATION OF IMMUNOLABEL FOR SIALYLTRANSFERASE IN RAT PROXIMAL COLONIC EPITHELIAL CELLS? Absorptive cell (n = 26) Golgi apparatus cistema”

Gold particle/pm 0.21 +/2.55 +/2.34 +/3.42 +/3.63 +/4.14 +/6.20 +/-

0.08 0.18 0.22 0.33 0.24 0.28 0.41

Total length (p) 115.5 134.3 138.4 132.1 126.8 124.1 125.7

Goblet cell ( n = 23) Gold particle/pm

Total length (pm)

0.45 +I- 0.24 0.16 +/- 0.08 0.15 +/- 0.08 0.16 +/- 0.09 0.18 +/- 0.09 0.18 +/-0.10 0.43 +/- 0.27 4.50 +/- 0.26 6.64 +/- 0.33

148.5 148.5 154.3 154.3 151.7 148.5 147.3 147.9 148.5

“From Roth er nl. (1986). ”Cistema 1 designates the fenestrated first cis-cisterna and the following numbers the subsequent cistemae toward the trans side of the Golgi apparatus.

observed on micrographs (Table 11). Moreover, it was apparent that although the labeling was diffuse throughout the Golgi apparatus of absorptive cells (with the exception of the fenestrated first cis-cistema), the labeling intensity increased gradually from the cis to the trans side. This was the first demonstration of an apparent lack of subcompartmentation for a glycosyltransferase within the Golgi apparatus cistemal stack. In the same study, we found that another terminal glycosyltransferase, the blood group A N-acetylgalactosaminyltransferase,was distributed in strikingly different patterns in the Golgi apparatus of absorptive versus goblet cells from human intestine, restricted to trans-cisternae in goblet cell Golgi apparatus, and diffusely localized throughout the absorptive cell Golgi apparatus (Fig. 16). In accordance with the matching labeling in the Golgi apparatus for sialyltransferase and sialic acid residues in rat intestine, the distribution of blood group A substance (detected with a monoclonal antibody) mirrored that of the blood group A N-acetylgalactosaminyltransferasein both absorptive and goblet cells. These results were then confirmed and extended in further studies of human intestinal cells (Roth er al., 1987, 1988~).Thus, for both N-linked oligosaccharide processing (sialyltransferase),as well as 0-linked (blood group A N-acetylgalactosaminyltransferase) a terminal glycosyltransferase was not distributed in the Golgi apparatus cisternal stack as would be predicted by the subcompartmentation model. The implications of these findings for the elaboration of oligosaccharide side chains of glycoconjugates in intestinal absorptive cells are not clear. Although the subcompartmentation of glycosyltransferases would serve to prevent competing reactions which could alter the normal processing of oligosac-

I72


FIG.16. Immunocytochemical localization of blood group A al.3-N-acetylgalactosaminyltransferase in absorptive cell Golgi apparatus from human ileum. Label is present throughout the cistemal stack and trans-tubular network of the Golgi apparatus. Note the complexity of the structures at the trans side of the Golgi apparatus. X 48,000.Bar = 0.2 pm. (Reproduced with permission from Roth tv a/., 1986.)

charides, it is not clear that normal processing requires such subcompartmentation. Other mechanisms such as the differential expression of the levels of two competing glycosyltransferases could favor one terminal glycosylation pattern over another. Besides, it is not known if the consequences of having oligosaccharides with one type of terminal structure would have functional significance over another for most proteins. It should be emphasized, though, that more insight into these questions awaits the immunolocalization of other glycosyltransferases in a variety of cell types. Indirect support for these results has emerged from recent lectin-binding investigations. Diffuse labeling throughout the Golgi apparatus cisternal stack has been observed with RCL I in mouse epididymal cells (Yokoyama et al., 1980) and rat absorptive intestinal cells (Pavelka and Ellinger, 1986). Hedman er al. ( 1986) observed label with LFL throughout the cisternal stack with the excep-


173

tion of one cis-cisterna in 3T3 cells. Similarly, Roth and co-workers (Lee et al., 1989) found that LFL labeled the entire Golgi apparatus cisternal stack in CHO cells. Moreover, in the same study (Lee et al., 1989) CHO cells were transfected with a cDNA coding for the P-galactoside a2,6-sialyltransferase. This enzyme competed with the endogenous P-galactoside a2,3-sialyltransferase for the termination of oligosaccharide chains. This competition was assessed by the binding of SNL I (specific for NeuSAc a2,6-Gal/GalNAc sequences) to sections from wild type and transfected cells. While SNL I did not stain wild type CHO cells, the Golgi apparatus of transfected cells was labeled throughout the entire cisternal stack. Thus, in both wild type and transfected cells, sialic acid residues were not restricted to trans-cisternae of the Golgi apparatus. Such lectin-binding studies identify any glycoconjugate in the Golgi apparatus carrying the required sugar residues. Although the nature and extent of recycling of glycoconjugates from the plasma membrane through the Golgi apparatus remains controversial (Farquhar, 1985; see Snider and Rogers, 1985, 1986; Neefjes et al., 1988; Reichner et al., 1988, for disparaging views) such recycling could at least in part explain the presence of complex-type oligosaccharide chains in the middle and cis regions of the Golgi apparatus cisternal stack. For this reason, we feel that it is most important to determine the intra-Golgi apparatus distribution of a particular glycosyltransferase before surmising that the pattern of glycoconjugate localization represents the site of glycosyltransferase activity.

3. Demonstration of Lectin-Binding Sites in lntestinal Cell Golgi Apparatus

In contrast to the relatively few investigations detailing the localization of glycosyltransferases within the intestinal cell Golgi apparatus, many studies have employed lectins for the demonstration of sugar residues therein (Pavelka, 1987). Preembedding methods employing peroxidase-conjugated lectins, as well as postembedding methods employing colloidal gold-labeled lectins and glycoproteins have been used. Although the methods and animal species investigated may differ among the various investigators, the lectin-binding patterns to intestinal goblet and absorptive cell Golgi apparatus may be summarized as in Table 111. As can be seen from the table, the interpretations of lectin-binding studies by various investigators tend to overlap, but also display variability. Such discrepancies may result from species variability, variability among intestinal segments as well as crypt versus villus regions, and methodology (preversus postembedding, tissue fixation and processing, probe preparation, etc.). Moreover, probably of equal importance is the very subjective nature of the interpretation of lectin labeling patterns within the Golgi apparatus. It may be rather easy to distinguish between cis- and trans-sides of the Golgi apparatus cisternal stack, yet what defines where the cis region ends and middle begins, or where middle ends and trans begins? How many cisternae compose the desig-


174

TABLE 111 LECTINBINDING TO GOLGIAPPARATUS IN INTESTWAL EPITHELIAL CELLS" Goblet cell Lectin ConA SBL PSL LCL HPL

cis nd

LFL UEL I O+,

Absorptive cell cis middle trans

+

+I-

-

+

+

-

nd

nd

nd

nd nd

nd nd

nd nd

+

+I-

-

-

+

+I+I-

+/-

nd

+

nd

nd

t

nd nd

nd nd

nd nd

-

+

+/-

t

GSL I RCL I

middle trans nd nd

+

-

+

+

+

-

+I-

+

-

+

-

-

-

-

-

+

-

-

nd

nd

nd

+

+I-

+

t

-

+I-

+ +

References Pavelka and Ellinger (1985) Tsuyama et al. (1986) Pavelka and Ellinger (1989a) Pavelka and Ellinger (1989a) Murata et a / .(1986) Pavelka and Ellinger (1985); Ellinger and Pavelka (1988b) Roth ( 1984) Ellinger and Pavelka (1988b) Pavelka and Ellinger (1985, 1989b) Tsuyama ef a / . (1986) Roth et al. (1986) Ellinger and Pavelka (1988a)

Staining present; -, not detected; nd, not determined.

nated cis, middle, and trans regions of the stack? In the absence of specific markers, these borders seem to be arbitrarily defined by individual investigators. The situation is further complicated by cell type variability. For instance, some cell types such as hepatocytes may contain Golgi apparatus with as few as three cistemae, whereas the Golgi apparatus of goblet cells may possess up to 20 cisternae. The number of cistemae within a given cell may also vary depending upon the functional condition of the cell. Finally, the plane of section must be considered when interpreting the number of cistemae within, as well as the orientation of the Golgi stack. This may necessitate examining serial sections in order to exclude the possibility of missing a particular region of the cisternal stack in a given section. For instance, Orci et al. (1986) performed a serial sectioning analysis of the transport of horseradish peroxidase from the cell surface to the Golgi apparatus in insulin-secreting B cells. Their results showed quite convincingly that what appeared to be a cis- or trans-cisterna in a random section could always be traced to a position in the Golgi stack intermediate (i.e., middle cistemae) between the cis and trans poles. Taking all of these points into consideration, and assuming some subjectivity on our part, we propose the following scheme for the localization of sugar residues within the intestinal cell Golgi apparatus. In absorptive cells, mannose/glucose residues are restricted to cis and middle portions of the cisternal stack (Pavelka and Ellinger, 1985, 1989a); N-acetylgalactosamine residues are concentrated in cis and trans regions (Pavelka and Ellinger, 1985; Ellinger and Pavelka, 1988b); galactose residues to


175

trans- and variably middle cistemae (Pavelka and Ellinger, 1985); fucose residues to middle/trans regions (Ellinger and Pavelka, 1988a); and sialic acid residues diffuse throughout the stack, but concentrated in trans-cistemae (Roth et al., 1986). In goblet cells, mannose/glucose residues are restricted to cis/middle portions of the stack (Tsuyama et al., 1986); N-acetylgalactosamineresidues to cis and trans regions (Roth, 1984); galactose residues to middle/trans portions of the stack (Pavelka and Ellinger, 1985, 1989b); fucose residues to trans-cisternae (Ellinger and Pavelka, 1988a); sialic acid residues to trans-cisternae (Roth er af., 1986); sialic acid a2,3-linked and a2,6-linked to galactose concentrated in trans-cisternae (Sata et al., 1989; Taatjes and Roth, 1990). In a single study, Ellinger and Pavelka ( 1988b) have reported that a-galactose residues as detected with GrifSoonia simplicifolia isolectin I-B4, are restricted to cis- cisternae in intestinal goblet cells. C. POST-GOLGI APPARATUS DISTRIBUTION OF GLYCOSYLTRANSFERASES: FACTOR ARTIFACT? It is well established that glycosyltransferases exist outside of their usual location as Golgi apparatus integral membrane proteins; specifically in cellular plasma membranes and in soluble form in a number of secretions, predominantly milk and colostrum (Andrews, 1970; Barker er al., 1972; Paulson er al., 1977), and serum (Hudgin and Schachter, 1971; Fujita-Yamaguchi and Yoshida, 1981; Kaplan el al., 1983). Immunocytochemical methods have indicated the presence of galactosyltransferase (Pestalozzi et a f . , 1982; Davis et af., 1984; Roth et al., 1985b; Shaper er al., 1985; Bayna er al., 1988), N-acetylgalactosaminyltransferase (Balsam0 er al., 1986), blood group A N-acetylgalactosaminyltransferase (Roth et al., 1987, 1988c), and sialyltransferase (Roth er al., 1986; Taatjes and Roth, 1988; Taatjes et al., 1988a) at the plasma membrane of many cell types. A detailed discussion of cell surface glycosyltransferases is beyond the scope of this review. However, interested readers should consult several excellent reviews of this area (Pierce er al., 1980; Strous, 1986; Shur, 1989). In this section we will focus on the evidence pertaining to the presence of glycosyltransferases outside of the Golgi apparatus in intestinal cells. Such evidence has been presented from three types of experiments: (1) measurement of glycosyltransferase activities in plasma membrane fractions; (2) autoradiographic detection of glycosyltransferase activity in plasma membranes; and (3) in situ immunocytochemical localization of glycosyltransferases. Many studies have reported the presence of glycosyltransferase activity in the plasma membranes of intestinal epithelial cells, and these results were already presented in Sections IV,A, 1 and 2. Briefly, activities for galactosyltransferase and sialyltransferase have been detected on the apical and basolateral plasma membranes of intestinal epithelial cells (Weiser, 1973a,b; Weiser er al., 1978).

176


Using a different methodology, Bennett ei al. (1987) have reported on the existence of an active sialyltransferase at the microvillar surface of rat intestinal absorptive cells. They injected CMP-['Hlsialic acid into the intestinal lumen, followed by visualization of autoradiographic products at the light microscopic level. Injection period was restricted to 5 min to ensure that reaction product reflected cell surface phenomena and not activity from the Golgi apparatus. They observed a moderate autoradiographic reaction at the microvillar surface of small intestinal absorptive cells, yet found no reaction at the luminal surface of epithelial cells from gallbladder, ciliary body, and iris. Likewise, the injection of UDP-['H]galactose resulted in no reaction at the cell surface of all these cells, including intestinal absorptive cells. They attributed these results to reflect the presence of a sialyltransferase capable of sialylating endogenous acceptors at the luminal surface of small intestinal absorptive cells. In light of immunocytochemical results to be discussed below, it would have been of interest if Bennett and co-workers had examined reaction in the large intestine as well. As mentioned previously, several immunocytochemical investigations at both the light and electron microscopic levels have reported the presence of immunoreactivity for glycosyltransferases at the plasma membrane, as well as other post-Golgi apparatus sites of intestinal epithelial cells (Pestalozzi et al., 1982; Roth et al., 1985b. 1986, 1987, 1988~;Taatjes and Roth, 1988; Taatjes er al., 1988a). Berger and co-workers (Pestalozzi ei al., 1982), observed at the light microscopic level label with an affinity-purified galactosyltransferase antibody at the apical, but not basolateral plasma membrane of human jejunal enterocytes. Roth ei al., (1985b) performed a similar investigation using postembedding protein A-gold immunocytochemistry at the electron microscopic level. An affinity-purified antibody against human milk galactosyltransferase was applied to thin sections from human duodenum embedded in Lowicryl K4M. Intense gold particle label was observed at the apical (brush border), as well as basolateral plasma membrane of enterocytes. The intensity of label decreased on the lateral plasma membrane as it approached the basal membrane. Staining was completely abolished by preabsorption of the antibody with purified galactosyltransferase antigen. However, the validity of these results as representing true cell surface, or ecto-galactosyltransferase has recently been challenged. First, Boyle ef al. ( 1986) using analytical subcellular fractionation techniques, reported that galactosyltransferase activity was confined to the Golgi apparatus fraction in human jejunal biopsy homogenates, with no significant amount detectable in the brush border membrane fraction. They postulated that the staining observed by Roth et al. (1985b) was probably due to contaminating immunoglobulins present in the milk used as the source of the galactosyltransferase antigen. An alternative explanation for their inability to detect significant amounts of galactosyltransferase activity in their plasma membrane fraction could have resulted from failure to block endogenous intestinal nucleotide py-

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rophosphatase. As reported by Lau and Carlson (1981) (and discussed in Section IV,A, 1), when measuring glycosyltransferase activities in tissues rich in nucleotide pyrophosphatase activity (such as intestinal mucosa), precautions must be taken, including inclusion of EDTA and soybean trypsin inhibitor, to ensure that glycosyltransferase degradation does not occur. A second, and perhaps more troublesome critique, has been the recent revelation that polyclonal antibodies raised against glycoproteins may contain clones directed against carbohydrate epitopes of the antigen (Feizi and Childs, 1987). Human milk galactosyltransferase possesses blood group-related carbohydrate structures as part of its oligosaccharide constituency. Indeed, Feizi and co-workers (Childs et al., 1986) have reported immunochemical data demonstrating that the affinity-purified antibody raised against the human milk galactosyltransferase used in the above-mentioned studies (Pestalozzi et al., 1982; Roth et al., 1985b) contains a minor population of antibodies directed against the blood group-related carbohydrate moiety of the enzyme. When used in immunofluorescence experiments, these antibodies against carbohydrate epitopes of galactosyltransferase intensely stained the brush border of intestinal epithelial cells (Childs et al., 1986). This staining could be abolished by preabsorbing the antibodies with blood group substances, suggesting that the staining did not reflect galactosyltransferase immunoreactivity at the brush border, but rather that of blood group substances. How do these results relate to those published by Roth et al. (1985b) described above? Perhaps a reevaluation is necessary, employing a galactosyltransferase antibody preabsorbed with blood group carbohydrate structures. Alternatively, antibodies could be raised against a deglycosylated form of the enzyme and used for immunocytochemistry.However, it is not clear what effect removal of the carbohydrate moieties would have on the folding and three dimensional conformation of the enzyme. Resulting antibodies could potentially recognize antigenic structures not present on the molecule in situ. Perhaps the best method for resolving this discrepancy would be to utilize the recent successful cloning of several galactosyltransferases (Narimatsu et al., 1986; Shaper et al., 1986, 1988; Masri et al., 1988; Nakazawa et al., 1988; D’Agostaro et al., 1989; Masibay and Qasba, 1989) to produce polypeptide epitope-purified antibodies (Taatjes et al., 1988a) recognizing only the protein portion of the enzyme as related below. During our studies on the subcompartmentation of sialyltransferase in the Golgi apparatus of intestinal epithelial cells, we noted predominant staining over a variety of post-Golgi apparatus structures, including plasma membrane and mucus droplets (Roth et al., 1986). In view of the findings of Childs et al. (1986) concerning the contamination of galactosyltransferase antisera with carbohydrate-directed antibodies noted above, we sought to determine if the immunolabeling we observed for sialyltransferase outside of the Golgi apparatus represented true sialyltransferase enzyme, or rather was due to nonspecific

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cross-reaction with carbohydrate antigens. For this purpose we took advantage of the recent cloning of the gene for this particular sialyltransferase (Weinstein er al., 1987) to prepare polypeptide epitope-purified polyclonal sialyltransferase antibodies (Fig. 17) by adsorption to a recombinant P-galactosidase-sialyltransferase fusion protein produced in Escherichia coli (Taatjes et al., 1988a). Because the fusion protein is nonglycosylated, the resulting purified antibodies recognize only protein epitopes of the sialyltransferase. Using these antibodies for immunoelectron microscopy, we observed immunoreactivity to the sialyltransferase polypeptide in several post-Golgi apparatus structures, in addition to the Golgi apparatus, in both absorptive and goblet cells from the rat colon (Taatjes et al., 1988a). In absorptive cells, labeling was found in the apical and basolateral plasma membranes, lysosomes, and multivesicular bodies, and at the

FIG. 17. Characterization of a P-galactosidase-sialyltransferase fusion protein epitope purified antibody ( h STI). SDS-polyacrylamide gels of rat liver Golgi apparatus (lanes 2 , 4 , 6 ) and purified Galpl.4ClcNAc a-2.6 sialyltransferase (lanes I . 3, 5 ) were stained by Coomassie blue (lanes I and 2) or processed as Immun-blot with fusion protein epitope-purified antibody (lanes 3 and 4) or by antibody mock-purified with P-galactosidase without fused sialyltransferase (h GTI I; lanes 5 and 6). The h ST1 antibody recognizes both the purified and Golgi apparatus forms of sialyltransferase, while the h GT11 control antibody shows only a background level of staining. (Reproduced with permission from Weinstein ef a/., 1987.)

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limiting membrane of apical cytoplasmic vesicles (Fig. 18). In goblet cells (Fig. 19a), label was detected in the apical and basolateral plasma membranes and in mucus droplets (both in the lumen and at the limiting membrane). Surprisingly, label was undetectable (including the Golgi apparatus) in all regions of the small intestine from the same animals (Fig. 19b), as presented in Table IV, and previously detailed in Section IV,A, 1.

FIG.18. Immunocytochemical localization of sialyltransferase with P-galactosidase-sialyltransferase fusion protein epitope-purified antibody in an absorptive cell from rat proximal colon. Sialyltransferase immunoreactivity is detectable in the apical plasma membrane (asterisk) and along the inner aspect of the limiting membrane of apical cytoplasmic vesicles (arrowheads). X 50,000. Bar = 0.2 pn. (Reproduced with permission from Taatjes et al., 1988a.)

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GLYCOSYLATION IN INTESTINAL EPITHELIUM TABLE IV DISTRIBUTION OF SIALYLTFWNSFERASES IN RAT INTESTINE' Intestinal segment Duodenum Jejunum Ileum Colon

Sialyltransferase activityb GalPl,3(4)GlcNAc a2.3-ST' GalPl,4GlcNAc a2.6-STd 0 0 0 12

0 0 0 14

"From Taatjes et al. ( 1988a). hActivity expressed as picomole of [14C]Neu5Actransferred per milligram of protein/hour for both sialyltransferases. 'A value of 0 indicated activity not detected with limit of detection at 10 pmol of ['4C]Neu5Ac/ milligram of proteinhour. dA value of 0 indicates activity not detected with limit of detection at 1 pmol of [14C]Neu5Ac/ milligram of protein/hour.

Thus, the powerful combination of molecular cloning and immunocytochemistry provided very strong support to the contention that glycosyltransferases are also housed in cellular locations distal to the Golgi apparatus. However, the question must be asked whether the post-Golgi apparatus localizations of sialyltransferase in rat intestine are functionally significant. If we consider first the label present in the mucus droplets, it is possible that this luminal sialyltransferase continues its function in the sialylation of glycoproteins. Recently, Paulson and co-workers (Colley et al., 1989) investigated the conversion of membrane-bound Golgi apparatus sialyltransferase to a secretory form of the protein. By replacing the NH,-terminal signal anchor with the cleavable signal peptide from y-interferon, and transfecting CHO cells with this sialyltransferase expression vector, they were able to show that this construct was secreted from the cell with a half time of 2-3 hr. Most importantly, this secreted form of sialyltransferase contained the catalytic portion of the enzyme and was enzymatically active. By analogy, the sialyltransferase located in the mucus may represent a form of the enzyme rendered soluble by cleavage of the NH,-terminal signal anchor by an endogenous protease in the trans region of the Golgi apparatus. Indeed, precedence for such a situation has been documented for the conFIG. 19. Immunocytochemical demonstration of sialyltransferase in goblet cells using P-galactosidase-sialyltransferase fusion protein epitope-purified antibody. (a) Gold particle label indicative of sialyltransferase immunoreactivity is found in trans-cistemae of the Golgi apparatus (facing the mucus droplets), in the mucus droplet lumen (md), and along their limiting membrane (arrowheads), and along the lateral plasma membrane (arrows) of goblet cells from rat proximal colon. In contrast, immunoreactivity for sialyltransferase is undetectable in both the Golgi apparatus and mucus droplets (md) of goblet cells from rat jejunum (b). X 50.000 (a and b). Bar = 0.2 pm. (Reproduced with permission from Taatjes et al., 1988a.)

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version of the blood group A N-acetylgalactosaminyltransferasefrom a membrane-associated to a nonmembrane-associated form in the trans-tubular network of human intestinal goblet cells (Roth et al., 1988~).In this case, active enzyme has been directly demonstrated in the mucin released from the goblet cells (Omtoft et al., 1987). Of course, even if the enzyme is active, continued glycosylation would require the transport of the appropriate nucleotide sugar from the cytoplasm into the lumen by a nucleotide sugar antiport protein (Hirschberg and Snider, 1987). Thus, whether or not the sialyltransferase plays a functional role in the mucus is still an open question. Once it is released with the mucin into the lumen of the gut, it most likely has no catalytic activity due to lack of substrates, and is unlikely to play any role in the physical properties of the much since it would be such a minor component of the total protein (estimated at less than 0.0001% by activity). The demonstration of sialyltransferase at the apical and basolateral plasma membrane of intestinal cells provides further evidence for the existence of ecto-glycosyltransferases. The unambiguous existence of cell surface glycosyltransferases has been difficult to establish since Roseman ( 1970) first proposed their role in cell recognition and adhesion. However, in recent years Shur and co-workers, in a series of elegant studies, have succeeded in demonstrating the role of cell surface galactosyltransferase in such diverse functions as fertilization, preimplantation embryonic development, implantation, mesenchymal cell migration on substrates, and growth control in normal, neoplastic, and metastatic cells (Shur, 1989). Presently, it is not clear if ecto-sialyltransferase plays a functional role in intestinal cells or if its occurrence simply reflects its less restricted distribution in the post-Golgi apparatus membranes of these cells. In this respect, it would be of interest to determine if other glycosyltransferases in these cells have similar or different distributions, since similar distributions would favor the view that their existence on the cell surface is a consequence of an alteration in the underlying mechanism which would restrict their subcellular localization to the Golgi apparatus. Indeed, this view is supported by the immunocytochemical localization of the blood group A N-acetylgalactosaminyltransferase in both the apical and basolateral plasma membrane of human intestinal epithelial cells (Roth et al., 1987). On the other hand, Lopez ez a/. (1989) have reported that at least in F9 embryonal carcinoma cells the levels of cell surface (ecto-galactosyltransferase) and Golgi apparatus galactosyltransferase change relative to one another during cell differentiation, suggesting that these functionally and distinct pools of galactosyltransferase are independently and differentially regulated. This would indicate that we should not necessarily think of ecto-glycosyltransferases as representing nonspecific vesicular transport of the Golgi apparatus form of the enzyme to the plasma membrane, but rather as an independently regulated entity of its own.


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V. Effects of Exogenous Agents on Intestinal Glycosyltransferase Activity and Glycosylation A. HORMONES In adult rats, a continuous subcutaneous administration of testosterone for fourteen days resulted in qualitative and quantitative changes in the glycosphingolipid composition of rat small intestinal mucosa (Dahiya et al., 1989). These changes were accompanied by increases in the enzymatic activities of CMP-Nacetylneuraminic acid : lactosyfceramide sialyltransferase and UDP-galactose : lactosylceramide galactosyltransferase. The authors proposed that testosterone induced the activities of the two glycosyltransferases to increase, resulting in changes in intestinal mucosa glycosphingolipid composition. In a similar investigation, Dudeja et al. (1988) analyzed the activities of the same two glycosyltransferases reported above, in Golgi apparatus membranes, in response to subcutaneous administration of the synthetic glucocorticoid dexamethasone. They found that the activities of both glycosyltransferases were elevated in response to dexamethasone administration. They speculated that the increase in galactosyltransferase activity may have resulted from an increased membrane fluidity caused by the dexamethasone. However, they could not attribute the increase in sialyltransferase activity to the same cause. Several investigations have been aimed at examining the effect of hormone administration on glycosylation activity in developing intestine. As described in Sections 1V,A,2 and 3, the postnatal development of rat small intestine is characterized by a decrease in sialyltransferase activity, with a concomitant increase in fucosyltransferase activity. A postnatal injection of cortisone caused precocious changes in the activities of sialyltransferase and fucosyltransferase in the mucosal fractions from 2-week-old rats (Chu and Walker, 1986). Specifically, cortisone administration resulted in a 50% decrease in sialyltransferase activity and an 8-fold increase in fucosyltransferase activity as compared to control animals. Likewise, glycosidic-bound sialic acid content was significantly decreased, while glycosidic-bound fucose content significantly increased in the hormone-treated animals. Walker and co-workers (Ozaki et al., 1989) have also shown that cortisone injection into suckling rats causes a precocious increase in the activities of two developmentally regulated galactosyltransferases:the UDPwas increased 2.7-fold and the UDPGal : GlcNAc (~1-4)-galactosyltransferase Gal : GalNAc(~1-3)-galactosyltransferaseactivity was increased 1.8-fold. In an earlier study, Mahmood and Torres-Pinedo (1985) injected suckling rats with cortisone, thyroxine, epidermal growth factor, or insulin and measured the effect on the intestinal microvillar membrane content of sialic acid and fucose, as well as subsequent lectin binding. Cortisone treatment was found to lower sialic acid content and raise fucose content of microvillar membranes, as well as

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increase the incorporation of ['Hlfucose into these membranes. These results were also reflected in the binding of '251-labeledlectins to purified microvillar membrane preparations; cortisone administration decreased the binding of WGA to microvillar membranes, while increasing the binding of UEL I and PNL. Thyroxine treatment had a similar effect as cortisone on membranous fucose content and UEL I binding, but did not alter the incorporation of ['Hlfucose into membranes or the sialic acid content of membranes. Epidermal growth factor and insulin did not affect any of these parameters. Thus, these results demonstrated that only cortisone administration to suckling rats induced precocious changes in sialic acid and fucose content of microvillar membranes normally associated with postnatal intestinal development. Kolinska e? al. (1988) examined the effect of hydrocortisone administration on sialyltransferase activity in the crypts versus villus of 10-day-old rat small intestine. They found that the decrease in sialyltransferase activity induced by hydrocortisone administration occurred mainly in the crypt cells. B. DRUGSAND OTHERNoxlOuS STIMULI

Treatment of rats with the microtubule-disrupting drug colchicine or with turpentine results in an increase in the serum level of sialyltransferase activity (Mookerjea et al.. 1977; Kaplan ez al., 1983). Ratnam et al. (1987) speculated that some of this increase in serum sialyltransferase may result from secretion from the small intestine. They injected rats with colchicine and then 4 hr later measured the activity of the a2,6-sialyltransferase in the homogenates from jejunal slices. They found that secretion of soluble sialyltransferase into the medium was elevated in the animals treated with colchicine, as compared to control animals. A similar increase in intestinal and serum sialyltransferase activity has also been shown to be induced by inflammation caused by a standardized 25% body surface area thermal injury in rats (Chu e? al., 1988). These results thus suggested that intestinal sialyltransferase may form part of the acute phase response to inflammation. However, we believe that this sialyltransferase originates from cells of the lamina propria, and not from intestinal epithelial cells. As pointed out in Sections IV,A,l and IV,C, immunoreactivity and enzymatic activity for the a2,6-sialyltransferase were undetectable in rat small intestinal mucosa. Colchicine is normally used as a depolymerizing agent for microtubules to study cellular processes which may be microtubule-dependent. Indeed, Hugon e?al. (1987) performed such a study on mouse jejunal epithelial cells to investigate the role of microtubules in the migration of glycoproteins from the Golgi apparatus to the apical and basolateral plasma membranes. They examined by autoradiography the incorporation of [3H]fucose into glycoconjugates in explants of mouse jejunum cultured in a medium containing colchicine. They

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found that colchicine inhibited the labeling of the brush border by 67%, while labeling of the basolateral plasma membrane increased 114%. Similar results were also obtained with the microtubule-disrupting drug nocodazole. These results suggested that some glycoproteins destined for the apical plasma membrane may be rerouted to the basolateral plasma membrane in the presence of colchicine and thereby suggests a role for microtubules in the transport of glycoconjugates from the Golgi apparatus to the apical plasma membrane in polarized intestinal absorptive cells. Similar effects of colchicine on glycoprotein migration were reported earlier for human jejunal biopsies in culture (Blok et af.,1981) and for rat small intestine (Ellinger et af., 1983). The effect of polyamine deficiency on Golgi apparatus membranes and galactosyltransferase activity in mouse small intestinal epithelial cells was studied by Sakamaki et al. (1989). They produced polyamine-deficient cells by injecting two inhibitors of polyamine synthesis, ethylglyoxal bis(guany1hydrazone) and a-difluoromethylornithine into mice. Polyamine deficiency produced swelling of the Golgi apparatus membranes (demonstrated by electron microscopy) accompanied by a decrease (to approximately 55% of the control value) in galactosyltransferase activity. These results suggested that galactosyltransferase activity is diminished in swollen Golgi apparatus membranes. Umesaki and Ohara (1989) investigated in detail treatments which lead to an increase in GDP-fucose : asialo GH,a(1-2)fucosyltransferase activity in rat small intestinal mucosa. The increase in this particular fucosyltransferase activity was manifested by alteration in the neutral glycolipids of the microvillar plasma membrane. Factors shown to cause an increase in fucosyltransferase activity were microbial contamination of germ-free mice, weaning (see Section IV,A,2), intraperitoneal injection of the protein synthesis inhibitors cycloheximide or emetine (although repeated injection of cycloheximide every 2 hr resulted in a repression of fucosyltransferase activity), injection of a soluble fraction from a small intestinal homogenate, and mechanical injury to the intestinal mucosa. They also analyzed the composition of the glycolipids in mucosal fractions after such treatments and found an increase in their fucose content. Finally, by separating crypt from villus cells, they found that fucosyltransferase activity was increased in villus cells as compared to crypt cells. They attributed these findings to indicate that the increase in fucosyltransferase activity in response to various stimuli is preferentially localized to the postmitotic epithelial cells located on the villus.

VI. Differentiation and Glycosylation in Intestinal Cell Culture Systems Although the morphology and physiology of the intestinal tract are quite amenable for studying differentiation events (Section I), pitfalls of using such an

I86


organ system in biochemical studies are numerous, i.e., obtaining pure cell populations, experimental manipulation of cells, and difficulty in administering exogenous agents, to name a few. For these reasons, alternative experimental systems such as intestinal organ (Quaroni, 1985) and cell culture (Rousset, 1986) have been introduced. The recent introduction of several stable cell lines derived from intestinal epithelial cells has made this a particularly fruitful avenue of research. The oligosaccharide composition of cell surface glycopeptides was investigated in confluent and subconfluent cultures of the rat small intestinal epithelial cell line IEC-6 by measuring the incorporation of D-[2-3H] mannose and by glycopeptide sensitivity to various oligosaccharide processing enzymes (Sasak et al., 1982). They found that confluent cells contained a much higher proportion of complex oligosaccharides in glycopeptides of the plasma membrane than did subconfluent cells. Only minor differences were observed between total mannose-labeled glycopeptides from confluent and subconfluent cultures, suggesting that the cell surface changes were mainly due to differences in biosynthesis of the carbohydrate moieties and not to the formation of different glycoproteins. Moreover, this alteration in oligosaccharide composition of cell surface glycopeptides was shown to be dependent upon cell density and not on the growth rate of the cells. Interestingly, Sasak et al. (1982) also were able to draw a correlation between degree of cell adhesion to the substratum and cell surface oligosaccharides: confluent cultures containing cell surface glycopeptides with complex-type oligosaccharide structures were more adherent than their subconfluent counterparts displaying more high mannose-type oligosaccharides. Several recent reports have documented the relationship between cell differentiation and the extent of processing of N-linked oligosaccharides in the human colon cancer cell line HT-29 (Trugnan et a / . , 1987; Ogier-Denis et al., 1988, 1989). HT-29 cells remain undifferentiated in media containing glucose, but undergo differentiation when glucose is removed from the media. Trugnan er al. ( 1987) examined the biosynthesis of sucrase-isomaltase, a microvillar membrane protein taken as a marker for differentiated intestinal epithelial cells in v i w , in both differentiated and undifferentiated HT-29 cells. In contrast to the normal processing and expression of this enzyme at the cell surface in differentiated HT-29 cells, in undifferentiated cells no enzyme was detectable at the plasma membrane. They showed that the failure to detect membrane expression was not due to lack of synthesis, but rather to abnormal posttranslational processing. Indeed, as compared to the enzyme synthesized and expressed in differentiated HT-29 cells, sucrase-isomaltase produced in the undifferentiated cells displayed ( I ) an impairment of the conversion from high mannose to complex form of the enzyme; (2) abnormal complex form glycosylation; and (3) rapid intracellular degradation of both high mannose-type and complex-type enzymes. In a subsequent paper (Ogier-Denis et al., 1988), this group investigated


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whether the impairment in glycosylation noted in undifferentiated HT-29 cells was specific for sucrase-isomaltase, or a general glycosylation defect. They found that there is an overall defect in the processing of N-linked oligosaccharides (Section 11) which is manifested by alterations in three processing steps: ( 1) incorporation of ~-[2-~H]mannose into glycoproteins; (2) conversion of high-mannose chains to complex-type N-linked glycans; and (3) trimming of high-mannose chains at the level of conversion of Man,,-GlcNAc,-Asn to Man,-GlcNAc,-Asn. This particular trimming reaction was elaborated on in another report (Ogier-Denis et al., 1989), where it was suggested that it may represent an important regulatory point in the conversion of undifferentiated to differentiated cells. These results, therefore, suggest that there is an impairment in the conversion of high mannose forms of N-linked oligosaccharides into complex-type in undifferentiated HT-29 cells. Future investigations on these cultured intestinal cells should help to unravel in more detail the cellular mechanisms involved in terminal differentiation as it relates to glycosylation.

VII. Concluding Remarks Although much effort has been directed toward elucidating glycosylation mechanisms and patterns in intestinal cells, unequivocal answers have not been forthcoming. This lack of emergence of a unifying concept underlying intestinal cell glycosylation may be the result of many divergent factors. Conflicting results concerning the activities of glycosyltransferasesin intestinal homogenates almost certainly results from variation in methodologies; i.e., (1) mucosal scrapings representing mostly epithelial cells versus homogenates containing submucosa and lamina propria; (2) failure to allow for endogenous intestinal enzymes which could potentially degrade glycosyltransferases; (3) variation in acceptor substrates employed, resulting in the measurement of different glycosyltransferases within the same class; and (4)different techniques for the separation of crypt versus villus epithelial cells. Similar technique-related problems could explain the variation in expression of intestinal carbohydrates. However, this is more likely due to the inherent variability in glycosylation expressed in a given cell type. Many detailed investigations have revealed a marked degree of glycoconjugate heterogeneity, not only among similar cell types from different species (Holthofer, 1983; Schulte and Spicer, 1983a,b; Spicer er al., 1987), but also among supposedly homogeneous cell populations within a given organ (Watanbe er al., 1981; Spicer et al., 1981; LeHir er al., 1982; Roth er al., 1983; Brown et al., 1985; Roth and Taatjes, 1985; Roth er al., 1988b; Taatjes et al., 1988b). Such heterogeneity may reflect blood group specificities, environmental or genetic variation, differentiation state of the cell, or pathological influences. However, a rapidly emerging concept suggests that the glycoconjugate repertoire displayed

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by a given cell reflects its endogenous expression of glycosyltransferases. This concept has recently been discussed in detail by Paulson (1989; Paulson et al., 1989) and by Rademacher et al. (1988), and will not be elaborated on here. Given the role played by terminal oligosaccharide structures in cell-cell recognition phenomena (Section I), the expression of glycosyltransferases would appear to occupy a key position in the posttranslational processing of glycoconjugates and thus influence cellular functions. Does this then mean that the carbohydrate portion of all types of glycoconjugates is important for their biological functioning? Certainly this is not the case for all glycoconjugates, and is an important area of concern in biotechnology. The importance of glycosylation in intestinal systems is mostly unknown at this point, although the well-documented shift from sialylation to fucosylation during rat postnatal development (Sections IV,A,2,4, and 6) has been attributed to represent the change in physiological functioning of the intestine during the weaning phase (Torres-Pinedo and Mahmood, 1984). It seems probable that the application of cDNA probes for various glycosyltransferases to intestinal systems (Paulson er a/., 1989) as well as the development of chimeric and transgenic mice (Gordon, 1989; Trahair et a/., 1989) will provide exciting opportunities in the future for the investigation of the importance of intestinal glycosylation in a myriad of functions. ACKNOWLEDGMENTS The original research described in this paper has received generous continual support from the Swiss National Science Foundation. We would like to thank Daniel Wey, Michele von Turkovitch, and Linda Barcornb for preparing the figures and photographs.

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INTERNATIONALREVIEW OF CYTOLOGY, VOL. 126

Physiological and Pharmacological Regulation of Biological Calcification DANIEL c . WILLIAMS*

AND CHARLES A. FROLIK~

*Bone Biology Research Group, Department of Connective Tissue and Monoclonal Antibody Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 f Department of Biochemistry Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

I. Introduction Calcification is essential to both the physiology and structural integrity of the human body. It is important for growth and maintenance of the skeletal system as well as for mineral homeostasis and several specialized functions. Understanding the mechanism by which calcium deposition and mobilization occurs in the skeletal system and being able to regulate it has important medical connotations. Osteoporosis, a disease characterized by low bone mass and increased bone fragility, affects over twenty-four million people in the United States and is recognized as one of the major concerns of modern medicine. The direct medical costs associated with osteoporosis in the United States alone in 1986 were estimated to exceed 5 billion dollars (Philips et al., 1988). In addition, millions of individuals are affected by improperly healing bone fractures, orthopedic replacement surgery, and ectopic calcification (e.g., kidney stones and calcified atherosclerotic plaques). Add to this the potential involvement of abnormal bone modeling in the pathology of osteoarthritis (over 15 million people) and rheumatoid arthritis (over 2 million people) (Lawrence et al., 1989) and it is clear that the medical implications of calcification disorders are enormous. In a broader sense, the deposition of calcium salts is a widespread phenomenon in biology. It occurs in living organisms ranging phylogenetically from bacteria to higher plants and animals. As a result, biomineralization processes can have profound effects on the environment we live in - modulating the chemistry of the oceans, the shape and composition of the land masses, and the survival of many biological species. The literature relating to the regulation of calcification, particularly in the vertebrate skeleton, has increased dramatically in the last two decades. As a practical matter, this article is limited primarily to regulation of calcification processes in vertebrate bone. Because of the large literature base, representative papers have been cited rather than providing a comprehensive list of references. In many cases, the most recent citations have been chosen so that the readers can 195 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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refer to those papers for a more complete survey of the literature in a particular topic area. A. FUNCTION OF BIOLOGICAL CALCIFICATION Calcification is used in biological systems for a number of purposes. Mineralization of the connective tissue matrix of the vertebral skeleton provides it with the rigidity necessary to support the body. Most of this structural support comes from the cortical (compact) bone which comprises 80% of the skeletal mass. In addition, the skeletal framework provides attachment sites for muscles and acts as levers in movement. The hard skeletal infrastructure also provides the sensitive soft tissues of the body with a significant degree of protection against injury. In a similar manner, the calcified egg shells common to avian and reptilian species serve to protect embryos during development. Calcium salts deposited in the teeth, tusks, horns, and antlers provide these organs with the rigidity and strength necessary to serve as tools and weapons. These basic functions of the vertebrate endoskeleton, in many cases, hold true as well for the calcified shells and exoskeletal elements of invertebrates. Calcified tissues are also involved in a number of specialized functions such as sound transmission (as illustrated by the otoliths of the middle ear) and maintenance of balance (e.g., the utoconia of the inner ear). The calcified skeleton is furthermore the major reservoir for ionic calcium, a key cation in physiological regulation of many body functions, and therefore an ion whose concentration in the body fluids and tissues is carefully regulated. The trabecular (cancellous) bone, constituting only 20% of the skeletal mass but more than 60% of the bone surface due to its spongelike macroscopic structure, is metabolically more active than cortical bone and therefore provides a greater contribution to this mineral homeostasis (Jee, 1983; Martin ct al., 1988). Additionally, bone serves as a maternal reservoir for skeletal salts used in both fetal and neonate development. In higher plants, calcification is most often viewed as a mechanism for removing metabolic products from the cellular milieu (Amott and Pautard, 1970; Smith, 1982), but also may function as a storage site for ionic material (Amott and Pautard, 1970) and in intracellular pH balance (Smith, 1982).

B. MECHANISMS OF CALCIFICATION I . Nudeution Theory

These are two general theories proposed for the mechanism of initiation of calcification in biological tissues. One concept, the nucleation theory (Glimcher, 1987, 1989), maintains that, in a biological system, there must be a highly

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ordered nucleation site present that places the substrates for the crystallization process in the correct spatial conformation necessary for the proper interactions to occur to allow crystal formation. The biological nucleator in bone is believed to be the collagen fibril. Indeed, under certain conditions, initial calcium phosphate crystals do appear to be deposited in the hole-zone region of these fibrils (Berthet-Colominaset al., 1978; Glimcher, 1989). While collagen may be a necessary component for formation of these crystals, the noncollagenous proteins appear to be required for control of the process. The nature and function of these proteins will be discussed in a later section.

2. Matrix Vesicle Theory The second theory concerning the initiation of calcification in biological systems involves matrix vesicles (Wuthier, 1988; Anderson, 1989). Matrix vesicles are small membrane-bound compartments believed to be primarily formed by the pinching off of the plasma membrane (Cecil and Anderson, 1978; Ralphs and Ali, 1986; Palumbo, 1986) and are found in hypertrophic cartilage (Anderson, 1969), primitive woven bone early in fetal osteogenesis (Bernard and Pease, 1969), and in some bone repair processes (Schenk et al., 1970), but rarely occur in more mature bone (Landis et al., 1977). The interior of these vesicles are enriched in specific enzymes, proteins, and lipids, many of which appear to be involved in maintaining a high intravesicular calcium and phosphate concentration (Peress et al., 1974; Bernard, 1979; Majeska et al., 1979). Matrix vesicles are postulated to function in the calcification process by: (1) releasing enzymes that remove inhibitors of crystal growth (for example, pyrophosphate and proteoglycans); (2) increasing calcium and phosphate concentrations to adequate levels for calcification to occur; (3) maintaining a protective environment for the formation of the more soluble octacalcium phosphate prior to its conversion to hydroxyapatite; and (4) providing for site-specific deposition of seed crystals into the hole zones of the collagen fibrils. While matrix vesicles may play an important role in initiating calcification (Ali, 1976; Hsu and Anderson, 1978; Wuthier, 1982), once it has begun, they no longer appear to be needed, and disappear. Opponents of the matrix vesicleinduced calcification theory point out that electron micrographs seem to indicate, as discussed above, that calcification first occurs in the hole zone of the collagen fibril, while the matrix vesicle theory has the initial crystals forming separately from the collagen fibrils. It is possible that in mature bone, calcification is initiated by heterogeneous nucleation while in cartilage and early embryonic bone, where there is no clear relationship between collagen fibrils and calcium phosphate crystals (Glimcher, 1989), matrix vesicles control the mineral formation process (Eanes, 1989).

DANEL C. WILLIAMS AND CHARLES A. FROLIK

c.

DIVERSITY OF BIOLOGICAL CALCIFICATION

1. Chemical Species and Crystal Forms About two-thirds of all biogenic minerals are salts of calcium with a diversified anion composition that includes salts of carbonates, phosphates, citrates, sulfates, and oxalates (Lowenstam, 1981). As a general rule, phosphate salts tend to be most common in vertebrate calcified tissues, carbonates in invertebrate tissues, and oxalates in tissues of higher plants. However, as with all “general rules” in biology, there are many exceptions, particularly when individual tissues are considered (e.g., both the otoconia of the vertebrate inner ear and the cystoliths of higher plants are carbonates). Lowenstam ( 1981) notes that two fundamentally different biomineralization processes occur in nature. In the more primitive systems, such as for certain bacteria and green and brown algae, mineralization occurs by bulk intracellular or extracellular crystal formation in the absence of a preformed organic matrix. This process is far less controlled than those associated with an organic matrix, and the mineral phase is similar to that produced by precipitation from inorganic solutions. In the second, more advanced form of biomineralization, an organism produces an organic matrix which serves as a framework for mineral deposition and crystal growth. In this latter case, the mineral type, crystal orientation, and microarchitecture are under genetic control.

2. Biological Diversity While the emphasis in this review is on the regulation of calcification in the vertebrate skeleton, an appreciation of the diverse species that utilize calcification and how they regulate this process is useful for understanding the common mechanisms employed in biomineralization throughout the biological kingdom. Calcification, even in “simple” unicellular organisms, is often a precisely regulated procedure that may result in the deposition of calcium salts in elaborately sculptured crystalline arrays (Pautard, 1970). Examples of some of the most highly regulated cellular calcification processes occur among microorganisms. The control of crystal shape by the coccolithophorid algae, where the organic matrix is laid down within the Golgi vesicles prior to calcification to form highly sculptured, interconnecting elements, is an elegant example of the role of the organic matrix providing a framework for mineral deposition and growth (Outka and Williams, 1971; Williams, 1974; van der Wal el al., 1983). Even in relatively primitive bacterial mineralizing systems, cellular regulation of calcification is evident. The biosynthesis of hydroxyapatite requires that calcium and phosphate be delivered to the site of membrane-associated nucleation, and that protons formed during mineralization be removed in order to maintain appropriate pH conditions. During calcification of the membrane of the oral bacterium Bacter-


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ionema matruchotii, bacteriorhodopsin serves this role, being an active proton pump whose action is facilitated by calcifiable proteolipids (Swain and Boyan, 1988; Swain et al., 1989). Despite the fact that calcification occurs throughout the plant kingdom, in comparison to what we know about animal calcification, relatively little is known about its regulation. There is, however, general agreement that mineral deposition in plants is a highly regulated function (Amott and Pautard, 1970; Smith, 1982). While deposits of calcium salts can be found in the plant cell wall, more typically they are located intracellularly, occurring predominantly in leaves and stems, but also found in roots, flowers, fruits, and seeds. The shape of calcium deposits and their location in a given species appears to be under genetic control. The diversity of mineralization among invertebrate animals, as discussed in three recent books (Leadbeater and Riding, 1986; Simkiss and Wilbur, 1989; Lowenstam and Weiner, 1989), is enormous. Nevertheless, clearly, there are common factors that tie the calcification process in these various species together. For example, there is a reliance on organic material to nucleate crystals, and to direct and control mineral growth (Weiner, 1986; Wheeler et al., 1988). Also, an important role for active ion transport mechanisms and hormonal regulation is evident in some invertebrate mineralizing systems (Cameron, 1989). Weiner (1986) views the phylogenetically diverse mineralizing tissues as part of a continuum, although the end members may differ markedly in the degree of control they exert over crystal growth.

11. The Vertebrate Skeleton A. BIOCHEMISTRY OF MINERALIZED TISSUES Healthy bone consists of an extracellular matrix that is spatially arranged in a highly organized pattern and is embedded with crystals of hydroxyapatite. While this hydroxyapatite is the major constituent of bone, accounting for 60-70% of the dry weight of cortical bone, there are well over 200 other macromolecules (Delmas et al., 1984) that have important roles in maintenance of bone structure and function. Of these molecules, the collagens play a major role in formation of the bone matrix, while the noncollagenous proteins are believed to regulate calcification and control matrix synthesis and degradation through actions on osteoclasts and osteoblasts. In this section, a brief review of the various components of bone will be presented. While emphasis will be placed on true bone and not mineralized cartilage, many of the molecules found in bone either occur or have related components in cartilage.

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1. Mineral Phase Although hydroxyapatite, Ca,,(PO,),(OH),, was first described as being the mineral component of bone over 60 years ago (DeJong, 1926; Roseberry et al.. 193I), the exact chemical composition and spatial arrangements of minerals in bone is still poorly understood. The mineral phase is not pure hydroxyapatite but contains small portions of carbonate, citrate, sodium, and magnesium as well as other trace metals and elements. Also, bone mineral is in a state of continual flux and the “natural” ions can be replaced by other ions that may be in the local environment at the time that mineralization is taking place. This may lead to unwanted side effects, such as those which occur due to the substitution of lead for calcium in lead intoxication, or to the incorporation of strontium-90 into bone after exposure to nuclear fallout. On the other hand, ion substitution may also have desirable properties. For example, in the prevention of dental caries, fluoride treatment leads to substitution of the fluoride ion for the hydroxyl ion with the subsequent formation of a less soluble fluoroapatite. During the process of mineralization there is still some question as to what is the initial physical state of calciumiphosphate in bone (for reviews see Eanes. 1985; R. G. G. Russell et al., 1986; Glimcher, 1987). Using in vitro and synthetic models to simulate the multistep process of mineralization, several precursors have been postulated to occur during the formation of hydroxyapatite. These include octacalcium phosphate [Ca,H,(PO,), 5H20] (Brown, 1966; Eanes and Meyer, 1977), amorphous calcium phosphate [Ca,(PO,),.,,(HPO,),, , XH,O] (Eanes er al., 19651, and dicalcium phosphate dihydrate [CaHPO, 2H,O] (Francis and Webb, 197 1). Whether these transient intermediates actually do occur in viw is still a matter of debate. 2 . Collagens The word “collagen” is derived from the Greek words kolla and genes, which are literally translated as meaning to produce glue. Collagen is indeed the “glue” produced by cells to provide the coherent structural element that forms the mature tissue. In addition. in developing tissue, collagen appears to have a directive role in morphogenesis. The basic unit of collagen (called tropocollagen) consists of three polypeptide chains wound around each other in a helical motif. There are currently 20 different types of chains known which are associated together in various ratios to form 11 types of collagen (for review see Miller and Gay, 1987). Type I collagen, and small amounts of type V collagen, account for approximately 90% of the proteins in bone. However, while type I cartilage is the predominate form of collagen found in many connective tissues, only in bone is it physiologically involved in the mineralization process. Therefore, the involvement of type I collagen in calcification is most likely not due to a characteristic


20 1

of the collagen backbone itself but probably occurs because of a posttranslationa1 modification of the collagen fiber (Mechanic et al., 1985) and/or to the presence or absence of other factors in the local bone environment (Termine et al., 1981b; Boyan, 1985).

3. Noncollagenous Proteins (NCP) While NCP represent only 10-15% of the organic phase of bone, their role in maintaining bone structure is essential (for reviews see Termine, 1988; Veis, 1988; Heinegard and Oldberg, 1989; Boskey, 1989a). For the purpose of this article, emphasis will be primarily placed on the involvement of the NCP in calcification. The NCP are believed to be associated with both the initiation of mineralization (through removal of inhibitors of nucleation and/or modification of the sites of nucleation) and, once crystallization has begun, with the regulation of the final shape and orientation of the crystal, and restricting the mineralization process within the proper boundaries. Table I lists the broad categories under which the NCP may be grouped. It should be remembered that many of the proteins may fall under more than one of the categories. Thus, osteopontin is not only a phosphoprotein but is also a glycoprotein. In the next few paragraphs, a brief description of the various classes of proteins that are found in bone will be presented along with examples of specific proteins in each class. a. Proteoglycans. The proteoglycans are proteins that contain one or more polysaccharide (glycosaminoglycan or GAG) sidechains (Scott, 1988) with chondroitin sulfate, dermatin sulfate, keratin sulfate, and sometimes heparin sulfate being found most often. The proteoglycans are thought to inhibit the process of mineralization (Howell et al., 1969; Cuervo et al., 1973; Boskey, 1989b), possibly through their ability to bind calcium, thus lowering the effective calcium concentration at the calcification site (Hunter, 1987). Classified according to their size, the very large proteoglycans are found only in calcified cartilage and make up 5-10% of the tissue weight. Their function appears to aid in tissue hydration (Campo, 1988), giving the cartilage resilience and providing a low friction joint surface, as well as to maintain matrix organization and regulate mineralization (Buckwalter et al., 1987). The large and small proteoglycans represent 4 1 0 % of the NCP in bone. In addition to being present in bone, they have a wide tissue distribution, occurring in most connective tissues. Three of these proteoglycans [PG I (also called PG-SI or biglycan), PG I1 (also named PG-SII or decorin), and fibromodulin] have extensive sequence homology (Fisher et al., 1989; Heinegard and Oldberg, 1989). They have been shown to bind to both collagen I and I1 (Hedbom and Heinegard, 1989) and are thought to modulate collagen fibril formation (Scott, 1988), thereby effecting mineral organization and calcification.

DANIEL C. WILLlAMS AND CHARLES A. FROLlK

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TABLE I NONCOLLAGENOUSPROTEINS OF BONE

Class

Proposed Role

Examples PG-I (PG-Si. biglycan) PG-I1 (PG-Sii, decorin) Fibromodulin

Proteoglycans

Regulate matrix organization and mineral imtion

Clycoproteins

Control extracellularcalcium Osteopontin (BSPI) Regulate crystal growth and shape Bone sialoprotein (BSPII) Provide cell adhesion to bone surface

Phosphoproteins

Control extracellularmatrix formation and mineralization

Osteonectin

y-Carboxyglutamicacidcontaining proteins

Regulate crystal growth Recruit osreoclasts

Osteocalcin (Bone GLA protein) Matrix GLA Protein

Proteolipids

Initiation of crystal formation Ionophores

Enzymes

Formation of mineral phase Bone resorption

Alkaline phosphatase Acid Phosphatase Proteases Collagenase Carbonic Anhydrase

Growth factors

Control of cellular proliferation

Transforming Growth Factor+ (TGFP) lnsulinlike Growth Factor-I,11 (IGF-I, IGF-11) Epidermal Growth Factor (EGF) Fibroblast Growth Factor (FGF) Platelet-DerivedGrowth Factor ( PDGF) Cytokines

b. Glycopr-oteinsand Phosphoproteins. It is difficult to separate the glycoproteins and the phosphoproteins into two different classes since many of the glycoproteins isolated from bone also appear to be phosphorylated. In general, these proteins are highly anionic and therefore bind both hydroxyapatite and calcium. As a result, they are able to either retard or promote mineral deposition on hydroxyapatite, depending on concentration, physical form, and secondary structure (Renugopalakrishnanet al., 1986). Bone sialoprotein (BSPII) was one of the first noncollagenous bone glycoproteins described (Andrews er al., 1967). While originally thought to be specific to bone, Northern blot analysis now is able to detect low levels of mRNA for BSPI1 in highly localized sites in other tissues as well (Fisher ef al., 1990). Bone sialoprotein is a 59,000 MW glycoprotein that contains a high content of sialic acid (Oldberg et al., 1988a; Fisher er al., 1990), and a high glutamic acid and


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phosphorylated serine content which appear to be responsible for its ability to bind calcium (Heinegard and Oldberg, 1989) and hydroxyapatite (Franzen and Heinegard, 1985; Fisher er al., 1987). The primary structure also includes an Arg-Gly-Asp cell-binding sequence typical of cell-adhesion peptides, enabling the protein to bind to osteosarcoma cells (Oldberg et al., 1988b). These characteristics may be partly responsible for the ability of osteoblasts to adhere to bone surfaces recently exposed by resorption. A second phosphorylated glycoprotein, osteopontin (also called BSPI or 2ar), shows many similarities to bone sialoprotein. While it has a lower carbohydrate content, it is highly phosphorylated on serine and contains the Arg-Gly-Asp sequence (Franzen and Heinegard, 1985; Oldberg et al., 1986). Like bone sialoprotein, osteopontin is not specific for bone but has also been detected in a number of other tissues (Nomura et al., 1988; Senger et al., 1989), and has been demonstrated to be synthesized in virro by several epithelial and fibroblast cell lines (Smith et al., 1987), as well as by osteoblastlike cells (Yoon et al., 1987; Mark et al., 1987). The presence of the cell-binding tripeptide sequence and the protein’s localization in bone at the clear zone where osteoclasts are binding to mineral (Heinegard and Oldberg, 1989), has led investigators to speculate that it may be involved in the binding of osteoclast precursor cells to the mineralized matrix of bone. In support of this concept is the observation that osteoclasts contain a high concentration of the vitronectin receptor, a member of the integrin family of receptors that recognizes the Arg-Gly-Asp sequence (Horton, 1988). A third member of this class, osteonectin (also called SPARC, BM-40, or 44K albumin-binding protein) is a 32,000 MW phosphorylated glycoprotein first isolated and characterized by Termine and co-workers in 1981 (Termine et al., 1981a,b; for review see Tracy et al., 1988). While it represents the most abundant noncollagenous protein in mineralized matrix (23% of the noncollagenous proteins) it is also widely distributed in nonmineralized tissues (Wasi et d., 1984; Young et a/., 1986; Holland et al., 1987) and platelets (Stenner er al., 1986). Osteonectin displays a high affinity for calcium, hydroxyapatite, and type I collagen (Termine et a1.,1981a,b; Romberg et al., 1985, 1986; Engel et al., 1987; Domenicucci er al., 1988). Examination of the primary sequence of human (Villarreal et al., 1989) and bovine (Bolander et al., 1988) bone osteonectin indicates several possible calcium binding domains in the amino terminal and carboxy terminal portions of the peptide similar to those observed in other intracellular calcium-binding proteins such as calmodulin (Persechini et al., 1989). Osteonectin may function in a general manner in controlling extracellular matrix formation. It has been shown to increase the binding of hydroxyapatite to collagen and to increase collagenlinked crystal formation from metastable solutions (Termine et al., 1981b). On the other hand, osteonectin at very low concentrations (lo-’ M) also inhibits hydroxyapatite crystal growth in vitro (Romberg ef al., 1985, 1986) and therefore could function to prevent mineralization of newly synthesized osteoid.

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c. yCarhoiyglutamic Acid-Containing Proteins. Another general class of proteins found in bone are those that contain the amino acid y-carboxyglutamic acid. To date, there are two bone-related proteins that fall into this category. The first, osteocalcin (bone Gla protein), represents 10-20% of the NCP in bone (Hauschka et al., 1975; Price er al., 1976a, 1982; for reviews see Lian and Gundberg, 1988; Price, 1988; Hauschka et al., 1989). It is a 5800 MW protein that contains three residues of y-carboxyglutamic acid (Price et al., 1976b) which are formed posnranslationally by a vitamin K-dependent carboxylation reaction. Osteocalcin mRNA is detected only in bone (Yoon et al., 1988: Fraser and Price, 1988) and appears to be synthesized exclusively in the osteoblast (Nishimoto and Price, 1980). It is expressed relatively late in bone development (Otawara and Price, 1986),appearing shortly after the initial deposition of bone mineral (Price el al., 198la; Hauschka e f al., 1983).The synthesisof osteocalcin is under the control of many of the factors that are known to affect bone, including vitamin D (Price and Baukol, 1980; Lian et al., 1985; Spiess ef al., 1986), transforming growth factor-p (TGFP) (Noda, 1989), and parathyroid hormone (PTH) (Noda et al.. 1988a). The protein has been detected in serum where levels appear to correlate with bone turnover, often being used as a diagnostic for bone diseases (Price et al., 1980; Delmas et al., 1983; Slovik et al., 1984)reflecting mainly, if not exclusively, bone formation (Price etal., 1981b; Brown etal., 1984; Riggs etaf., 1986). As with many of the NCPs studied to date, osteocalcin binds both to hydroxyapatite (Poser and Price, 1979) and to calcium (Svard et al., 1986). However, the protein does not appear to be involved in the initial deposition of bone mineral (Boskey et al., 1985) but instead may be involved in inhibition of hydroxyapatite crystal growth (Price et al., 1982; Romberg et al., 1986). This might occur through the binding of osteocalcin to various lipids, making them unavailable for their proposed role in calcification (Gendreau et al., 1989). However, osteocalcin does not appear to be absolutely necessary for normal mineralization, since administration of warfarin to rats (which inhibits osteocalcin synthesis and blocks y-carboxylation) still results in normal bone formation (Hauschka and Reid, 1978; Price et al., 1982). In addition to a proposed regulatory role in mineralization, osteocalcin may also recruit osteoclasts, resulting in an increase in bone resorption (Malone et al., 1982; Lian e f al., 1984; Glowacki and Lian, 1987). Finally, recent evidence indicates that this protein is able to inhibit the activation of prothrombin in vitro, thereby perhaps serving as a natural anticoagulant within the Haversian system or on the endosteal surfaces (Gendreau ef al., 1989). A second y-carboxyglutamic acid-containing protein, matrix Gla protein, has been isolated from bone, dentin, and cartilage (Price et al., 1983). Amino acid sequence analysis shows it to be a peptide of 84 amino acids (10,612 kDa) with five y-carboxyglutamic acid moieties (Price et a/., 1987; Kiefer et al., 1988).


205

The C-terminal domain has a 20% homology with osteocalcin (Kiefer et al., 1988). It represents approximately 2% of the NCP and accounts for 10-20% of the Gla-containing protein in adult bone (Otawara and Price 1986). While the protein occurs in calcified tissues, it is also found in noncalcified cartilage (Hale et al., 1988) and mRNA levels for matrix Gla protein are 10-fold higher in lung and heart and 5-fold higher in kidney than in bone, although these tissues do not accumulate much of the protein (Fraser and Price, 1988). As with osteocalcin, 1,25-dihydroxyvitamin D3[1,25-(OH),-D,] stimulates expression of the protein (Fraser et al., 1988). Matrix Gla protein is tightly associated with the organic phase of bone and cartilage (Price et al., 1983) and appears before the onset of mineralization (Otawara and Price, 1986). However, because of its widespread occurrence, its function does not appear to be specific for calcified tissues. It has recently been postulated that matrix Gla protein may be involved in inhibition of the mineralization process and that it is synthesized in those soft tissues that are susceptible to ectopic calcification (Fraser and Price, 1988). d. Proteolipids. Another class of proteins, the proteolipids, also appears to be intimately involved in the calcification process. The proteolipids are hydrophobic membrane proteins which tend to be complexed with various acidic phospholipids, the most common being phosphatidylserine. They are able to cause hydroxyapatite deposition both in vitro (Ennever et al., 1978) and in vivo (Raggio et al., 1986). The proteolipids are found in high concentrations in matrix vesicles (Boyan-Salyers et al., 1978) where they may act as ionophores, serving to export protons and import calcium and phosphate into the vesicle. (Sapirstein and Rounds, 1983; Swain and Boyan, 1988). They are also involved in formation of calcium phosphate-phospholipid complexes (Cotmore et al., 1971; Boskey and Posner, 1976), perhaps functioning to adjust the conformation of the phosphatidylserine to allow it to interact with the ions. These complexes are believed to be involved in the initial formation of hydroxyapatite crystals (Posner, 1985). While the phospholipids, as indicated above, are often found complexed with proteins, they also occur by themselves in close association with the mineral phase (Shapiro et al., 1966) and are enriched in matrix vesicles (Majeska et al., 1979) where they are involved in formation of the lipid bilayer membrane necessary for forming the internal environment of the vesicle. Phospholipid metabolism is influenced by 1,25-(OH),-D,, which increases phosphatidylserine levels in human osteoblastlike cells (Haining et al., 1988), and in UMR-106 rat osteosarcoma cells (Matsumoto et al., 1985). It has therefore been postulated that at least part of the effects of 1,25-(OH),-D3 on bone may be to increase the components necessary for formation of calcium phosphate-phospholipid complexes and, secondarily, hydroxyapatite formation.

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DANLEL C. WILLIAMS AND CHARLES A. FROLIK

e. Enpmes. Of the enzymes involved in the calcification process, only alkaline phosphatase has been studied extensively. The alkaline phosphatases are glycoproteins that are attached to the cell membrane via interaction with phosphatidylinositol (Low and Zilversmit, 1980). There are several isozymes of alkafine phosphatase that have been separated into three classes (liver, kidney, bone; placental; intestine) based on immune cross reactivity (McKenna et al., 1979). Recently, using specific antibodies, it has become possible to specifically detect bone alkaline phosphatase in mixtures of the other isozymes (Hill and Wolfert, 1989). In general, the enzymes are metalloproteins containing zinc and magnesium with broad substrate specificities and alkaline pH optima. The possible role of alkaline phosphatase in mineralization is supported by a number of observations. First, it was demonstrated quite early that the enzyme is able to cause precipitation of calcium phosphate in solutions of calcium salts and phosphate esters (Robison, 1923). Second, alkaline phosphatase concentration increases at the site of mineralization in bone and cartilage (Martland and Robison, 1924; Follis, 1949: Sandhu and Jande, 1982) and is present at high concentrations in matrix vesicles (Ali et al., 1970; Majeska and Wuthier, 1975). Finally. inhibitors of alkaline phosphatase will also block matrix vesicle mineral ion uptake (Register and Wuthier, 1984). It must be kept in mind, however, that alkaline phosphatases are widely distributed in the body, including locations in tissues that do not calcify. Therefore, although these enzymes may have a role in the calcification process in bone and cartilage, they must also have functions not associated with mineralization. A number of mechanisms have been proposed to explain the participation of an alkaline phosphatase in formation of the mineral phase. First, the enzyme may be involved in removal of phosphorylated inhibitors of crystallization (i.e., pyrophosphate and ATP) (Fleisch and Neuman, 1961; Fleisch and Bisaz, 1962). Second, during the hydrolysis of the phosphorylated inhibitors, inorganic phosphate becomes available for use as a substrate in the crystallization process. However, the levels of phosphorylated substrates found in cartilage extracellular fluid are considered to be too low to supply the necessary phosphate concentration needed at the nucleation site (Wuthier and Register, 1985). Alkaline phosphatase has also been implicated in the transport of inorganic phosphate across the cell membrane (Petit-Clerc and Plante, 1981; Letellier e l al., 1982), though this role has been questioned, since selective removal of alkaline phosphatase from renal tubule brush border membranes actually stimulates phosphate transport (Yusufi et al.. 1983). However, alkaline phosphatase may play a modulatory role as part of a multiprotein membrane complex, perhaps involving phosphorin, a proteolipid postulated to be involved in phosphate transport (Kessler and Vaughn, 1984). In addition to its possible function in controlling phosphate concentrations, the enzyme also has calcium binding properties (Vittur and

REGULATION OF CALCJFICATION

207

deBemard, 1973) and Ca-ATPase activity (Haussler et al., 1970) although these properties may be due to other components that copurify with the enzyme. Finally, alkaline phosphatase has been reported to have phosphotyrosyl protein phosphatase activity (Swamp et af.,1981). While selective removal of a phosphate group from tyrosine may be involved in controlling many of the responses of growth factor stimulation, the role of alkaline phosphatase in this pathway is still uncertain. In support of this concept, levels of the enzyme rise at confluency when cell growth slows, growth factors are able to stimulate alkaline phosphatase activity (Wuthier and Register, 1985) and factors that suppress alkaline phosphatase activity are able to stimulate cellular proliferation (Carpenter, 1981). B. MODELING AND REMODELING IN SKELETAL TISSUES

In the face of a burgeoning literature that has emphasized the physiology and metabolism of individual cellular components of the skeletal system (e.g., osteoblast, osteoclast, chondrocyte, etc.), Frost and others have emphasized a holistic approach to understanding the biogenesis and maintenance of calcified tissue in the vertebrate skeleton (Frost, 1988, 1989a-d). In order to understand how cells function to form skeletal tissues, and how these tissues interact to form and maintain a functioning skeletal system, it is necessary to realize that the processes which occur during skeletal growth and development, and those that are responsible for skeletal maintenance, often involve the same cellular players. However, these cells may function by a different set of regulatory rules under different physiological conditions (Jee, 1983; Eriksen, 1986; Frost, 1988; Burr and Martin, 1989). Bone modeling is a term used to describe the processes involved in bone growth and changes in bone shape (some investigators, e.g., Frost, 1988, prefer to consider growth and modeling as mechanistically separate). Bone formation and resorption are not “in balance” either locally or systemically during the process of modeling, and both formation and resorption may be occurring simultaneously in different regions of the same bone. While modeling is primarily associated with the immature, growing skeleton, changes in bone shape can also occur in the mature skeleton in response to appropriate stimuli (Frost, 1988). On the other hand, bone remodeling refers to the processes by which bone is maintained in nongrowing tissue. Resorption and formation occur sequentially in a local region of the skeleton and are quantitatively in balance. This equilibrium between bone formation and resorption is often referred to as “coupling” (Frost, 1963; Martin et al., 1988). Errors in the coupling of bone formation and resorption during remodeling can lead to skeletal disease (Eriksen, 1986; Marcus, 1987; Burr and Martin, 1989).

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DANIEL C. WILLIAMS AND CHARLES A. FROLlK


209

C. CELLSOF BONE 1. Osteoblasts

Osteoblasts are derived from noncirculating mesenchymal cells called osteoprogenitors. These progenitor cells are found in the soft connective tissue of the periosteum, endosteum, and Haversian canals of bone, and among the stromal cells in the trabecular bone marrow adjacent to the bone (J. Russell, et al., 1986; for review see Marks and Popoff, 1988). Four differentiation stages can be defined morphologically in the osteoblast lineage-the preosteoblast, the osteoblast, the lining cell, and the osteocyte. These cells are recognized by their morphology, by their position in bone, and by their proliferative capacity. Under well-defined conditions within an organized tissue environment, preosteoblasts can be recognized by morphological criteria such as their localization in soft tissues adjacent to bone, their nuclear size and morphology, and their glycogen and alkaline phosphatase content (Scott, 1967; Roberts et al., 1982). Efforts are also underway to define these cells by antigenic characteristics (Nijweide and Mulder, 1986; Nijweide el al., 1988; Nakano el al.,l989; Peny et al., 1990). These endeavors are particularly important in order to better understand the properties of osteogenic cells in culture where their characteristic morphology and tissue relationships may not be evident. Osteoblasts are observed histologically as cuboidal cells lying on the bone surface (Figs. 1 and 2). They are morphologically and functionally polarized cells with abundant rough endoplasmic reticulum and a prominent Golgi region, i.e., characteristics of a secretory cell. Their main product, type I collagen, is assembled extracellularly to form the primary organic constituent of osteoid, the matrix which is mineralized to form the rigid structural elements of bone. The FIG.1. Light micrograph of a section through the metaphysis of a rat long bone showing the classic morphology of osteoblasts (Ob) on the bone surface that are actively secreting bone matrix. The cells are cuboidal to columnar in shape with an acentric nucleus and a prominent central secretory region. Also shown in this section are osteocytes (0)located within the bone matrix, and an osteoblastic transition cell (T) which is located just below the surface layer of osteoblasts. This cell is an osteoblast that is being surrounded with bone matrix and becoming an osteocyte. Bar = 10 pm. FIG.2. Transmission electron micrograph of a demineralized bone preparation showing a section of an osteoblastic (Ob) transition cell in canine rib bone. This cell lies at the surface of the bone matrix (BM), and has been almost completely surrounded by loose collagen fibrils. It has extended a cellular process through the canalicular canal (C) in the bone matrix which allows it to form contacts with osteocytes, and also maintains contact with the overlying osteoblasts by close cellular contacts (arrows) that form gap junctions between the cells. Bar = 1 pm. FIG.3. Light micrograph of a section through a rat tibia1 metaphysis. A large, multinucleate osteoclast (arrow) can be seen on the surface of a bone aabecula. Bar = 10 pm. FIG.4. Transmission electron micrograph showing a large multinucleate osteoclast (Oc) forming a resorption lacuna (RL) between itself and the surface of the bone matrix (BM). The numerous cellular processes extending into the resorption lacuna form the osteoclastic ruffled border. Bar = l pm.

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shape and density of the mineralized matrix varies depending on its location within a bone and on the location of the bone within the skeletal framework. The regulatory steps that determine these characteristics are poorly understood. In adult mammalian bone, osteoblasts lay down collagen fibrils in a parallel manner producing a lamellar arrangement. Individual lamellae are typically 3-7 pm thick and appear to be stacked together to form the bony tissue. The collagen fibers in adjacent lamellae are not parallel with each other and the lamellae are separated by interlamellar “cement.” In embryonic bone, fracture healing, or certain disease states, osteoblasts produce a less organized woven bone in which the collagen fibers are nonparallel and irregularly arranged. Cultured osteoblastic cells have been derived from a variety of species and have proven to be valuable tools in understanding osteoblastic function (e.g., Peck et al., 1964: Wong and Cohn, 1975; Kodama et al., 1981; Williams et al., 1980; Partridge ef al., 1981; Robey and Termine, 1985; McCarthy ef al., 1988). Both primary and longer term cultures of cells obtained from normal tissues have been employed. However, information acquired from primary cultures must generally be considered as being derived from a heterogeneous mix of bone cells, whereas, in long-term cultures, there frequently appears to be a change of phenotypic expression associated with extended culture which can serve to complicate interpretation of data (e.g., Aubin et al., 1988). In addition, a number of the osteoblastic cell lines used as models for bone metabolism studies have been derived from osteosarcomas [e.g., UMR (Partridge et al., 1981) and ROS (Majeska er al., 1980) cell lines]. The extension of information from these cancer cells to normal tissue metabolism must be viewed cautiously. Typical phenotypic markers used to identify osteoblastic cells include type I collagen synthesis, production of alkaline phosphatase, and response to the calciotropic hormone FTH (G. A. Rodan, er at., 1989). Mineralization in vim may also be considered to be an important phenotypic marker (Williams ef al., 1980; Sudo er al., 1983). lntegrens (cell surface receptors for a range of cell and extracellular matrix proteins) on osteoblasts do not seem to differ radically from other stromal or fibroblastic cells (Horton and Davies, 1989). Several animal models have been used to investigate the effects of mechanical loading in modulating bone formation and osteoblast activity. Suppression of weight bearing or complete inactivity will lead to loss of bone, whereas physical activity increases relative levels of bone mass (Schoutens et al., 1989). Experiments applying dynamic loads to functionally isolated bones of skeletally mature roosters also indicate that removal of load bearing results in reduced bone mass, while cyclic loading prevents resorption and causes bone formation (Rubin and Lanyon, 1984). A number of studies, performed on animals in real or simulated space flight conditions, have demonstrated that osteopenia can result from exposure to microgravity. The mechanism of this osteopenia appears to be a transient inhibition of osteoblastic function (Morey and Baylink, 1978;


21 1

Wronski and Morey, 1983; Jee et al., 1983; Simmons et al., 1983) in the presence of unchanged levels of bone resorption (Wronski and Morey, 1983; Jee et al., 1983; Cam and Adachi, 1983; Bikle et al., 1987; Vico et al., 1988). Taken from another view, Pead et al., (1988) have demonstrated that a single short period of dynamic loading is capable of transforming quiescent adult periosteal cells on the bone surface to active bone-forming cells. Studies with bones in culture have shown that intermittent compressive force, applied to mouse bone rudiments, stimulates matrix mineralization and inhibits osteoclastic invasion and resorption of mineralized bone (Burger et al., 1989). Osteoblastic cells in culture will respond to cyclical mechanical deformation by activation of both prostaglandin synthesis and CAMPand phosphodiesterase pathways, by increases in cell division rates, by changes in cell orientation on the culture substrate, and by release of bone resorbing agents (Buckley et al., 1988; Sandy et al., 1989a,b). Bone cells will also respond to electrical stimulation with an enhanced osteogenesis. This response has been used clinically to treat problem fractures (Haupt, 1984). Similarly, a number of recent studies in animal models suggest that electrical (or electromagnetic) stimulation of osteogenesis might find utility in the treatment of osteoporosis. The effects of electrical stimulation in these models appears to be associated with increased bone formation which may or may not involve effects on bone resorption (e.g., Brighton et al., 1988, 1989; Rubin et al., 1989). Pulsed electrical stimulation has also been shown to stimulate DNA synthesis in cultured osteoblastic cells (Ozawa et al., 1989). Finally, a study of the effects of surface charge on beads injected intrafemorally into the medullary cavity of chicken hatchlings indicated that new osteogenesis was found to be preferentially associated with positively charged beads. These positively charged beads at neutral pH created a negative counterionic environment, thus making the finding consistent with the observation that osteogenesis is associated with the cathodes of surgically implanted electrodes (Krukowski et al., 1988). In addition to their role in bone formation per se, osteoblasts (and/or bone lining cells) play a crucial role in bone resorption (see discussions in Rodan and Martin, 1981; McSheehy and Chambers, 1986a; Kahn and Partridge, 1987; Martin et al., 1988). Osteoblasts, but not osteoclasts, have receptors to several resorption-promotingagents, and osteoblasts release factors capable of stimulating bone resorption. Several osteoblast products have been identified which may serve to mediate both the short and long-term osteoblast-osteoclast interaction. These include granulocyte-macrophage colony-stimulating factor (GM-CSF) (Shiina-Ishimi et al., 1986; Felix et al., 1988; Horowitz et al., 1989a,b), prostaglandins (Nolan et al., 1983; MacDonald et al., 1984; Feyen et al., 1984), interleukins (Feyen et al., 1989), TGFP (Centrella and Canalis, 1987; Robey et al., 1987), and a number of less well-characterized factors (e.g., McSheehy and

212


Chambers, 1986b; Abe et al., 1988; Sandy et al., 1989b). Osteocalcin, synthesized exclusively by osteoblasts, may be involved in osteoclast recruitment and/or differentiation (Malone ef al., 1982; Lian er al., 1984; Glowacki et al., 1989). In addition, osteoblasts produce a neutral collagenase capable of rernoving the nonmineralized osteoid from the bone surface so the osteoclast can resorb the mineralized bone (Chambers and Fuller, 1985). Finally, the osteoblasts produce plasminogen activator, an enzyme hypothesized to be involved in the activation of collagenase, as well as inhibitors capable of blocking both collagenase and plasminogen activator activity (Kahn and Partridge, 1987; Martin et al., 1988). Osteoblasts also may regulate osteoclast access to the bone surface through changes in cell shape (Rodan and Martin, 1981). Changes in osteoblast cell shape have been observed by several groups in response to either PTH or prostaglandin E, (PGE,) (Jones and Boyde, 1976; Miller et al., 1976; Jones and Ness, 1977; Shen et al., 1986). Ali et al. (1990) found, however, that osteoblasts from 2-week-old rats, growing on a natural substrate, change shape in response to PTH but not PGE,, though both agents are known to induce bone resorption in neonate calvarial culture. These observations, they suggest, indicate that osteoblast shape change may not be essential for bone resorption.

2 . Bone Lining Cells Bone lining cells are thin, flat elongated cells containing few organelles (Luk et al., 1974a,b; Miller et al., 1980; Bowman and Miller, 1986). These cells cover the bone surface (Figs. 5 and 6), appearing to be metabolically inactive with respect to bone formation or resorption, and are often joined to each other by gap junctions. While viewed by many as “inactive” osteoblasts, Miller and Jee (1987) have proposed that the lining cell represents a separate phenotype. Evidence exists for an estrogen-induced transition between the lining cell and osteoblast in avian medullary bone induction (Bowman and Miller, 1986). However, relatively little is known about the function and regulation of lining cells because they are found primarily in nongrowing adult bone, while the vast majority of cell biology studies are carried out in fetal tissues where these cells do not play a significant role (Nijweide et al.. 1986). It has been speculated that they may have one or more of the following functions-progenitors for osteoblasts, selective barriers for mineral exchange between bone and extracellular fluid, regulation of crystal growth, regulation of hemopoiesis, and/or physical barriers to osteoclastic bone resorption (Rodan and Martin, 1981; Miller and Jee, 1987; Marks and Popoff, 1988). 3. Osteorytes

Osteocytes are derived from osteoblasts. They lie within lacunar spaces embedded within the mineralized bone (Figs. 1, 5, and 7). Osteocytic lacunae


213

are connected to each other, and to cells on the bone surface, by a complex canalicular lattice (Figs. 2, 5, and 7). This canalicular network allows the osteocytes to communicate with each other and the bone surface via thin cytoplasmic processes which form gap junctions with other cells. The exact function of the osteocyte is not known. Some investigators maintain that the osteocyte can both form and resorb bone (Belanger, 1969; Marks and Popoff, 1988), although this opinion is controversial (Sissons el al., 1984, 1990; Mercer and Crenshaw, 1985). An alternative view is that the osteocyte may be involved in sensing mechanical force on bone and relaying this information to the bone surface where resorptive or formation responses can be initiated (Martin et aZ.,1988). Osteocytes, as well as periosteal cells, will respond to mechanical loading (strain) by rapid changes in glucose-6-phosphatedehydrogenase (G6PD) activity, but not glyceraldehyde-3-phosphate dehydrogenase or aldolase activity, and by a slower (24 hr) increase in RNA synthesis ([3H]uridine uptake). While the functional significance of these changes is not currently known, the increase in the number of osteocytes demonstrating G6PD activity has been shown to be proportional to the mechanical strain applied to the bone, and related to increased bone formation at the bone surface (Skeny er al., 1989). 4. Osteoclasts

Osteoclasts are large multinucleated cells, located on the bone surface, which resorb mineralized bone and cartilage (Figs. 3 and 4). Active osteoclasts have two unique, specialized cell surface regions, a ruffled border, and a clear zone. The ruffled border is a highly folded region of the plasma membrane adjacent to the resorbing bone that has prominent cytoplasmic vacuoles (primary and secondary lysosomes) associated with it. The clear zone is an actin-rich region of the cell cytoplasm next to the ruffled border region and is thought to be involved in cell-substrate adhesion (Martin et al., 1988; Marks and Popoff, 1988). Though for many years osteoclasts and osteoblasts were thought to be derived from a common progenitor cell population, osteoclasts are now generally regarded to be derived from hemopoietic cells. A variety of in vivo and in vitro models have been used to support this hemopoietic origin. Studies using osteopetrotic rodents have proven to be particularly informative. Osteopetrosis is an inherited bone disease in which defective bone resorption results in excessive accumulation of bone throughout the skeleton. Walker (1975) first showed that osteopetrosis in rodents could be reversed by transplantation of hemopoietic tissue. In vitro, osteoclastlike cells can be formed in a variety of marrow cultures (MacDonald et al., 1987; Takahashi el a/., 1988a-c) as well as from circulating mouse blood leukocytes (Helfrich et al., 1989) and spleen cells (Takahashi el al., 1988b; Udagawa et al., 1989). Akatsu et al. (1989) have shown that prostaglandin (especially PGE, and PGE,) can stimulate the formation of osteoclastlike cells from mouse bone marrow cultures, apparently through a

DANIEL C. WILLIAMS A N D CHARLES A . FROLIK


215

CAMP-associated mechanism. PTH and 1,25-(OH),-D, can also induce the formation of osteoclastlike cells in these cultures (MacDonald er d., 1987; Takahashi et al., 1988~).In several of these in v i m models, osteoclast formation can be inhibited by calcitonin (e.g., McDonald er al., 1987). The formation of osteoclasts in marrow preparations and in spleen cell preparations is enhanced by the presence of osteoblastic or marrow stromal cells (Takahashi er al., 1988b; Wdagawa et al., 1989). Many workers propose that the osteoclast shares a common origin with phagocytic monocytes and macrophage. The work of Schneider and Relfson ( 1988), however, suggests that osteoclast precursors co-isolate with granulocyte colony-forming cells (G-CFC) but not macrophage colony-forming cells (M-CFC). Granulocyte-macrophage-colony forming cells (GM-CFC) populations were intermediate in their ability to reverse osteopetrosis in rats. These data suggest that the immediate precursors to osteoclasts and monocyte/macrophage are not the same, but do not necessarily indicate that the granulocyte lineage is the source of the osteoclast precursor. A more detailed discussion of the history of osteoclast lineage can be found in the reviews of Nijweide et al. (1986) and Marks and Popoff (1988). Osteoclasts induced to form in mouse marrow preparations are typically characterized by being multinucleate, and containing tartrate-resistant acid phosphatase. Some studies also evaluate the ability of the cells to resorb bone and look for induction of calcitonin receptors in association with osteoclast differentiation (Hattersley and Chambers, 1989; Taylor er al., 1989). Other studies look for evidence of immunologic relationships between cells formed in culture and osteoclasts from bone (Kukita and Roodman, 1989; Kukita ef al., 1989). In addition, integrens, which on mature osteoclasts differ from other hemopoietic cell types (i.e., large amounts of vitronectin receptor and collagen type I receptor), perhaps reflecting their specialized role in bone (Horton and Davies, 1989), may be useful in characterizing osteoclastic cells derived from marrow cultures. Bone resorption occurs in a specialized environment that is under the control of the osteoclast. This cell attaches to the bone surface to form an enclosed space, the resorption lacuna (Fig. 4), which becomes acidified (e.g., Fallon, FIG.5. Light micrograph showing the thin, flat nuclei (arrows) of the cells (lining cells) that line the medullary cavity of a quail long bone diaphysis. Also shown are osteocytes (Oc) and the network of canaliculae that connect the osteocytes to each other and the cells on the bone surface. Bar = 10

w.

FIG. 6. Transmission electron micrograph of a section through lining cells (LC) on the surface of trabecular bone in dog rib. The lining cells form a continuous cellular boundary separating the bone surface from the cells in the marrow cavity. Bar = 1 pn. FIG.7. Transmission electron micrograph of an osteocyte (0)in dog rib bone. The osteocyte is located in a lacunar space that is surrounded by bone matrix. Several thin cellular processes can be seen projecting into canalicular canals (C) that form pathways for cell-cell contact through the dense bone matrix. Bar = 1 pm.

216


1984; Baron et al., 1985) through the action of a proton pump located in the apical membrane (Anderson et al., 1986; Baron, 1989; Blair et al., 1989). Ultrastructural and immunocytochemical evidence suggests a role for cytoplasmic microfilaments in forming the tight membrane-cytoplasmic adhesion needed to maintain the acidic environment in the resorption lacunae (King and Holtrop, 1975; Turksen et al., 1988; Lakkakorpi et al., 1989). The attachment of the osteoclast and formation of the resorption lacuna defines the morphological and metabolic polarity of the active osteoclast (Baron, 1989). The apical pole of the cell is the region attached to the extracellular matrix, and the apical membrane forms the clear zone and ruffled border. Lysosomal enzymes are vectorially transported to the apical end of the cell in association with mannose-6-phosphate receptors (Baron er al., 1988). Upon fusion with the apical cell membrane and exposure to the acid extracellular environment of the resorption lacuna, the enzymes dissociate from the mannose-6-phosphate receptor and are released into the resorption lacuna. Osteoclastic acidification of the resorption lacuna is stimulated by PTH and inhibited by calcitonin (Baron et al., 1985; Hunter el al., 1988). The acidification of this space is mediated through the action of a proton pump located in the ruffled border membrane adjacent to the resorption lacuna (Baron ef af., 1985; Baron, 1989). The identity of the proton pump is not clear, and evidence for a H+-K+-ATPasepump (Baron et al., 1985; Tuukkanen and Vaananen, 1986; Anderson et al., 1986), a Na+-H+-antiporter(Hall and Chambers, 1990b), and an electrogenic H+-ATPase pump (Blair et ul., 1989; Baron, 1989) have been reported. A significant body of evidence suggests that carbonic anhydrase may also play a role in osteoclast acidification and bone resorption. Carbonic anhydrase is the enzyme responsible for the reversable hydration/dehydration reaction converting carbon dioxide to carbonic acid. Carbonic anhydrase has been localized histochemically to the apical region of the cell (Anderson et al., 1982; Cao and Gay, 1982), and inhibitors of carbonic anhydrase inhibit the acidification process (Hunter et a/., 1988). Carbonic anhydrase levels in osteoclasts can be modulated by FTH (Hall and Kenny, 1985; Silverton eta/. , 1987), and its cellular localization regulated by calcitonin (Anderson ef al., 1982; Cao and Gay, 1982). Thus the H' ions produced as a result of the action of carbonic anhydrase in the apical domain of the osteoclast are transported into the resorption lacuna via the proton pump. Evidence for a complementary CI--HCO,- anion transport system to remove carbonate ions from the cells, and thus balance the intracellular pH, has also recently been reported (Teti et al., 1989; Hall and Chambers, 1989; Klein-Nulend and Raisz, 1989). The cellular localization of this transport system has not been defined, but is assumed to be in the basolateral domain (Baron, 1989). Baron (1989) suggests that the basolateral cell membrane is also enriched with respect to sodium pumps (Na+,K*-ATPase)which may supply the primary force for proton transport, though others suggest an apical localization


217

for this ATPase activity (Akisaka and Gay, 1986). A calcium pump (Ca-ATPase) has been shown to be localized on the marrow (basal) face of the osteoclast and may be associated with removing excess calcium derived from resorbed bone out of the intracellular environment (Akisaka et al., 1988). The osteoclast can thus be viewed as a highly organized enzymatic factory in which hydrolytic enzymes are synthesized in the endoplasmic reticuludGolgi apparatus and transported in secretory vesicles (primary lysosomes) to the apical domain of the cell where they are released into a closed, acidic resorption lacuna formed by the tight adhesion of the osteoclast to the bone surface. The products of bone resorption in the resorption lacuna are taken up by the osteoclast by the process of endocytosis (phagocytosis).The osteoclast contains a variety of acidification and ion transport cellular machinery, localized in different functional domains of the cell.

111. Physiological Regulation of CaIeification in the Vertebrate SkeIeton

In the skeletal system, there are many factors that affect the initiation and rate of calcification. These biological regulators include collagen (BerthetColominas et al., 1979; Glimcher, 1989) pyrophosphates (Fleisch and Bisaz, 1962). proteoglycans (Chen et al., 1984), osteocalcin (Menanteau et al., 1982; Boskey et al., 1985), phosphoproteins (Fujisawa et al., 1987), and osteonectin (Menanteau et al., 1982; Romberg er al., 1986). It is clear that anything that increases or decreases the extracellular matrix concentration of these proposed regulators could affect the calcification process. Indeed, several hormones and growth factors are able to modulate the levels of these inhibitors and stimulators of crystal growth (Tables I1 and 111). In this section, these physiological regulators will briefly be discussed. While the focus of this article is on the cellular and biochemical processes directly involved in the formation and maintainence of calcified tissues, it is clear that associated mechanisms, such as the absorption, excretion, and transport of calcium and phosphate, also play an important role in the calcification process. A. ENDOCRINE FACTORS

1 . Parathyroid Hormone (PTH) There are several hormones that act on bone in order to maintain normal blood calcium homeostasis. Included in this group is PTH, a hormone secreted from the parathyroid gland in response to low serum calcium. In addition to its actions on bone, PTH accelerates calcium absorption in the intestine and kidney. In bone, PTH interacts primarily with the osteoblast and chondrocyte which


218

TABLE I1 ENDOCRINE REGULATORS OF BONEMETABOLISM Hormone

Proposed Role

Parathyroid hormone (PTH) Increase bone formation and resorption

Interaction with Other Factors Increases release of IGF-1, IGF-11, and GMCSF Modulates activity of TGFP. EGE and IL- I Increases levels of 1,25-(OH),-D, Effects inhibited by estrogen and testosterone

Calcitonin

Decrease bone resorption and increase bone formation

Increases levels of I ,25-(OH),-D, Effects enhanced by testosterone Secretion modulated by estrogen

Vitamin D

Regulation of calcification process

Decreases PTH levels Increases EGF receptor number Increases TGFP secretion Increases activity of IGF Modulates response to gonadal steroids Receptors regulated by retinoic acid and glucoconicoids Estrogen increases 1.25-(OH)Z-D, production

Vitamin A (retinoids)

Alter extracellular matrix synthesis Modulates PTH secretion and effects Controls I .25-(OH)2-D,activity and receptor levels Increases growth hormone production

Estrogens

Suppression of bone turnover

Modulates PTH and calcitonin Increases I .2S-(OH)z-D, production Effects modulated by 1,25-(OH),-D, Increases synthesis and secretion of IGF-I and TGFP Increases release of growth hormone

Androgens

Maintenance of bone mass Increase in bone formation

Inhibits PTH response Increases growth hormone and IGF-I production

Growth hormone

Increase in bone formation

Increases synthesis of IGF-I Production increased by retinoids, estrogen and androgens


219

TABLE I11 OF BONEMETABOLISM AUTOCRIN~~ARACRUE REGULATORS Growth Factor

Proposed Role

Interaction with Other Factors

IGF

Increase bone matrix production Increase osteoprogenitorcell proliferation

Secretion regulated by growth hormone, estrogen, F'TH, 1,25-(OH)2-DS, ~-miC@Obulin, and glucocorticoids Decreases secretion of growth hormone Regulated 1,25-(OH),-D, levels Synergizeswith EGF, FGF

TGFP

Regulates embryonic formation of cartilage and bone Modulates extracellular matrix production Modulates bone resorption

Activity increased by FGF, IL- I , 1,25-(OH),-DS,estrogen and decreased by calcitonin Modulates effects of FGF, EFG, IL-1, and I,25-(OH),-D3

Bone inductive proteins (BMPs, OF, osteogenin)

Induction of mesenchymd cell differentiation

OIF synergizes with TGFP Osteogenin activity modulated by PDGF and vitamin D BMP activity regulated by cytokines

EGF (TGFa)

Increase osteoblast/osteoclast precursor pool

Receptors modulated by TGFP, FTH Synergizes with IL-1

FGF

Increase osteoprogenitorcell proliferation

Increases TGFP levels Potentiated by TGFP, IGF

PDGF

Stimulatesosteoblast proliferation Stimulatesbone resorption

Effects modulated by IGF-I

Cytokines (IL-1, IL-3, GMCSF, TNF)

Regulation of matrix producing cells Modulation of bone resorption

Effects modulated by TGFP, FGF, 1,25-(OH),-D3 Regulates actions of EGF, TGFa, PTHrP Interacts with estrogen GMCSF induced by PTH

have been shown to possess high affinity receptors for the hormone (Silve et al., 1982; Pliam et al., 1982; Newman el al., 1989; Enomoto et al., 1989). The receptor is a glycoprotein (80,000 kDa with a 59,000 kDa polypeptide backbone) and is localized on the cell surface plasma membrane (Shigeno er al., 1988a,b). Parathyroid hormone will induce receptor down-regulation (Yamamoto er al., 1988; Abou-Samra et al., 1989b) with expression of the receptor being under the control of inhibitory guanyl nucleotide regulatory proteins (Abou-

220


Samra et al., 1989b). In rat long bones, PTH receptors have been localized to the osteoblast and a preosteoblastlike cell with little or no binding to the osteoclast (Rouleau et al., 1988). This supports the view that PTH acts indirectly on the osteoclast through interaction with the osteoblast, causing release of a soluble factor which then acts on the osteoclast to increase bone resorption (Rodan and Martin, 1981; McSheehy and Chambers, 1986a,b; Perry ef al., 1989). While the primary action of PTH on bone is the stimulation of resorption, (for review see Martin ef al., 1985), effects on bone formation have also been noticed. In viw, administration of physiological amounts of PTH results in gain of bone (Podbesek et al., 1983; Slovik et a[., 1986) due to increases in both cortical and trabecular bone mass (Hock ef al., 1988a). Subcutaneous injections of PTH into rats over a period of 12 days causes an increase in total bone calcium, dry weight, and bone-forming surfaces. These effects were not dependent on bone resorption (Hock et al.,l989a). In v i m , PTH can stimulate the proliferation of chick calvaria cells in culture (Farley ef al., 1988a) and human trabecular bone cells, cultured at high cell density, but has no effect on human skin fibroblasts (MacDonald et al., 1986a). The effect of PTH on bone formation may be through an increase in both the number and activity of osteoblasts, resulting in an increase in the extent of bone-forming surfaces and in the rate of mineral and matrix apposition. Parathyroid hormone causes an increase in the steady state levels of osteocalcin mRNA (Noda et al., 1988a), stimulates amino acid transport in neonatal mouse calvaria (Yee, 1988; Hall and Yee, 1989), DNA synthesis in fetal rat calvaria (Canalis et al., 1989a) and in embryonic chicken chondrocytes (Schluter et al., 1989) and, with transient treatment, increases type I collagen synthesis (Canalis et al., 1989a). The mechanism whereby PTH causes these anabolic effects is not known. They do not appear to be mediated by prostaglandins (Gera et af., 1987) or CAMP (Schluter et al., 1989). Recently, the involvement of the insulinlike growth factors (IGFs) has been suggested. Parathyroid hormone will stimulate the release of IGF-I and IGF-I1 from neonatal mouse calvaria (Linkhart and Mohan, 1989), and from fetal rat calvaria (Canalis et af., 1989a), and will stimulate the synthesis of IGF-I in osteoblastlike cells (McCarthy ef al., 1989a). Also, the effect of PTH on collagen synthesis in fetal rat calvaria is blocked by IGF-I antibodies, although the antibodies will not inhibit the mitogenic effect of PTH (Canalis et al., 1989a). Finally, infusion of IGF-I or PTH alone into the hind limb of a rat will not affect the rate of trabecular bone apposition but, when given together, will cause a significant rate increase (Spencer ef af., 1989). Therefore, at least some of the effects of PTH may arise from an autocrine mechanism through the release of the IGFs or a potentiation of their effects. In addition to IGF, PTH is known to interact with several other growth factors and hormones. In osteoblastlike cells, PTH modulates the activity of TGFP and enhances its cellular binding (Centrella ef al., 1988). The opposite effect has


22 1

been shown for epidermal growth factor (EGF), where PTH decreases both the number of EGF receptors and the responsiveness of the MC3T3-El osteoblastlike cell line to EGF (Ohta et al., 1989). Parathyroid hormone synergizes with interleukin-1 (IL-1) in stimulating bone resorption both in v i m and in vivo (Dewhirst et al., 1987; Sat0 et al., 1989) and induces the secretion of GM-CSF from bone cells (Weir et al., 1989; Horowitz et al., 1989a). Since GM-CSF is able to increase the number of osteoclasts (MacDonald et al., 1986b; Horowitz et al., 1989a), this factor may be the osteoblast-derivedcomponent that mediates PTH-stimulated effects on osteoclast and bone resorption. The effects of PTH on bone resorption are further modulated by the steroid hormones. Estrogen inhibits PTH stimulated bone resorption and PGE, release in the neonatal mouse calvaria (Pilbeam et al., 1989) and PTH-stimulated adenyl cyclase activity in primary cultures of calvaria and trabecular bone cells (Emst et al., 1989), and in SaOS-2 human osteosarcoma cells but not in ROS 17/2.8 rat osteosarcoma cells (Fukayama and Tashjian, 1989a). Testosterone and dihydrotestosterone also inhibit the cAMP response to PTH in SaOS-2 cells (Fukayama and Tashjian, 1989b). The net effect of these actions would be to decrease bone resorption. Finally, PTH plays a role in the metabolism of vitamin D by increasing the 25hydroxyvitamin D,- la-hydroxylase activity in the renal cortex of the kidney, thereby controlling the levels of 1,25-(OH),-D,, the active metabolite of vitamin D (Siege1etal., 1987). Parathyroid hormone may have a dual pathway for generating changes in the osteoblast. It was known quite early that PTH can activate adenyl cyclase and increase cellular levels of cAMP (Chase and Aurbach, 1970). More recently, it has been demonstrated that PTH is also able to stimulate the inositol phosphate pathway in calvarial osteoblastlike cells (Somjen et al., 1987; Farndale et al., 1988; Dunlay and Hruska, 1990). In this pathway, hydrolysis of phosphatidylinositol-4,5-bisphosphateby phospholipase C releases inositol 1,4,5-trisphosphate (which mediates the release of intracellular calcium) and diacylglycerol (which activates protein kinase C) (Berridge and Irvine, 1984). Indeed, both protein kinase C (Somjen et d., 1987; Abou-Samra et al., 1989a) and calcium mobilization (Lowik et al., 1985; Somjen et al., 1987; Civitelli et al., 1989b; Schluter er al., 1989) have been implicated in the PTH response. While it is tempting to speculate that the bone-resorbing effects of PTH may be mediated primarily through the cAMP pathway (Civitelli et al., 1989a), while its anabolic effects occur through its interactions with the phosphoinositide pathway (Somjen et al., 1987; Dunlay and Hruska, 1990), certain PTH analogs that are able to stimulate resorption without increasing cAMP concentrations (Lowik et al., 1985) indicate that it may not be as simple as this. A second protein with PTH-like properties has been found in several different tumor cells (Rodan et al., 1983; Moseley et al., 1987). The first 13 N-terminal amino acid residues of this PTH-related peptide (PTHrP) shares a 62%

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homology with PTH while the remaining sequence is markedly different (Suva et al., 1987). Parathyroid hormone-related peptide binds to the same receptor as PTH (Horiuchi et al., 1987; Abou-Samra er al., 1989c; Klein-Nulend et al., 1990). Its effects on bone and kidney are similar to those seen for PTH (Horiuchi et al.. 1987; Klein-Nulend et al., 1990), although it is perhaps slightly less potent (Fukayama et al., 1988; Hock et al., 1989b). Like parathyroid hormone, PTHrP modulates the effects of locally produced growth factors. For example, in osteoblast enriched cultures, PTHrP, while having no effect by itself, enhances the effects of TGFP on DNA and collagen synthesis and matrix turnover (Centrella er al., 1989a). The normal physiological function of PTHrP is unknown. Its presence in tumors (Moseley et al., 1987), and the fact that it produces hypercalcemia in vivo (Horiuchi et al., 1987) has lead to the suggestion that it may be responsible for the hypercalcemia observed in malignancy. 2 . Calcitonin Calcitonin, secreted from the C-cells of the thyroid gland, acts as an antagonist to PTH and decreases serum calcium levels (for reviews see Fischer and Born, 1987; Breimer et al., 1988). It is a 32-amino acid peptide that is the product of a gene complex that produces either calcitonin or calcitonin gene-related peptide (Fischer and Born, 1987; Breimer et al., 1988). The major action of calcitonin is to decrease bone resorption and to enhance kidney production of 1,25(OH),-D,. In modulating resorption, calcitonin appears to act directly on the osteoclast. Calcitonin receptors have been detected on this cell (Warshawsky et al., 1980; Rao et al., 1981; Nicholson et al., 1986) and the hormone abolishes cytoplasmic mobility and spreading of isolated osteoclasts in culture (Chambers and Magnus, 1982; Chambers et al., 1986; Nicholson et al., 1987). The effects of calcitonin may be partly mediated through the production of CAMP(Murad et al., 1970; Heersche ef al., 1974; Rodan and Rodan, 1974), although calcium may also play a modulatory role (Malgaroli et al., 1989). In addition to its effects on inhibition of resorption, calcitonin will also stimulate growth and maturation of embryonic chick cartilage in vitro (Burch, 1984) and bone formation in vivo (Weiss et al., 1981). Although calcitonin is not currently believed to have a significant etiological role in the development of osteoporosis (McDermott and Kidd, 1987; Prince et al., 1989), it has been used as a therapeutic agent in osteoporosis to increase trabecular bone volume and bone mineral content (Gruber et al., 1984; Mazzuoli et al., 1985; Civitelli et al., 1988). These anabolic effects of calcitonin appear to be a result of a direct interaction with the osteoblast. High affinity calcitonin receptors have been detected on a bone stem cell line (Eilon et al., 1983), and on osteoblastlike cells (Findlay and Martin, 1986). Calcitonin is able to stimulate the differentiation of an osteoblastlike cell line, MC3T3-E 1, and the production of alkaline phosphatase


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(It0 et al., 1987). These anabolic effects occur at a similar dose level that causes an inhibition of bone resorption (Farley et al., 1988b). Although not extensively investigated, calcitonin does appear to interact with other factors. Testosterone enhances the hypocalcemic effect of calcitonin in castrated rats (Ogata et al., 1970), while estrogen has been reported to modulate calcitonin secretory capacity, with lower calcitonin levels found in the postmenopausal woman (Reginster er al., 1989). However, other investigators have not observed a direct effect of estrogen on calcitonin secretion and postulate that any observed variations may be due to the effects of estrogen on bone metabolism (Body et al., 1989). In the mouse marrow culture system, 1,25(OH),-D, stimulates the formation of calcitonin-responsive cells and the appearance of calcitonin receptors (Takahashi et al., 1988a; Hattersley and Chambers, 1989). In return, calcitonin is able to act on the kidney to increase the production of 1,25-(OH),-D, (Rasmussen et al., 1972). These higher levels of 1,25-(OH),D, are then able to stimulate intestinal absorption of calcium, thus providing additional motivation for the decrease of bone resorption.

3. Vitamin D Vitamin D and its metabolites, 1,25-(OH),-D3 and 24,25-(OH),-D,, are another group of factors that interact with bone and are involved with calcium homeostasis (for reviews see DeLuca, 1988; Reichel et al., 1989). Specific, high affinity receptors for 1,25-(OH),-D3 have been detected in bone (Kream et al., 1977; Chen et a [ . , 1979), being localized on the osteoprogenitor cells and the osteoblast and osteocyte (Narbaitz et al., 1983; Boivin et al., 1987). The number of receptors in bone decrease with age (Horst et al., 1990). This decrease correlates with a decrease in the response of the osteoblast to 1,25-(OH),-D, (Chen et al., 1986) and is therefore perhaps partly responsible for the age-related changes observed in bone. Although the major site for the synthesis of 1,25-(OH),-D, is in the kidney, human bone cells have been shown to contain both 1- and 24hydroxylase activity (Howard et al., 1981), and evidence exists for the production of 1,25-(OH),-D, in bone cells (Lohnes and Jones, 1987; Sempere er al., 1989). 1,25-(OH),-D, is thought to play an important role in the calcification process by modulating the levels of a number of the substrates and regulators of mineralization. Thus, in osteoblastlike cell lines, 1,25-(OH),-D, has been reported to increase phosphatidylserine levels (Matsumoto et al., 1985; Haining et al., 1988), a constituent of the calcium-phospholipid-phosphate complex formed during the crystal formation process, and to reduce the synthesis and increase the degradation of proteoglycans (Takeuchi et al., 1989), thereby removing a potential inhibitor of mineralization. The synthesis of another component of the calcification process, type I collagen, has been reported to be both stimulated and inhibited by vitamin D. In certain systems employing human bone cells

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(Beresford et al., 1986), or a human osteosarcoma cell line (Franceschi et al., 1988), 1,25-(OH)?-D,enhanced type I collagen synthesis. In other systems that utilized bone cells from rat sources, 1,25-(OH),-D, inhibited type I collagen production (Rowe and Kream, 1982) through the inhibition of the transcription of the type I collagen gene (Harrison et a[., 1989). This regulation was found to be bone-specific, occurring in bone cells but not in fibroblasts (Lichtler et al., 1989). Whether this variation is due to a difference in species or to other factors remains to be investigated. The osteocalcin gene also contains a vitamin Dresponsive promoter region (Lian et al., 1989; Demay et al., 1989) that is controlled by the vitamin D receptor (McDonnell et al., 1989). Binding of the receptor to this promoter causes an increase in the transcription of the gene (Yoon et af., 1988) leading to an increase in the synthesis of osteocalcin (Price and Baukol, 1980; Pan and Price, 1984; Lian et al., 1985). Matrix Gla protein synthesis is likewise stimulated by 1,25-(OH),-D, (Fraser ef al., 1988). It has also been postulated that some of the effects of vitamin D on mineralization may be indirect through an increase in the absorption of calcium and phosphorus from the intestine (Underwood and DeLuca. 1984). Yet another mechanism for the involvement of vitamin D in calcification of bone would be through an increase in the number or activity of differentiated osteoblast cells capable of producing the extracellular matrix required for the formation of crystals. While 1,25-(OH),-D, has been shown to inhibit bone cell proliferation, it increases alkaline phosphatase activity (Majeska and Rodan, 1982; Beresford et al., 1986), a marker for the differentiated osteoblast. In v i m , 1,25-(OH),-D, prevents the dedifferentiation of fetal rat calvarial cells, maintaining the expression of alkaline phosphatase (Fritsch et al., 1985). In vivo, in dogs, the vitamin reportedly increases the activity but not the number of osteoblast cells in bone (Malluche et al.. 1986). In contrast, dihydrotachysterol, an analog of vitamin D, supposedly acts on bone through the activation of an osteoprogenitor cell population with a subsequent increase in the number of osteoblasts (Tabuchi et al., 1989). In chondrocytes, 1,25-(OH),-D, has an effect on the matrix vesicle, causing a greater increase in alkaline phosphatase activity in the vesicle compared to the plasma membrane (Schwartz et al., 1988a; Boyan et al., 1989). Matrix vesicles from growth cartilage chondrocytes display a stimulation of not only alkaline phosphatase activity but also 5’-nucleotidase, phospholipase A2, and sodium/potassium ATPase activity upon treatment with 1,25(OH),-D, (Boyan et al., 1988; Schwartz ef al., 1988b). Other metabolites of vitamin D, in particular 24,25-(OH),-D,, have been suggested to have an effect on bone formation and mineralization (Omoy el al., 1978; Tam et al., 1986). This idea has, however, been questioned by data indicating that 24,24-difluoro-25-(0H),-D3, an analog that could not be metabolized to the 24,25-dihydroxy compound, was still able to enhance mineralization (Tanaka et al., 1979). In contrast to 1,25-(OH),-D,, the 24.25-dihydroxy


225

metabolite has no effect on phospholipid metabolism (Haining et al., 1988), on stimulation of alkaline phosphatase activity in human bone cells (Beresford er al., 1986), or on osteocalcin synthesis (Lian et al., 1985). In the matrix vesicle, the response to 24,25-(OH),-D, depends on the stage of chondrocyte differentiation. While it does not have much effect on growth zone cartilage matrix vesicles, it is able to stimulate alkaline phosphatase activity and inhibit phospholipase A, activity in the resting zone matrix vesicle (Schwartz et al., 1988a,b; Boyan ef al., 1988). A second natural metabolite of vitamin D, 1,25-(OH),-D326,23-lactone also appears to have unique properties in bone. It is able to inhibit 1,25-(OH),-D3-induced bone resorption while it has no effect on PTH-induced resorption (Kiyoki et al., 1985). This response is thought to occur through an inhibition of the induction of osteoclastlike cells by 1,25-(OH),-D3 (Ishizuka et a[., 1988a). The lactone metabolite is also able to increase alkaline phosphatase activity and collagen synthesis in osteoblastlike cells (Kiyoki et al., 1985; Ishizuka et al., 1988b), and to stimulate bone formation as evidenced by an increase in matrix production and mineral uptake in vivo during ectopic bone formation (Shima et al., 1990). Vitamin D interacts with a number of other factors to modulate its own response or the response of these factors. For example, 1,25-(OH),-D, blocks transcription of the PTH gene (J. Russell et al., 1986), thereby decreasing levels of pre-proPTH mRNA both in v i m (Silver et al., 1985) and in vivo (NavehMany et al., 1989). In addition, it blocks the synthesis and secretion of PTH from the parathyroid cell in culture (Cantley et al., 1985; Chan et al., 1986) as well as from the parathyroid gland in vivo (Delmez et al., (1989). Vitamin D metabolism has been reported to be regulated by the gonadal steroids. At low levels of estrogen, 25-hydroxyvitamin D is metabolized predominantly to 24,25(OH),-D, in the premenopausal woman, but at higher estrogen concentrations, the formation of 1,25-(OH),-D, is favored (Buchanan er al., 1986a). In postmenopausal women, estrogen increases the levels of circulating biologicallyactive free 1,25-(OH),-D, (Cheema et al., 1989). 1,25-(OH),-D3 also has been reported to selectively affect the biological response of skeletal tissues to the gonadal steroids (Somjen er al., 1989a). Finally, estrogen blocks the 1,25-(OH),D,-stimulated increase in the secretion of the IGFs from the rat osteosarcoma cell line, UMR-106 (Gray er al., 1989a). Receptors for 1,25-(OH),-D3 are regulated by a number of factors, including retinoic acid and glucocorticoids. In two different rat osteosarcoma cell lines, the receptor number is increased by retinoic acid treatment while in normal rat bone derived cells it is decreased (Petkovich et a1.,1984; Lee et al., 1988). This difference in response to retinoic acid is also observed when mouse is compared to rat. 1,25-(OH),-D, receptor number increases after retinoic acid treatment in mouse osteoblasts but decreases in rat osteoblasts (Chen and Feldman, 1985). This is in contrast to glucocorticoids which decrease the number of receptors in the mouse osteoblast and increase it

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in the rat osteoblast (Chen er al., 1983). These changes in receptor levels parallel the observed response to 1,25-(OH),-D, in induction of the 25-hydroxyvitamin D-24-hydroxylase (Chen and Feldman, 1985) and in the modulation of procollagen mRNA levels (Kim and Chen, 1989). In addition to changes in its own receptors, 1,25-(OH),-D, can increase the number of receptors for EGF on osteoblasts (Petkovich et al., 1987). It also increases the secretion of TGFP-like activity from osteoblasts (Petkovich et al., 1987) and enhances the activity of IGF in stimulating alkaline phosphatase activity and collagen synthesis (Kurose et al., 1989). This interaction with various growth factors may help to explain some of the effects of vitamin D on bone growth and differentiation. In addition to its effects on bone formation and mineralization, vitamin D also has both short- and long-term effects on the resorption process. In the short term, l.25-(OH),-D3 has been shown to increase the release of calcium from fetal rat long bone in culture (Raisz et al., 1972). This presumably occurs through the stimulation of osteoblasts to release a factor that, in turn, stimulates the osteoclast (McSheehy and Chambers, 1987; Key et af., 1988). Its long-term effects on resorption appear to be through an increase in the number of osteoclasts (Holtrop er al., 198 I ; Roodman et al., 1985). 4 . Vitamin A (Retinoids)

The retinoids also appear to be involved in regulation of bone formation and resorption. In general, retinoid excess causes a decrease in osteoblast activity resulting in a decrease in formation, while a deficiency results in an increase in the number of osteoblasts and the production of new bone. Retinoids can act directly on bone-forming cells to alter extracellular matrix synthesis. Retinol specifically inhibits collagen synthesis in embryonic chick and neonatal mouse calvaria (Dickson and Walls, 1985) and in a rat osteosarcoma (ROS) cell line (Nishimoto et al., 1987). In ROS 17/2.8 cells, retinoic acid inhibits both alkaline phosphatase production and PTH-stimulated adenyl cyclase without an effect on proliferation (Imai et al., 1988). In addition, retinoid toxicity may result in increased bone resorption (Fell and Mellanby, 1952; Hough et al., 1988). In cartilage, chondroitin sulfate proteoglycan synthesis is inhibited by retinol (Solursh and Meier, 1973). Likewise, retinoic acid stimulates the loss of proteoglycans from adult bovine articular cartilage with the larger proteoglycan being lost at a greater rate than the small proteoglycan (Campbell and Handley, 1987). Finally, retinoic acid rapidly reduces cartilage matrix synthesis in chick sternal chondrocytes by selectively changing the normal pattern of gene expression from synthesis of chondrocyte specific proteoglycan and collagen II to the production of fibronectin and collagen I11 (Horton et al., 1987). The retinoids have been implicated in the normal development of mammalian bone. While specific intracellular cytosolic binding proteins for retinol or retinoic acid could not be detected in UMR-106 osteoblastlike cells in culture


227

(Oreffo et al., 1985), retinoic acid receptor mRNA is expressed in the calcifying fronts of developing mouse finger bones (Noji et al., 1989). In embryonic and fetal rat calvaria bone cells, retinoic acid causes increases in both FTH-stimulated adenyl cyclase and alkaline phosphatase activity and in type I procollagen mRNA, all considered to be markers of a more differentiated state (Ng et al., 1988; Heath et al., 1989). As indicated in the discussion on vitamin D, the retinoids are able to modulate 1,25-(OH),-D,- receptor levels. Retinol and retinoic acid have also been reported to stimulate the activity of 25-hydroxyvitamin D 1-hydroxylase in primary cultures of chick kidney cells (Trechsel and Fleisch, 1981) and to enhance the activity of 1,25-(OH),-D, in the stimulation of osteocalcin in ROS 17/23 cells (Nishimoto et al., 1987). In addition to its effects on vitamin D metabolism and response, retinoic acid is able to control growth hormone production in pituitary cells in vitro, acting synergistically with thyroid hormone and glucocorticoids, (Bedo et al., 1989), and selectively increasing mRNA levels for the hormone (Morita et al., 1989). Retinoids are also able to stimulate the release of PTH from the parathyroid gland (Chertow et al., 1977), and testosterone production in rat Leydig cells (Chaudhary et al., 1989). 5 . Steroid Hormones

The steroid hormones, estrogen and testosterone, also appear to have significant effects on bone turnover and mineralization. The effects of estrogen are most clearly demonstrated in those situations where hormone levels are decreased by ovariectomy in the rat and by bilateral oophrectomy or menopause in the human. In the rat, removal of the ovaries results in enhanced bone turnover with increases in both the rates of bone apposition and removal (Wronski et al., 1985, 1988; Turner et al., 1987a). Estrogen treatment suppresses this turnover with declines in formation and resorption (Cruess and Hong, 1979; Turner et al., 1987a; Wronski et al., 1988). In the human, accelerated bone loss resulting in low bone mass and osteoporosis is found in postmenopausal women (Riggs et al., 1982; Nilas and Christiansen, 1987) as well as in the patient who has undergone bilateral oophrectomy (Aitken et al., 1973; Lindsay et al., 1980). Again, estrogen replacement therapy helps to prevent this loss (Meema et al., 1975; Lindsay et al., 1976, 1980). For a long time, the effects of estrogen on bone were considered to be indirect effects. This concept was supported by the fact that estrogen receptors, considered to be essential for the action of estrogen, could not be detected in bone. More recently, however, osteoblasts have been shown to contain estrogen receptors and estrogen-receptor mRNA (Komm et al., 1988; Eriksen et al., 1988; Kaplan et a[., 1988). In support of this data, autoradiographic evidence indicates receptors for estrogen in the periosteum, perichondrium, chondrocytes, and chondroblasts of adult baboon (Aufdemorte et al., 1988). Receptors have

228

DANIEL C. WlLLlAMS AND CHARLES A. FROLIK

likewise been observed in articular chondrocytes from both rabbit (Rosner ef al., 1982) and dog (Young and Stack, 1982), but not in epiphyseal chondrocytes from these two species (Kan et al., 1984). Finally, nuclear binding of estradiol has been detected in several different normal human osteoblastlike cell strains, suggesting that the action of estrogen on bone can occur through the classical receptor-mediated mechanism observed in other estrogen-responsive tissues (Colvard et al., 1989a). Additional evidence indicating the direct involvement of estrogen in bone formation comes from in vitro experiments. In neonate rat calvaria cells, estrogen increases cell proliferation and the steady state levels of type I procollagen mRNA while it decreases PTH-stimulated adenyl cyclase activity (Emst et al., 1988, 1989). In UMR- 106 osteosarcoma cells, estrogen decreases proliferation and increases alkaline phosphatase activity (Gray er al., 1987). Avian bone responds to estrogen treatment by inducing synthesis of proteoglycan (Hunter and Schraer, 1983) and of a calcium-binding glycoprotein with alkaline phosphatase activity (Stagni er al., 1980). in the diaphyseal bone of weanling rats, females show an increase in creatine kinase activity after estrogen treatment, while males do not respond to estrogen (although they will respond to androgens) (Somjen rt al., 1989b). Also, differentiation and mineralization are increased by estrogen and progesterone in the matrix-induced endochondral bone formation assay (Bumett and Reddi, 1983). Finally, in an in vivo experiment, local infusion of estradiol into rat bone inhibited bone resorption and stimulated bone formation with no effect on uterine or body weight (TakanoYamamoto and Rodan, 1990). The action of estrogen in cartilage appears to be dependent on the system being studied. While it has been suggested that proliferation and proteoglycan synthesis in bovine articular chondrocytes are not under the control of physiological levels of estrogen (Mackintosh and Mason, 1988), in rabbit cartilage, estrogen has been reported to both suppress proteoglycan synthesis and decrease proteoglycan turnover in vivo (Rosner et al., 1979), and to increase proteoglycan synthesis in vitro (Corvol et al., 1987). Although some of this difference may simply be due to an in vivo versus an in virro response, a portion of it could also be due to the age and sex of the animal from which the cells were derived. In fetal rabbit chondrocytes, estrogen will stimulate proteoglycan synthesis, while in chondrocytes from 5-30 day old animals no response to estrogen was detected. In older animals, the stimulation returned but it was less in the male compared to the female (Corvol er al., 1987). Finally, in the epiphyseal cartilage from weanling rats, estrogen stimulated creatine kinase activity and DNA synthesis (Somjen et al., 1989b). Originally, the effects of estrogen on bone resorption were thought to be indirect. Estrogen was not able to inhibit bone resorption either in fetal rat long bones (Caputo et al., 1976) or in neonatal mouse vertebral bones in culture


229

(Stewart and Stern, 1987). Instead, the effects of estrogen were thought to be mediated through an interaction with PTH and/or calcitonin. To get the observed decrease in resorption with estrogen administration requires either a decrease in PTH or an increase in calcitonin levels, or a change in tissue response to these factors. In support of this concept, it has been reported that there is an increased sensitivity to PTH in bone from ovariectomized rats (Orimo et al., 1972). In contrast, however, parathyroidectomized and ovariectomized rats have been reported to respond to estrogens with a conservation of bone to the same extent as sham-operated controls (Goulding and Gold, 1989). Similarly, the involvement of PTH in the in vivo response to estrogen in humans is not clear. While estrogen has been reported to increase PTH secretion from parathyroid tissue from bovine (Greenberg et al., 1987) or human (Duarte et al., 1988) sources, it does not change plasma levels of the hormone in postmenopausal women (Selby et al., 1985). Similarly, in normal premenopausal women, PTH levels remain constant during the menstrual cycle in spite of sharply fluctuating estrogen levels, although increased estrone levels might be associated with reduced circulating PTH (Buchanan et al., 1986b). In fact, early effects of estrogen on bone resorption in postmenopausal women have been reported to be independent of PTH, while later effects are only variably associated with increased PTH (Stock et al., 1989a). More recently, direct effects of estrogen on bone resorption have been observed. In neonatal mouse calvaria, estrogen will inhibit PTH-stimulated PGE, release and PTH-stimulated bone resorption (Atkins et al., 1972; Pilbeam et al., 1989), while in a human, but not a rat osteosarcoma cell line, it will inhibit PTH-stimulated CAMPproduction (Fukayama and Tashjian, 1989a). The mediation of the effects of estrogen on bone resorption through calcitonin have not been as thoroughly studied. In vitro, estrogen will stimulate the secretion of calcitonin from rat thyroid C cells (Greenberg et al., 1986). Also, some in vivo studies indicate that estrogen treatment of postmenopausal women will give rise to a sharp increase in plasma calcitonin (Whitehead er al., 1981; Stevenson et al., 1983; Civitelli et al., 1988). However, other in vivo studies have not demonstrated this effect (Selby et al., 1985; Body et al., 1989). Again, thyroidectomized rats show the same response to estrogen as sham-operated controls, implying the effects of the steroid hormones are independent of calcitonin (Goulding and Gold, 1989). Therefore, the involvement of calcitonin in mediating the effects of estrogens on bone resorption is still open to debate. Besides PTH and calcitonin, estrogens interact with a number of other factors involved in bone metabolism and calcification. The effects of estrogen on vitamin D metabolism and the possible control by 1,25-(OH),-D, of the estrogen response in rat long bone have already been discussed. Estrogens have also been shown to interact with several growth factors in bone and part of the skeletal effects of estrogen may be mediated via regulation of these factors. In rat calvaria cells, estrogen will increase the steady state level of IGF-I mRNA (Emst et

230


al., 1989) and stimulate the release of IGF-binding proteins (Schmid et al., 1989a). while UMR-106 osteoblastlike cells respond to estrogen with an increase in the secretion of IGF-I and I1 (Gray et al., 1989a). In bone, TGFP may also be under the control of estrogen. In rat and human osteosarcoma cells, the level of TGFP mRNA is increased by estrogen (Komm et al., 1988), which is translated into an increased production of the growth factor itself (Gray et al., 1989b). There is conflicting data on the role of IL-1 in mediating the action of estrogen on bone resorption. In one study, treatment of postmenopausal women with estrogen did not affect the spontaneous release of IL-I from peripheral monocytes in vitro (Stock et al., 1989b). in a second study, the levels of IL-1 in osteoporotic and nonosteoporotic women correlated inversely with vertebral mineral density. Treatment with estrogen/progesterone caused a significant decrease in interleukin activity (Pacifici et al., 1989). Additional studies will have to be done before a conclusion can be reached concerning the role of cytokines in the action of estrogen on bone. Finally, estradiol has been reported to amplify the neuroendocrine regulation of pulsatile growth hormone release with decreased circulating estradiol concentrations being correlated with decreased growth hormone levels (Ho et al., 1987). Indeed, the growth hormone response to levodopa is depressed in postmenopausal osteoporosis (Rico et ul.,l979). Estrogen administration to the postmenopausal woman results in enhanced growth hormone secretion and increased serum concentrations of growth hormone (Duurama et al., 1984; Dawson-Hughes et al., 1986). In addition to estrogens, the androgens, in particular testosterone and dihydrotestosterone, are involved in bone growth and mineralization. Their role best becomes evident in those situations where a deficiency or excess of the hormone exists. In rats, castration (Wink and Felts, 1980; Verhas et al., 1986) or administration of an antiandrogen (Feldmann et al., 1989) leads to reduced bone mass, as does hypogonadism in the human male (Finkelstein et al., 1989). In the female, androgen excess can cause increased trabecular bone density (Buchanan et al., 1988a) and can maintain normal bone mass even in the presence of undetectable estrogen levels (Dixon et al., 1989). Adrenal androgens have been suggested to have a role in maintaining bone mass in normal postmenopausal women (Devogelaer et al., 1987). In the young, normal woman, the androgens and estrogens appear to function as independent and additive determinants of peak trabecular bone density (Buchanan ef al., 1988b). Some studies have demonstrated a positive correlation between serum testosterone or dehydroepiandrosterone sulfate levels and bone density in aged men (Foresta et al., 1984) and women (Brody et al., 1981; Wild et al., 1987; Deutsch et ul.. 1987; Steinberg et al., 1989), while other studies have not been able to make this association (Meier et a[., 1987). Androgens, like estrogens, interact with their target tissues through a receptormediated mechanism. Indeed, receptors for testosterone have been detected in


23 1

human osteoblastlike cells, with cells from male and female sources containing similar receptor concentrations (Colvard et al., I989a,b). Human fetal epiphyseal chondrocytes have also been shown to contain high affinity binding sites for dihydrotestosterone (Carrascosa et al., 1990). In many tissues that respond to androgens, dihydrotestosterone is considered to be the compound that actually generates the biological response. Whether this androgen is the active metabolite in bone is still an unanswered question. Testosterone can be converted to dihydrotestosterone in bone (Vittek et al., 1974; Schweikert et al., 1980) and in cartilage (Audi et al., 1984), with no differences being observed in osteoporotic bone versus normal bone, or in male verses female. While the possibility exists that the androgens can act in bone by being first converted to estrogen (Frisch et al., 1980), the presence of receptors in bone seem to indicate that at least some of the actions of the androgens are through a direct interaction with the bone cells. Androgen treatment appears to result in an increase in bone formation. Administration of testosterone will produce an increase in skeletal growth both in the experimental animal (Jansson et al., 1983) and in man (Rosenfeld, 1986). In castrated mice, testosterone restores total bone protein in the femoral diaphyses to normal (Broulik et al., 1976). Similarly, direct injection of testosterone into the tibia1 epiphyseal growth plate of castrated rats causes a significant increase in the growth plate width when compared to control (Ren et al., 1989). In vitro, dihydrotestosterone enhances the proliferation of mouse and human osteoblastlike cells and increases the number of cells that produce alkaline phosphatase (Kasperk et al., 1989), although no effect on collagen or DNA synthesis was noted in fetal rat calvaria cultures (Canalis and Raisz, 1978). Using the bone-matrix-induced endochondral bone formation model, androgens markedly elevate the levels of alkaline phosphatase and calcium in the implant and cause an induction of mineralization (Kapur and Reddi, 1989). As is observed for estrogen, fetal rabbit epiphyseal articular chondrocytes respond to testosterone with an increase in proteoglycan synthesis, while chondrocytes from 5-30 day old animals lose their response, which is regained in older animals (Corvol et al., 1987). Testosterone deficiency in males is also associated with increased bone resorption (Stepan et al., 1989). Testosterone and dihydrotestosterone are believed to have no direct effect on resorption in fetal rat long bone in culture (Caputo et al., 1976). Therefore, its effects on inhibiting bone resorption appear to be through PTH and calcitonin. Evidence that supports this concept indicates that testosterone will inhibit the PTH-induced release of calcium from neonatal mouse calvaria (Atkins and Peacock, 1975) and the PTH-stimulated increase in CAMPin human osteosarcoma cells (Fukayama and Tashjian, 1989b). Although testosterone has no effect on the release of calcitonin from rat thyroid C cells in culture (Greenberg et al., 1986), decreased calcitonin levels were found in hypogonadal, osteoporotic young men and testosterone therapy resulted in a

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return to normal values (Foresta et al., 1987). It has also been postulated that an enhanced calcitonin response observed in normal men compared to women may be partially due to the higher testosterone levels found in men (GarciaAmeijeiras et al., 1987). Androgens may also mediate some of their effects on bone through increases in serum levels of IGF-I (Parker et al., 1984; Jasper, 1985; Rosenfeld and Furlanetto, 1985). Part of this increase may be through increased secretion of growth hormone (Parker et al., 1984; Craft and Underwood, 1984; Copeland et al., 1984; Mauras er al., 1987, 1989) which in turn stimulates IGF-I production (Green et a!., 1985; Isaksson er a/., 1985; Nilsson et at., 1986a). However, testosterone can also stimulate growth in the absence of growth hormone (Young et al., 1989), perhaps by directly regulating levels of IGF-I (Wilson, 1986). 6 . Growth Hormone

Before leaving the endocrine factors that regulate skeletal growth and mineralization, the actions of growth hormone on bone should be considered. It has been known for some time that growth hormone is involved in skeletal growth (Raben, 1958). Since many of the effects of growth hormone may be mediated through the IGFs, the interaction of these two factors will be deferred to the discussion of the IGFs. There is evidence, however, that growth hormone may also have direct effects on bone. For example, when continuous intravenous infusion of growth hormone to mutant growth hormone-deficient rats is compared to the infusion of IGF-I, greater bone growth is observed for the growth hormonetreated rats, implying that the two factors may differ in their mechanism of actions (Skottner er al., 1989). While it has been reported that growth hormone may act on the skeleton without changes occurring in the circulating level of IGF-I (Young er al., 1989), this may simply be due to the hormone causing local stimulation of IGF-I, which may not be observable systemically (Green ef al., 1985; Isaksson er al., 1985; Flint and Gardner, 1989). The direct interaction of growth hormone with bone is supported by the detection of specific binding sites for the protein on cultured rabbit chondrocytes (Eden et al.. 1983). Similarly, in young rabbits (20-50 days old), growth hormone receptors were localized on reserve and proliferative chondrocytes in the tibia1 growth plate, while in older animals, where the growth plate was closed. no receptors could be detected (Bernard et al., 1988). Also, treatment of mouse condylar cartilage cells in vitro with growth hormone results in stimulation of cell proliferation, differentiation, and extracellular mineralization (Maor er al., 1989). With fetal mouse calvaria osteoblastlike cells, growth hormone appears to preferentially act on the more differentiated cell resulting in an increase i n the number of osteoblasts (Slootweg er al., 1988). In addition to its interaction with the IGFs, growth hormone secretion may be regulated by several other factors that interact with bone, including retinoids,


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androgens, and estrogens. Since these factors have already been discussed, the reader is referred to those sections in this article that cover these interactions. B. PARACRINE/AUTOCRINEFACTORS 1. lnsulinlike Growth Factors (IGFs)

In addition to the hormonal factors that are produced elsewhere in the body and camed to bone via the circulatory system, there are numerous factors that are now known to be produced in the local bone environment that may play major roles in regulation of bone cell growth and differentiation and, ultimately, the calcification process. Included in this category are the IGFs. Because the IGFs occur in the serum at high concentrations, it was originally thought that the bone was responding to these circulating factors (Salmon and Daughaday, 1957). However, available evidence now seems to indicate that it is probably the locally produced IGFs that are important in stimulating bone growth and mineralization (Skottner et al., 1987). For example, a chronically elevated serum concentration of IGF-I in transgenic mice resulted in an increase in body weight without an apparent effect on skeletal growth (Mathews et a[., 1988). Similarly, infusion of IGF-I into diabetic rats caused no change in bone growth (Carlsson et al., 1989), while infusion into mutant growth hormone-deficient dwarf rats caused smaller changes in bone growth than that observed for growth hormone by itself (Skottner et al., 1989). Studies with hypophysectomized rats are not as clear. Several studies indicate that infusion of IGF-I or IGF-I1 into these animals results in increases in tibial epiphyseal plate width and bone growth similar to that observed with growth hormone (Schoenle et al., 1982; Guler et al., 1988; Shaar et al., 1989) while other studies demonstrate that although infused IGF-I and IGF-I1 are able to increase the width of the tibial epiphysis, growth hormone showed greater changes, again implying that endogenously produced IGFs are more effective than exogenously supplied material (Schoenle et al., 1985). The importance of the IGFs in bone is supported by the occurrence of these factors and their receptors and binding proteins in the bone environment. Skeletal tissues are a rich source of both IGF-I and IGF-I1 (Frolik et al., 1988; Mohan et al., 1988). That these factors are being produced in bone rather than being picked up from the serum is indicated by experiments demonstrating the synthesis of IGF-I in cultured fetal rat calvaria or tibia (Stracke et al., 1984; Canalis et al., 1988a), in embryonic chick pelvic cartilage (Burch et al., 1986) and in osteosarcoma cells (Gray et al., 1989a). Similarly, IGF-I1 is a product of human osteoblastlike cells in culture (Wergedal et al., 1986; Gray et al., 1989a). Using labeled growth factor or immunohistochemical staining techniques, IGF-I has been localized to chondrocytes in the proliferative and hypertrophic zones of porcine (Simon and Cooke, 1988), rat (Nilsson et al., 1986a; Hansson et al.,

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1988), and bovine (Trippel et al., 1986) epiphyseal growth plate cartilage. Activity, however, was not observed in the reserve zone. During rat embryogenesis, the IGF-I1 gene is expressed in chondrocytes and declines substantially prior to ossification (Stylianopoulou et al., 1988). Even though IGF is produced in bone and is present at high concentrations, in order for it to have an effect on bone formation, the cells involved in the actual process need to have the proper receptors. Indeed, specific, high affinity receptors for both IGF-I and IGF-I1 have been detected on osteoblastlike cells from the newborn mouse and embryonic chick calvaria (Mohan et al.. 1989a) and from fetal rat calvaria (Bennett et al., 1984; Cantrell et al., 1989; Centrella et al., 1990). Bovine (Watanabe et al., 1985) and rabbit (Jansen et al., 1989) articular chondrocytes and rat costal chondrocytes (Schalch et al., 1986; Sesseins et al., 1987) also contain IGF-I and I1 receptors. Interestingly, there are 2-6 times more IGF-I receptors on chondrocytes from the bovine growth plate than on articular chondrocytes (Trippel et al., 1983) which is not unanticipated if IGF-I is involved in skeletal growth. In agreement with the immunohistochemical staining experiments, chondrocytes from the growth zone had a higher concentration of IGF-I receptors than those from the resting or hypertrophic zones (Makower et al., 1989a). Insulinlike growth factor-binding proteins, that apparently function to modulate the activity and availability of the IGFs, have been described in a number of tissues (for review see Ooi and Herington, 1988) with bone being no exception. Conditioned medium from rat osteoblastlike cells (Schmid et al., 1989a,b) and from human osteosarcoma cells (Mohan er al.. 1989b). as well as from human bone extracts (Frolik and Black, 1989), contains IGF-binding protein activity that appears to be different from activity found in other tissues. IGF-binding proteins are also secreted by both epiphyseal and articular chondrocytes from rabbit (Froger-Gaillard er al., 1989). chick (Burch et al., 1990), and human (Hill et al., 1989) sources. While the exact function of these binding proteins is still unknown, there is one report that the human amniotic fluid 25-kDa binding protein will inhibit IGF-I stimulated growth of chick embryo pelvic cartilage (Burch et al., 1990). implying that the binding proteins may serve to down regulate the activity of the IGFs. The IGFs have been postulated to have a role in the calcification process directly through increasing bone matrix production by the differentiated osteoblast, and indirectly by increasing the osteoprogenitor cell population and enhancing their differentiation into mature osteoblasts (Guenther et al., 1982; Schmid et al., 1984; Hock et al., 1988b). Thus, IGF-I will stimulate DNA synthesis in rat calvaria (Canalis, 1980; Schmid et al., 1984) and in osteoblastlike cells (Kurose et al., 1989). Similarly, IGF-I1 stimulates DNA synthesis in both rat and chick calvaria cells and in human bone cells (Canalis and Raisz, 1979a; Schmid er al., 1983; Farley e t a / . , 1986). In the osteoblast, the IGFs will stimu-


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late collagen synthesis (Canalis et al., 1977; Canalis, 1980; Linkhart et a/., 1986; Hock el a/., 1988b) by increasing mRNA levels for type I collagen and decreasing collagen degradation (McCarthy et a / . , 1989b; Schmid et al., 1 9 8 9 ~ ) . Insulinlike growth factor-I1 has also been shown to stimulate protein phosphorylation in the chick calvaria (Lau et al., 1988a). As discussed in an earlier section, both of these effects can play a major role in the modulation of the calcification process. In cartilage, besides having a possible role in the in v i m proliferation of the chondroblast (Makower et al., 1989b), the IGFs appear to play a major role in the maintenance of the fully differentiated chondrocyte, especially in regard to the synthesis of proteoglycans (Stevens et al., 1981; Guenther et al., 1982; Hiraki et a/., 1985; McQuillan e t a / . , 1986). As with the collagens, under certain conditions, the IGFs not only increase the synthesis of the proteoglycans, but also decrease their degradation (Tyler, 1989). Under other conditions, they help to maintain an equilibrium between proteoglycan biosynthesis and catabolism (Luyten et al., 1988). The IGF stimulation of proteoglycan synthesis occurs principally in the proliferative zone chondrocyte (Makower et al., 1988; Trippel et al., 1989) resulting in an increase in the size of the proteoglycan monomer (Makower et al., 1988). As already mentioned, the action of growth hormone on bone appears to be intimately intertwined with the IGFs. Treatment with growth hormone in vivo causes increased levels of IGF-I mRNA in rat rib growth plates (Isgaard et a/., 1988a,b), and in rat calvaria (McCarthy et al., 1989a), and of IGF-I protein (Stracke et al., 1984; Orlowski and Chernausek, 1988). The action of growth hormone in stimulation of both long bone growth in the hypophysectomized rat (Russell and Spencer, 1985; Isgaard et a/., 1986) and of isolated osteoblastlike cells in culture is blocked by IGF-I antibodies (Schlechter el al., 1986; Ernst and Froesch, 1988). In vitro, growth hormone stimulates the effects of IGF-I on extracellular matrix synthesis (Smith et al., 1989) and the release of IGF-binding protein (Schmid et al., 1989a,b). A current hypothesis for the mode of action of growth hormone and IGF in cartilage is that growth hormone causes differentiation of a precursor cell (the prechondrocyte)to a mature cell that is more responsive to the IGFs, while the IGFs interact with the cell at a later stage in development causing an increase in number or activity of the differentiated cell (Zezulak and Green, 1982; Nilsson et al., 1986a; Lindahl et a/., 1987). Finally, in perhaps a feedback type control loop, IGF-I has been reported to suppress the expression of the growth hormone gene and the secretion of the protein from the rat pituitary cell (Yamashita et a/., 1987; Namba et al., 1989). Growth hormone is not the only biological factor that controls IGF action. Glucocorticoids have been shown to inhibit the growth hormone induction of IGF-I mRNA levels in bone. This effect has been postulated to be a mechanism of action that can explain the observed growth retardation caused by glucocorti-

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coids (Luo and Murphy, 1989; McCarthy et al., 1990). By themselves, glucocorticoids have been reported to increase the concentration of IGF-I receptors in rat osteoblasttike cells (Bennett et al., 1984) and to inhibit the synthesis of IGF-I (McCarthy et a1.,1990). On the other hand, while glucocorticoids by themselves inhibit collagen synthesis in bone cell populations, they are able to enhance the responsiveness of the cell to the anabolic effects of IGF-I. Therefore, calvaria treated with cortisol and IGF-I showed a marked increase in collagen synthesis compared to cells treated with cortisol alone (Kream et al., 1990). Other factors that increase the concentration of IGF include estrogen (Gray e f al., 1989a; Schmid et al.. 1989a; Emst et al., 1989), 1,25-(OH),-D, (Gray et al., 1989a), PTH (McCarthy et al., 1989a; Linkhart and Mohan, 1989). and P,-microglobulin (Centrella et al., 1989b). Insulinlike growth factor-] has also been postulated to be involved in regulating serum levels of 1,25-(OH),-D, (Gray, 1987; Halloran and Spencer, 1988). Finally, fibroblast growth factor (FGF) and EGF have been reported to act synergistically with IGF-I in the stimulation of DNA synthesis in cultured rabbit costal chondrocytes (Hiraki ef al., 1987). 2 . Transforming Gronith Factors (TGFs) Transforming growth factor P(TGFf.3) is a second growth factor that appears to be intimately involved in modulating the cells that participate in the calcification process. Transforming growth factor f3 exists as a family of proteins (Roberts and Spom, 1988), many of which are found in cells of the skeletal system. From bone, it was originally isolated as cartilage inducing factor A and B (Seyedin et al., 1985) which were later shown to be identical to TGFP-1 (Seyedin et ol., 1986) and TGFP-2 (Seyedin ef ai., 1987). Primary chick embryo chondrocytes have been shown to contain TGFP-3 and -4 (Jakowlew er al.,

1988a,b) while the bone morphogenetic proteins also share sequence identity with the TGFs (Wozney cr al., 1988). The TGFs are thought to be involved in embryonic formation of cartilage and bone, controlling both endochondral and intramembranous ossification (Heine et al., 1987; Pelton et al., 1989).

Employing a model where endochondral bone is generated by implantation of demineralized bone matrix into the muscle of rats, TGFP was localized to the chondrocytes of the calcifying cartilage and to the osteoblasts and mineralized bone matrix. The concentration of TGFP was highest when cartilage was being replaced by bone, again suggesting a role for TGFP in the ossification process (Carrington et al., 1988). That the TGFs are actually being produced in bone has been shown by a number of investigators (Centrella and Canalis, 1985, 1987; Robey et al., 1987; Guenther et al., 1988; Sandberg et al., 1988; Pelton et af., 1989) and specific, high affinity receptors for TGFP have been detected on osteoblastlike cells (Segarini et al., 1987; Robey er al., 1987). In addition to the conventional receptors, TGFP also binds to a high molecular weight proteoglycan component that is found on primary fibroblasts, osteoblasts and chondro-


237

blasts but not on epithelial, endothelial or lymphoid-derived cells (Segarini et al., 1989). The function of this binding component is unknown. It does not appear to play a role in any of the known responses to the TGFs. Finally, in other systems, the TGFs have been shown to be produced in a latent form and require activation prior to the generation of their response (Roberts and Sporn, 1988). Similarly, in bone matrix and conditioned medium from calvaria organ cultures, the TGFP is found associated with a high molecular weight glycoprotein that masks the TGFP activity (Jennings and Mohan, 1990; Pfeilschifter ef al., 1990). While the biological mechanism for the activation of this latent TGFP is not clear, in the skeleton, osteoclasts have been reported to be capable of performing this function (Oreffo et al., 1989). The effects of TGFP on bone cell function and mineralization seem to depend on the species being studied, the state of differentiation of the target cell, and the experimental conditions employed. In vivo, TGFP is able to increase periosteal woven bone formation in rats (Noda and Camilliere, 1989). In vitro, it is able to both stimulate (Centrella et al., 1986; Datta et al., 1989; Jennings et al., 1988; Hock et al., 1990) and inhibit osteoblast cell proliferation (Noda and Rodan, 1986, 1987; Elford et al., 1987; Pfeilschifter et al., 1987; Guenther et al., 1988). It is also able to regulate extracellular matrix production. TGFP stimulates expression of the type I collagen gene (Centrella et al., 1987a; Noda and Rodan, 1987), possibly acting through a nuclear factor binding site in the a2(I) collagen promoter (Rossi et al., 1988), as well as the osteonectin (Noda and Rodan, 1987) and osteopontin (Noda et al., 1988b) genes. The response of alkaline phosphatase to TGFP varies, with the activity being stimulated in ROS17/2.8 cells (Noda and Rodan, 1987; Pfeilschifter et al., 1987) and inhibited in MC3T3-El and primary rat osteoblastlike cells (Noda and Rodan, 1986; Elford et al., 1987; Rosen el al., 1988; Wrana et al., 1988; Ibbotson et al., 1989). Finally, the expression of the fibronectin and the osteocalcin genes are decreased by TGFP (Noda and Rodan, 1987; Noda, 1989). These effects on cell function are independent of the effects of TGFP on cell proliferation (Centrella et al., 1987a; Wrana et al., 1988; Ibbotson et al., 1989). In cultured fetal rat calvaria, using histomorphometry and autoradiography, TGFP was shown to have mitogenic effects on all cell zones of the pericranial periosteum with an increase in the number of osteoblasts and a decrease in the number of osteoclasts. The study concluded that the effects of TGFP on matrix protein synthesis was partly due to increased cell proliferation and partly to increases in bone cell differentiation and function (Hock et al., 1990). TGFP also changes the morphological appearance of the cell (Noda and Rodan, 1986; Pfeilschifter et al., 1987; Rosen et al., 1988). This modified behavior of the cell may be due to a change in binding specificity of the integrins that are expressed by the cell after TGFP treatment (Heino and Massague, 1989).

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While TGFP is able to act on the osteoblastlike cell, a significant amount of evidence exists to indicate that it also has a role in cartilage. TGFP mRNA has been shown to be expressed in cartilage during murine embryogenesis (Pelton et al., 1989) with the highest concentration occurring in the growth plate (Sandberg et al., 1988). Immunohistochemical staining techniques support the involvement of TGFP- 1 in cartilage, detecting the factor intracellularly in the chondrocyte and extracellularly in the cartilage matrix (Thompson et al., 1989). TGFP was initially isolated from bone based on its ability to cause differentiation of the neonatal rat muscle mesenchymal cell into chondrocytes and to stimulate production of cartilage-specific proteoglycans and type I1 collagen (Seyedin er d., 1985). In addition to the stimulation of proteoglycan synthesis in cartilage (O'Keefe er al., 1988a; Redini et al., 1988), TGFP inhibits both the degradation of newly synthesized proteoglycans (Morales and Roberts, 1988) and the IL-1stimulated release of proteoglycans (Andrews et al., 1989). Finally, TGFP is able to stimulate DNA synthesis in chick epiphyseal chondrocytes (O'Keefe et al., 1988b). However, TGFP also can inhibit the phenotypic expression of cultured chondroblasts, resulting in a decrease in the production of type I1 collagen and cartilage proteoglycan (Rosen ef a!., 1988) and an inhibition of in vitro cartilage-induced mineralization (Kato et al., 1988). In addition to its effects on bone and cartilage formation, TGFP also has effects on bone resorption. In neonatal mouse calvaria in culture, TGFP stimulates resorption, perhaps through a PGE,-related mechanism (Tashjian et al., 1985). Transforming growth factor-p is able to stimulate the synthesis and release of PGE, from MC3T3-El cells (Sumitani ef a!., 1989) and from fetal rat calvaria (Centrella ef a/., 1986). On the other hand, in cultured fetal rat long bone, TGFP causes a decrease in resorption (Pfeilschifterer af., 1988). This effect of TGFP may be due to inhibition of the proliferation of the osteoclast precursor (Chenu er a/., 1988). Transforming growth factor-P may also have effects on connective tissue degradation. It has been reported to inhibit the IL-I-stimulated production of neutral protease activity by rabbit articular chondrocytes (Chandrasekhar and Harvey, 1988) and to stimulate the synthesis of the collagenase inhibitor tissue inhibitor of metalloprotease (TIMP) and of PAI- I , a plasminogen activator inhibitor, while decreasing collagenase production by normal fetal rat calvaria bone cells thereby suppressing matrix degradation and removal (Overall er al., 1989). The role of TGFP in the regulation of bone growth and calcification may be modified by the action of other agents (Centrella et al., 1987b). Factors that will increase TGFP activity in bone include FGF (Noda and Vogel, 1989), 1,25(OH),-D, (Petkovich er al., 19871, IL-1 (Pfeilschifter and Mundy, 1987), and estrogen (Komm el al.. 1988; Gray et al., 1989b) while calcitonin will decrease TGFP activity (Pfeilschifter and Mundy, 1987). Parathyroid hormone has been reported to increase TGFP activity in the mouse or rat calvaria (Pfeilschifter and Mundy, 1987) but to decrease it in a rat osteosarcoma cell line (Noda and Vogel,


239

1989). These factors not only modify the levels of TGFP, but can also regulate its effects. For example, FTH will enhance the binding of TGFP to fetal rat calvaria osteoblastlike cells in culture and decrease the response of the cells to TGFP (Centrella et al., 1988). In contrast, in the same cells, PTH-related peptide has been reported to enhance the effects of TGFP on DNA and collagen synthesis (Centrella et al., 1989a). In rabbit articular chondrocytes grown in soft agar, TGFP has no effect on DNA synthesis unless grown in the presence of EGF (Skantze et al., 1985). While some factors are able to control the activity of TGFP, the converse is true for other factors whose actions in bone are modulated by TGFP. For example, TGFP potentiates the effects of FGF on bone cell proliferation (Hiraki et al., 1988; Globus et al., 1988; Iwamoto et al., 1989). Similarly, chondrocytes, grown in the presence of TGFP and FGF, will express the proteoglycans specific for a differentiated chondrocyte (Inoue et al., 1989). In rat long bone, resorption stimulated by IL-1 or 1,25-(OH),-D3 is inhibited by TGFP (Pfeilschifter et af., 1988). Other effects of TGFP on IL-1 activity have already been described. Interestingly, while short term treatment of MC3T3-E1 cells with TGFP causes an increase in the number of EGF receptors and decreases the rate of DNA synthesis, longer term treatment decreases the number of receptors with an increase in DNA synthesis (Uneno et al., 1989). It should be apparent, from this discussion, that the effects of TGFP on bone involve a complicated interaction with a wide range of other bone regulatory molecules and that the observed response depends on the differentiated state of the bone cell at the time the factor is in the local environment of that cell.

3. Bone tnductive Proteins Another group of factors, the bone inductive proteins, appear to be related to the TGFs in structure but not necessarily in function. They are postulated to be responsible for the differentiation of mesenchymal cells into osteoprogenitor cells, while other factors are involved in the proliferation of these cells (Urist et al., 1983). These proteins were originally discovered, and have been characterized, using the ectopic bone formation assay where extracts from demineralized bone induce new bone when implanted at an ectopic site (Urist, 1965). The sequence of events that occurs during this process are:.(l) the migration and attachment of mesenchymal cells into the implant and their proliferation; (2) by days 5-7, the appearance of chondroblasts and formation of a cartilaginous template: (3) at days 10-14, hypertrophy of the cartilage and vascularization of the extracellular matrix; and (4) removal of the cartilage and replacement with bone, complete with marrow, by day 21 (Reddi and Huggins, 1972; Firschein and Urist, 1972; Reddi, 1981). There are a number of proteins that appear to initiate some of, or all, of these events. Bone inductive proteins that have been purified and at least partially sequenced include the bone morphogenetic proteins (BMPs), the osteoinductive factor (OIF), and osteogenin.

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Bone morphogenetic protein, purified from bovine bone matrix, is an acidic protein of approximately 18.5 kDa with a pl of 4.9-5.1 (Urist et al., 1984; Bessho et al., 1989). Besides being found in bone matrix, it also occurs in dentine, enamel, and cementum (Kawai and Urist, 1989), as well as in different osteosarcomas (Hanamura et al., 1980; Bauer and Urist, 1981; Takaoka et al., 1989). Three different human BMPs have been cloned and complete amino acid sequence obtained (Wozney et al., 1988). Two of the proteins, BMP-2A and BMP-3. are members of the TGFP family, with 34 to 38% sequence homology to TGFP-1 and TGFP-2, while the third, BMP-1, is a unique protein. Initial reports indicated that these proteins, by themselves, will form cartilage when placed ectopically, but the cartilage will not be replaced by bone. However, more recent reports indicate that purified recombinant human BMP-2A is able to induce bone formation by itself in the rat ectopic bone formation assay (Wang et al., 1990). Although TGFP is related to these proteins, subcutaneous injection of TGFP by itself results only in formation of granulation tissue with no appearance of cartilage or bone (Roberts et al., 1986). A fourth member of this group, OIF, has also been isolated from bovine bone and has been shown to be a glycosylated protein of 22-28 kDa with a protein backbone of 12 kDa (Bentz et al., 1989). Although purified OIF is able to produce ectopic bone, its activity is substantially increased by the coadministration of TGFP-1 or TGFP-2. The complete OIF amino acid sequence shows no homology with other reported proteins, including those with osteoinductive activity (Bentz et al., 1990). Finally, osteogenin has also been found in bovine bone matrix (Luyten ef al., 1989) and in rat tooth (Katz and Reddi, 1988). It is a 22-kDa protein whose partial amino acid sequence shows homology to BMP-3 (Luyten et al., 1989). However, unlike BMP-3, it is able to initiate both cartilage and bone formation in vivo and its activity is enhanced by type 1 collagen (Muthukumaran et al.. 1988). In vitro, osteogenin is able to stimulate the expression of the osteogenic phenotype in periosteal cells and osteoblasts as measured by increased alkaline phosphatase activity, collagen synthesis, and PTH-stimulated adenyl cyclase activity, and of the chondrogenic phenotype of chondroblasts as measured by increased production of sulfated proteoglycans. The effects seem to be somewhat specific, as NIH 3T3 fibroblasts do not respond to the factor (Vukicevic et al., 1989). In addition to possible interactions with TGFP, some of the bone inductive proteins may be dependent on vitamin D, since it has been reported that bone matrix from vitamin D-deficient rats have reduced levels of osteogenin (Sampath et al., 1984). The ectopic bone forming activity of osteogenin is enhanced by platelet-derived growth factor (PDGF) while EGF, FGF, and insulin have no effect (Howes ef al., 1988). A role of IL-1 in ectopic bone formation, and perhaps fracture healing, has also been suggested by experiments that show greater amounts of heterotopic ossification induced by BMP in the presence of this cytokine (Mahy and Urist, 1988). Also antiinterleukin antibodies are able to


24 1

abolish the activity of BMP (Mahy and Urist, 1988). Another cytokine, tumor necrosis factor-a, is able to inhibit ectopic bone formation, presumably by preventing mesenchymal cells from differentiating into chondrocytes (Yoshikawa et al., 1988). It would therefore appear that these bone inductive factors may be important for the initial phases of differentiation, after which other factors are necessary to carry out the entire osteogenic process to the final conclusion of the generation of mature bone. 4. Epidermal Growth Factor (EGF)

EGF is another growth factor shown to affect the skeletal system and mineralization. Specific, high affinity receptors for EGF have been found on human osteosarcoma cells (Shupnik and Tashjian, 1981, 1982) and on newborn murine calvaria cells (Shupnik et al., 1980; Ng et al., 1983). These EGF receptors were localized to preosteoclastlike cells, that were found in close association with osteoclasts in the regions of bone or cartilage where matrix was being actively resorbed, and to preosteoblastlike cells in the metaphysis. Mature osteoblasts and osteoclasts themselves were not labeled. (Martineau-Doizeet al., 1988). The number of EGF receptors are modulated by TGFP (Uneno et af., 1989), PDGF (Shupnik et al., 1982), PTH (Ohta et al., 1989), and by agents that increase protein kinase C activity (Borst and Catherwood, 1989). One of the possible roles for EGF in bone is to increase the osteoblast/osteoclastprecursor pool. Evidence in support of this role demonstrates that EGF receptors occur on a population of immature cells that were sequentially released from the proliferative zone of newborn mouse calvaria by enzymatic digestion (VandePol et al., 1989). These same cells also responded to EGF by an increase in proliferation. As observed for TGFP, the response to EGF depends on the conditions of the experiment. Epidermal growth factor has been reported to either have no effect (Shupnik et al., 1980; Shupnik and Tashjian, 1981) or to stimulate (Canalis and Raisz, 1979b; Hiramatsu et al., 1982; Ng et al., 1983; Partridge et af., 1985) DNA synthesis in different in vitro bone systems. In addition to its possible effects on DNA synthesis, EGF is able to stimulate cartilage proteoglycan synthesis (Makower et al., 1989b; Osborn et al., 1989), inhibit type I collagen synthesis in MC3T3-El cells (Hiramatsu et al., 1982; Hata et af., 1984) and in fetal rat calvaria (Canalis and Raisz, 1979b; Canalis, 1983), and inhibit alkaline phosphatase activity (Canalis, 1983; Ibbotson et al., 1986). It also has a differential effect on prostaglandin synthesis, causing stimulation (Shupnik and Tashjian, 1981, 1982; Feyen et al., 1984), no effect (Shupnik and Tashjian, 1982), or an inhibition (Partridge et al., 1985). In general, EGF may have a function in bone formation partly through increasing the number of early preosteoblasts and, at the same time, blocking the more differentiated function of the later preosteoblastlike cell and/or the mature osteoblast. This hypothesis is supported by experiments with the UMR-106 cell where EGF and TGFa block

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a differentiated function of the osteoblastlike cell, that of the PTH induction of adenyl cyclase activity (Gutiemez et al., 1987). A dual role in controlling the process of bone mineralization is emphasized in experiments with fetal rat calvaria cells grown under conditions where bone nodules are able to form. In this situation, EGF can inhibit or stimulate nodule formation, depending upon the time and duration of exposure to the growth factor (Antosz et a1.,1989). This has also been shown in in vivo experiments where short-term administration of physiological levels of EGF causes a transient reduction of endosteal matrix apposition and mineralization, while longer term treatment resulted in an increase in periosteal bone formation (Marie et al., 1990). Again, the effects of EGF on bone resorption are dependent on the system being btudied. Epidermal growth factor and TGFa (a member of the EGF family; for review see Gill et al., 1987)are able to raise the systemic plasma calcium levels in intact mice (Tashjian et al., 1986). In vifro, in neonatal mouse calvaria, these factors stimulate bone resorption (Tashjian and Levine, 1978). most likely through a PGE2-mediatedmechanism (Stem et al., 1985; Ibbotson er af., 1986 Tashjian et al., 1988). However, in fetal rat long bone, while EGF and TGFa are still able to increase bone resorption (Raisz et al., 1980; Ibbotson et al., 1983, 1985, 1986), it does not appear to be through the prostaglandin pathway (Raisz et a[., 1980; Stem et ul., 1985), but rather through a mechanism that results in an increase in the number of osteoclasts (Takahashi et al., 1986). Interestingly, the response in fetal rat long bone could be made prostaglandin-dependent by blocking DNA synthesis (Lorenzo et a/., 1986), or by treatment with IL-1 (Lorenzo et at., 1988). Interleukin-1 also acts synergistically with TGFa in fetal rat calvaria to increase prostaglandin synthesis and inhibit collagen synthesis (Hurley et al., 1989). 5. Fibroblast Gniwrh Factor (FGFI

Fibroblast growth factor (FGF) has been postulated to play a role in bone formation by increasing the number of osteoprogenitor cells that are then available for differentiation to mature bone forming cells capable of synthesizing matrix proteins (Canalis et al., 1987, 1988b). Fibroblast growth factor is able to stimulate the formation of bonelike mineralized nodules in an in vitro bone marrow cell system, presumably through enhanced proliferation and differentiation of an osteoprogenitor cell population (Noff et al., 1989). Factors that have characteristics similar to FGF have been found in bone extracts (Hauschka et al., 1986) and both acidic FGF and basic FGF are produced by cultured fetal bone cells ( G l o b u s et al., 1989). Because t h e FGFs are able to bind to heparin (Gospodarowicz et al., 1984), and since the osteoblasts produce heparin sulfate proteoglycans (Beresford et ul., 1987), it is thought that, once produced, the FGFs bind to the extracellular mamx proteoglycans (Globus et af., 1989). They could then be released and activated by heparinaselike enzymes produced by cells in the immediate environment (Baird and Ling, 1987).


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In addition to their proposed role in the stimulation of precursor cell division, these factors have been shown, in vitro, to increase proliferation in rat osteoblastlike cells (Canalis and Raisz, 1980; Rodan et al., 1987; Globus et al., 1988; Noda and Vogel, 1989; Shen et al., 1989), being more mitogenic for bone cell populations with fewer osteoblastlike characteristics (McCarthy et al., 19894. It also decreases the expression of the osteoblast phenotype, causing diminished levels of alkaline phosphatase, type I collagen, osteocalcin, and PTH responsive-adenyl cyclase activity (Canalis and Raisz, 1980; S . B. Rodan et al., 1989; Noda and Vogel, 1989; McCarthy et al., 1989c; Shen er al., 1989), although it enhances osteopontin ( S . B. Rodan et al., 1989) and, in bovine bone cells, osteocalcin (Globus er al., 1988). In cartilage, FGF stimulates proliferation of chondrocytes in vitro (Kato and Gospodarowicz, 1985) and in vivo (Cuevas e f al., 1988), resulting in increases in the formation of extracellular matrix. In addition to its effects on cell division, FGF is also able to inhibit the terminal differentiation of rabbit growth-plate chondrocytes to hypertrophic cells which results in a decline in cartilage calcification (Kato and Iwamoto, 1990). Part of the effects of FGF on bone may be through enhancement of expression of the TGFP gene (Noda and Vogel, 1989) which, in turn, is able to potentiate the mitogenic effects of FGF (Globus er al., 1988). Insulinlike growth factor-I also is able to enhance the mitogenic effect of FGF on osteoblastlike cells (Rodan et al., 1987). Finally, there is a report that FGF is able to cause a prostaglandin-dependent stimulation of bone resorption in fetal rat long bones (Shen et al., 1989) although the physiological importance of this observation is still to be determined. 6 . Platelet-derived Growth Factor (PDGF)

Although PDGF, or closely related proteins, have been isolated from bone matrix (Hauschka et al., 1986), the role of PDGF in bone has not been extensively studied. While PDGF was not detected in the conditioned medium of normal fetal rat bone cultures (Centrella and Canalis, 1985), it has been shown to be synthesized by human osteosarcoma cells (Heldin et a1.,1986), and by normal human adult bone-derived cells (Graves et al., 1989). Platelet-derived growth factor receptors have been detected on osteoblasts (Centrella et al., 1 9 8 9 ~ ) which respond to the factor with increases in DNA synthesis (Canalis, 1981; Graves et al., 1989; Centrella et al., 1989c; Canalis et a[., 1989b) and general protein synthesis (Centrella et al., 1989c; Canalis et al., 1989b). In addition to its effects on osteoblasts, PDGF also stimulates collagen degradation and bone resorption (Tashjian et al., 1982; Canalis er al., 1989b), perhaps due partly to an increase in prostaglandin synthesis (Tashjian et al., 1982; Habenicht et al., 1985), and partly to an increase in collagenase production (Bauer er al., 1985). These effects of PDGF on collagen degradation are prevented by IGF-I, while the two factors show additive effects on DNA synthesis (Canalis et al., 1989b).

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7. Cytokines The role of the cytokines in bone formation and resorption is just now becoming appreciated. Interleukin- 1 is known to stimulate the proliferation of osteoblastlike cells (Rifas et al., 1984; Gowen el al., 1985a,b; Canalis, 1986; Smith et al., 1987). At the same time, it may also inhibit the differentiation of bone- or cartilage-forming cells, as suggested by decreases in the synthesis of type I collagen (Gallagher et al., 1985; Canalis, 1986; Smith et al., 1987), and proteoglycan (Tyler, 1985), as well as osteocalcin and alkaline phosphatase (Gallagher et al., 1985). A decrease in functional matrix-producing cells predicts that the calcification process would be diminished. Indeed IL-1 has been reported to prevent the formation of mineralized bone nodules by fetal rat osteoblasts in vitm (Stashenko et al., 1987). However, one group has reported an association between an increase in IL-I production by circulating monocytes and bone formation rates (Pacifici et al., 1987). In addition to its possible role in bone formation, IL-1 also stimulates bone resorption in vitro (Gowen et al., 1983; Thomson et af., 1986). Indeed, human osteoclast activating factor has been shown to be identical to IL-lp (Dewhirst et a/., 1985). This effect of IL-I on resorption is apparently mediated through the osteoblast (Thompson et al., 1986). While IL-I induces production of prostaglandins (Sato et al., 1986a,b; Bandara et a1.,1989), its effects on resorption are not caused only through this increase in prostaglandin synthesis. This is evidenced by experiments demonstrating that stimulation of resorption is only partially blocked (Sato et d., 1986a) or unaffected (Gowen and Mundy, 1986) by indomethacin. Interleukin- 1 may also stimulate resorption through activation of matrix degrading enzymes (Krakauer et al., 1985) such as neutral proteinases (Bandara et al., 19891, collagenase (Lin et al., 19881, and phospholipase A2 (Lyons-Giordano et al., 1989) and/or through the enhanced production of macrophage-colony stimulating factor (M-CSF), which may then potentiate resorption through the recruitment of osteoclasts (Sato et al., 1986b). The factor does not appear to act on the osteoblast through a calcium-mediated pathway (Pacifici ec al., 1988). Interleukin-1 will also stimulate resorption in vivo (Sabatini et al., 1988). When administered subcutaneously directly over the calvaria, the early effects of IL-I, which were independent of prostaglandin synthesis, resulted in increased bone resorption while the longer term effects, that appeared to be dependent on prostaglandin production, showed osteoclast-mediated resorption with the resorbed bone replaced by increased amounts of new bone (Boyce et al., 1989). These effects of IL-1 on resorption could also be observed when the factor was membrane-bound, implying that the resorption that occurs in inflammatory diseases such as rheumatoid arthritis and peridontal disease could be due to macrophage membrane-bound IL- 1 (Sabatini et al., 1988). As with the other factors described, IL-I interacts with a number of other regulators of bone metabolism. Several factors have been shown to modulate


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the effects of IL-1 in bone. For example, TGFP opposes the actions of IL-1 on enhanced proteoglycan synthesis and degradation in cartilage and thus may have a protective effect in inflammatory diseases (Andrews et al., 1989). On the other hand, FGF will enhance the IL-1-stimulated production of neutral proteases by chondrocytes (Phadke, 1987) perhaps through an increase in the number of IL-1 receptors in these cells (Chandrasekhar and Harvey, 1989). Finally, 1,25-(OH),-D, will increase the production of IL-1 in macrophage cells. It has therefore been postulated that some of the effects of vitamin D on bone may actually be mediated through the immune system (Hodler et al., 1985). The actions of other factors in bone are, in turn, modified by IL- 1. For example, the resorptive response to TGFa or to EGF is enhanced by IL-1 while it inhibits the mitogenic response to these factors (Lorenzo et af.,1988). Finally, IL-1 acts synergistically with TGFa in fetal rat calvaria to increase prostaglandin synthesis and inhibit collagen synthesis (Hurley et al., 1989), and with PTHrP to stimulate bone resorption both in virro and in vivo (Sato et al., 1989). As indicated earlier, the interaction of IL-1 with estrogen is not clear. In one report, treatment of postmenopausal women with estrogen did not affect the spontaneous release of IL-1 from the peripheral monocytes and it was concluded that the action of estrogen on bone resorption was not mediated by effects on IL-1 production (Stock et al., 1989b). In a second report, estrogen administration caused a significant decrease in IL-1 activity in blood from postmenopausal women, and the opposite conclusion was reached; the inhibition of bone loss by estrogen may, at least in part, be mediated through a decrease in IL-1 (Pacifici er af.,1989). Additional experiments are needed to determine the role of IL-1 in the response to estrogen. Other cytokines are also able to regulate osteoblast activity and, therefore, calcification. GM-CSF is produced by neonatal mouse calvaria and by calvarial osteoblastlike cells (Felix et al., 1988). This production is induced by PTH (Horowitz et al., 1989a,b; Weir er af., 1989). Because GM-CSF is able to induce baboon bone marrow cultures to produce cells with osteoclastlike features (MacDonald et al., 1986b), it has been postulated to be the factor, produced by osteoblasts in response to PTH, that in turn stimulates the osteoclast (Weir et al., 1989). However, in fetal mouse long bone cultures, GM-CSF decreases the development of osteoclasts (Lorenzo et al., 1987). The reasons for the difference in these two systems still needs to be investigated. In addition to its proposed role on osteoclast development, GM-CSF is able to modulate human trabecular osteoblastlike cells, stimulating their proliferation, but antagonizing their differentiated function (Evans et al., 1989). Similar to GM-CSF, IL-3 is able to induce the formation of osteoclastlike cells in bone marrow cultures (Barton and Mayer, 1989). The response to both of these factors is suppressed by TGFP (Sing et al., 1988). Finally, tumor necrosis factor (TNF) is able to stimulate bone resorption in nude mice (Johnson er al., 1989), perhaps through the stimulation of prostaglandin (Tashjian et af., 1987; Gowen et af., 1988) or elastase (Redini et

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al., 1988) synthesis. Tumor necrosis factor has also been reported to inhibit chondrocyte differentiation in the process of ectopic bone formation in mice (Yoshikawa et al., 1988). In vifro,TNF is able to stimulate bone resorption in cultures of fetal rat long bones and to inhibit collagen synthesis in calvaria (Bertolini et a/.. 1986), and proteoglycan synthesis in cartilage explants (Saklatvala, 1986). Like several other factors that stimulate bone resorption, the effects of TNF on the osteoclast appear to be mediated through the osteoblast (Thomson et al.. 1987). Besides stimulating the osteoblast to produce an osteoclast activating factor, TNF is also able to increase bone cell proliferation resulting in an increased number of osteoblastlike cells, although, at the same time, decreasing their differentiated phenotype (Canalis, 1987; Gowen et a/., 1988). Osteoblasts are not only able to respond to TNF but also synthesize this factor in response to IL- 1 or GM-CSF stimulation (Gowen et al., 1990). The role of the immune system and its cytokines in normal bone formation and resorption still needs further experimentation in order to be defined. However, it is not too difficult to expand the effects observed to date and postulate the involvement of these factors in various inflammatory diseases where increased bone resorption occurs.

iV. Pharmacological Regulators of Calcification In this section a structurally diverse group of compounds will be discussed that can be loosely classified as pharmacological regulators of calcification. Using either in vitro or in viva models, these agents have been shown to affect some facet of bone metabolism. In a few cases, the mechanism of action for a compound in bone has been evaluated experimentally. In other examples, the mode of action i s surmised based on the agent’s known biochemical mechanism i n different biological systems. In many instances, however, agents can be shown to act on bone, but t h e biochemical mechanism is unknown. Pharmacological agents which affect bone metabolism may prove not only to be useful therapeutically, but may also be valuable tools in elucidating the details of the calcification process. This survey of pharmacological effectors of calcification in skeletal tissues is not meant to be exhaustive, but rather to provide the reader with a sampling of active agents. The inclusion of particular agents in specific categories (e.g., enzyme inhibitors) is primarily for the convenience of discussion. The reader should recognize that many of these pharmacological agents have multiple sites of action in biological systems. The category in which agents are included may represent only one of the mechanisms by which they can modulate the calcification process.


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A. ENZYME INHIBITORS

I. Carbonic Anhydrase Inhibitors As discussed earlier in this article, carbonic anhydrase I1 plays an important role in osteoclastic bone resorption. Several inhibitors of this enzyme [(acetazolamide, ethoxzolamide, 5-(3-hydroxybenzoyl)2-thiophenesulfonamide (HTS)] have been used to block bone resorption either in vitro or in vivo (e.g., Kenny, 1985; Raisz et al., 1988). One difficulty with the therapeutic use of carbonic anhydrase inhibitors is associated with the wide spread involvement of the bone isozyme in the physiology of other tissues (Pierce and Waite, 1987; Raisz et a[., 1988). An approach to overcoming the problem of selectivity has been to target an inhibitor of the enzyme, e.g., acetazolamide, to bone, by coupling it to a bone-seeking agent such as tetracycline (Pierce and Waite, 1987).

2. Collagenase Inhibitors Collagenases of osteoblastic origin are believed to be important in the process of bone remodeling (e.g., Kahn and Partridge, 1987). Natural inhibitors of collagenases have been characterized from several skeletal tissues (Sellers and Reynolds, 1977; Sellers et al., 1980; Sakamoto et al., 1981; Cawston et al., 1981, 1983; Nagayama et aZ., 1984). However, the role of these enzymes in osteoclastic-mediated bone resorption is not well defined, and collagen breakdown in the acidic resorption lacuna may be mediated through a cysteine proteinase or other lysosomal enzymes, rather than collagenases (Delaisse et al., 1987). C1- 1 [N-(3-N-benzyloxycarbonyIamino- 1-R-carboxypropy1)-L-leucyl-0methyl-L-tyrosine N-methylamide] is reported to be a specific and potent inhibitor of tissue collagenases (Delaisse et af., 1985). In cultured calvaria, a complex system in which both osteoblasts and osteoclasts play a role in the resorption process, C1-1 inhibited PTH-induced resorption of bone matrix (Delaisse et al., 1985). It was, however, ineffective in blocking resorption by isolated chick osteoclasts cultured on a dentin surface, whereas inhibitors of cysteine proteinases were effective inhibitors in this model (Delaisse et al., 1987). 3. Phosphodiesterase Inhibitors

A number of important physiological regulators of bone metabolism, including PTH, calcitonin, and prostaglandins, utilize cAMP as an intracellular second messenger. The intracellular concentration of cAMP is dependent both on the rate of formation via adenyl cyclase and on the rate of degradation via phosphodiesterase (PDE). It is therefore not surprising that PDE inhibitors affect bone metabolism in a complex manner. The PDE inhibitor, theophyllin, retards the fall in serum calcium that follows acute parathyroidectomy in rats, and causes

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elevation of serum calcium in rats parathyroidectomized for 4 days (Wells and Lloyd, 1967), thus implicating PDE in the mechanism of action of PTH and the regulation of blood calcium. In short-term organ culture (less than 24 hr), the PDE inhibitors, theophylline and 3-isobutyl-methylxanthine(IBMX) can inhibit mineral and lysosomal enzyme release from unstimulated or PTH-stimulated mouse calvaria (Lerner, 1980), whereas the nonxanthine PDE inhibitors rolipram (an imidazole) or Ro 20-1724 (a pyrrolidone) did not reduce PTH-stimulated mineral release (Lerner et al., 1986). In longer term cultures (24-120 hr), PDE inhibitors of several types (e.g., xanthine, aminophyllin, theophylline, IBMX, and the two nonxanthine PDE inhibitors, Ro 20-1724 and rolipram) will stimulate the release of calcium, phosphate, and lysosomal enzymes from cultured calvaria (Herrman-Erlee and van der Meer, 1974; h e y et al., 1976; Lemer et al., 1986). This effect is not observed if endogenous prostaglandin production is inhibited, implying that PDE inhibition of resorption is only effective in the presence of stimulators of adenylate cyclase (Ransjo et al., 1988). Chambers and Ali (1983) reported that theophyllin and IBMX increased the activity of prostaglandins and calcitonin in inhibiting the motility of isolated osteoclasts. Similarly, Allan et al., (1986) indicated that IBMX enhanced the activity of PTH, prostaglandins, and calcitonin in stimulating tissue-type plasminogen-activator activity in different strains of the UMR osteosarcoma cell line. Finally, Robin and Ambrus (1982) evaluated two imidazoquinazolinone PDE inhibitors and found that they were able to prevent heparin-induced osteoporosis. The cardiotonic agents amrinone and milrinone have positive ionotropic and vasodilatory effects. Their mechanism of action in the heart may be related to PDE inhibitory effects on alterations in calcium balance (Schneeweiss, 1986). In this regard, amrinone will stimulate bone resorption when given alone, and inhibit bone resorption induced in cultured bone by PTH, 1,25-(OH),-D,, and PGE, (Krieger and Stern, 1982). More recently, the cardiotonic agent milrinone has also been shown to stimulate bone resorption in neonate mouse calvaria organ culture and, at the same time, to inhibit collagen synthesis (Krieger et al., 1987, 1988). Milrinone, but not amrinone, has structural homology to thyroxine (T4) (Davis et al., 1987), which is a well documented agent that stimulates bone turnover both in vivo and in vitro. In addition, thyroid hormones have been shown to have PDE-inhibitory effects (Marcus, 1975). However, milrinone is not effective in competing for thyroid hormone binding sites in bone cultures, thus suggesting that this is not its probable mechanism of action in bone (Krieger et al., 1988). 4 . Prostaglandin Synthesis Inhibitors

Prostaglandins have diverse effects on bone. Their production by osteoblasts in response to a variety of bone-stimulating agents indicates that they may play a key role in mediating the activity of several different physiological regulators of


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bone metabolism. Agents which interfere with prostaglandin synthesis have been used both to discern the mechanism of prostaglandin action on bone, and to evaluate how they might be used in treating diseases of the skeletal system. The effects of prostaglandins, and of the compounds that inhibit their synthesis on bone metabolism are complex. The most consistent observations show that the nonsteroidal antiinflammatory drugs (NSAIDS) that inhibit the cyclooxygenase pathway of prostaglandin synthesis will also inhibit prostaglandin-mediated bone resorption both in v i m (Sandberg et al., 1977; Tashjian and Levine, 1978; Katz et al., 1983) and in vivo (Williams et al., 1985, 1988; Jee et al., 1988; Thompson and Rodan, 1988; Li et al., 1989; Hayward et af., 1989). In addition, cyclooxygenase inhibitors have been shown to modulate osteoblast function with both inhibition and stimulation of DNA synthesis and alkaline phosphatase being reported in v i m (Khokher and Dandona, 1988; Fujimori et al., 1989), and bone formation in vivo (Jee et af., 1988; Li el al., 1989). Nonsteroidal antiinflammatory drugs have been used to prevent the induction of ectopic bone in animal models (Nilsson et af., 1986b). They have also been employed therapeutically to reduce ectopic bone formation following orthopedic surgery (Ritter and Sieber, 1985; Elmstedt et al., 1985), and to diminish alveolar bone loss associated with periodontal disease (Williams er al., 1989). AND ANTAGONISTS B. ESTROGEN AGONISTS

Significant interest has recently been focused on the modulation of bone metabolism by a group of compounds that elicit mixed agonist/antagonist actions on estrogen target tissues (e.g., tamoxifen, clomiphene), and are commonly referred to as antiestrogens (Jordan, 1984). This interest in regard to the biology of bone has been stimulated by recent reports that these compounds may mimic the activity of estrogen in preventing the development of osteopenia in ovariectomized rats (Beall et al., 1984; Jordan et af., 1987; Turner et al., 1987b, 1988), and neurectomized rats (Wakley et al., 1988), and may inhibit bone resorption in v i m (Stewart and Stem, 1986). Feldmann and co-workers (1989), on the other hand, found that antiestrogens caused osteopenic changes in intact rats which were similar to those associated with surgical castration, though one of the compounds tested, tamoxifen, did show some indication of agonist effects at the highest dose when given to ovariectomized rats. Kusuhara and Ishida (1986) evaluated the effect of tamoxifen on capons in which medullary bone had been induced by treatment with estrogens and androgens for 40 days. They found that tamoxifen treatment during the last 10 days of this period appeared to antagonize the effects of the estrogenlandrogen treatment and prevented osteoblast formation and increased osteoclast number. Turken et al. (1989) have reported, in preliminary studies evaluating the therapeutic potential of this class of compounds, that in women being treated with tamox-

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DANIEL C . WLLIAMS AND CHARLES A. FROLIK

ifen for breast cancer, the compound may reduce bone loss associated with menopause. C. IMMUNE MODULATORS Numerous reports in recent years have demonstrated that the immune/hemopoietic system is closely associated with the generation of osteoclasts, and that cytokine-mediated responses can be observed in both bone-forming and resorbing models. This has led a number of investigators to study the effects of immune modulators, such as cyclosporin A (CsA), on bone metabolism. ?he cyclosporins are potent immunosuppressive agents (Bore], 1989; Kahan, 1989) that also have effects on bone. CsA inhibits bone resorption induced by a variety of stimulating agents (e.g., PTH, IL- 1, PGE,, 1.25-(OH),-D3, lipopolysacchandes or thrombin) in both long bone and calvarial organ culture (Skjotd et al., 1985; Stewart et a/., 1986; Klaushofer et al., 1987; Stewart and Stem, 1988, 1989a,b; Sasagawa et al., 1989). It also antagonizes IL-1-mediated stimulation of PGE, synthesis and inhibition of osteocalcin synthesis in human osteoblastlike cell cultures (Skjodt et a/., 1985). An immunosuppressive analog of CsA, cyclosporin G (CsG), was also effective in inhibiting bone resorption (Stewart and Stern, 1989a), whereas nonimmunosuppressive analogs (cyclosporin F, cyclosporin H) or a weakly suppressive analog (cyclosporin D) were ineffective in blocking resorption in tdtm (Stewart and Stem, 1988, 1989a). Moreover, CsA, given concomitantly with calcitonin, delayed or eliminated the escape phenomenon which is typical of calcitonin inhibition of bone resorption in v i m (Stewart and Stem, 1989b). Sasagawa et al. (1989) examined additional classes of immunosuppressants (lobenzarit, traxanox, mizoribine) in addition to CsA, and found that they too inhibited resorption stimulated by a variety of factors. CsA and mizoribine also inhibited basal resorption in the calvarial cultures. The effects of CsA in iivo are less clear. The compound administered to either male or oophorectomized female rats results in severe osteopenia associated with enhanced bone remodeling (Movsowitz et al., 1988, 1989; Schlosberg et al., 1989). Withdrawal of CsA results in a partial reversal of the osteopenia (Schlosberg et ul., 1989). Orcel et a/. (1989) evaluated the effect of CsA in weanling rats and, in agreement with the studies of Movsowitz and co-workers, found histomorphometric evidence for an increase in bone formation. In contrast to the studies of Movsowitz et al., however, Orcel and co-workers reported that treated rats had reduced bone resorption. These apparent differences can be partially resolved in that, at comparable doses and treatment time, both groups saw increased parameters of bone formation. and either no change, or reduced bone resorption. It was only at higher doses or longer treatment time that Movsowitz and co-workers noted increased resorption.


25 1

D. ION FLUXEFFECTORS A number of agents that affect the passage of ions in and out of cells have been shown to have a direct effect on bone. While there is some controversy in the literature about the effects of calcium ionophores in bone, a majority of investigators have reported that the calcium ionophores (A23 187 and/or ionomycin) stimulate bone resorption (e.g., Dziak and Stem, 1975; Lorenzo and Raisz, 1981; Rabadjija et al., 1987). This stimulated resorption was found to be associated with prostaglandin synthesis (Rabadjija et al., 1987). The calcium antagonists, verapamil and diltiazem, inhibit bone resorption stimulated in vitro by PTH or vitamin D compounds (Herrmann-Erlee et al., 1977; Lerner and Gustafson, 1982; Ly et al., 1985). Guggino et al. (1988, 1989) identified a phenylalkamine (verapami1)-sensitive calcium channel in osteoblastlike osteosarcoma cells that could be stimulated by the dihydropyridine calcium agonist BAY K 8644. Desmethoxyverapamil inhibits PTH-stimulated calcium uptake, osteocalcin synthesis, and bone resorption, while BAY K 8644, on the other hand, stimulates osteocalcin synthesis and bone resorption. Dietrich and Duffield (1979) have shown that verapamil inhibits the synthesis of collagen and noncollagen proteins in vitro in skeletal tissue. Indirect effects of calcium antagonists have also been demonstrated, particularly with respect to modulation of PTH levels, although both increases and decreases in PTH levels have been reported (e.g., Fox, 1988; Seely et al., 1989). Using a variety of pharmacological agents including ouabain, monovalent ionophores, vanadate and dichlorobenzamil, a role for sodium, sodium-potassium and sodium-calcium exchanges in bone resorption has been suggested using in vitro organ culture bone resorption experiments (Krieger and Tashjian, 1980, 1981, 1982, 1983; Krieger and Kim, 1988). The proton pump mechanism in the osteoclast ruffled border likely involves a maleimide-inhibited kidney-type electrogenic proton pump (Baron, 1989; Blair et al., 1989). Omeprazole, an inhibitor of the hydrogen-potassium ATPase also inhibits resorption (Tuukkanen and Vaananen, 1986), suggesting a role for this proton pump as well. Disulfonic stilbene inhibitors of chloride-bicarbonate exchange have been used to inhibit bone resorption both in organ culture (Klein-Nulend and Raisz, 1989) and with isolated osteoclasts (Hall and Chambers, 1989), indicating that anion exchange, presumably associated with the need to regulate intracellular pH in the acid-secreting osteoclast, plays an important role in bone resorption. As might be expected, ion flux i n osteoblasts has also been implicated in the mineralization process (Anderson et al., 1984a) and the effects of hormones and metabolic inhibitors on, these fluxes have been described in osteoblastlike cells (e.g., Anderson et al., 1984b; Reid et al., 1988).

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E. OTHERORGANIC AGENTS I . Bisphosphoncites An extensive literature on the effects of bisphosphonates (formerly referred to as diphosphonates) on biological calcification and calcium metabolism has developed over the past twenty years (for review see Fleisch, 1989). The initial interest in these compounds was based on the suggestion that pyrophosphates and other condensed phosphates may have a role in regulating the physiological formation and dissolution of calcium phosphate crystals (Fleisch et al., 1966; Fleisch. 1989; Blumenthal, 1989). Bisphosphonates are stable analogs of pyrophosphates in which the P-0-P group is replaced with P-C-P. These compounds, while retaining a high affinity for calcified tissues, are resistant to enzymatic hydrolysis. A variety of bisphosphonates have been synthesized by changing the structure of the carbon side chains or by esterification of the phosphate groups, resulting in a biologically diverse group of compounds (Fleisch, 1989; Papapoulos et al., 1989). Bisphosphonates have been shown to inhibit the process of calcification in \Yvo in both skeletal and ectopically mineralizing tissues (Fleisch et al., 1970; Casey et al., 1972; Fraser et al., 1972; Schenk et al., 1973; Levy et al., 1985). This effect is presumably associated with their ability to inhibit crystal growth in solution, rather than being associated with specific cellular effects (Fleisch, 1989). The bisphosphonates are also potent inhibitors of bone resorption both in vifro and in i*iw(e.g., Fleisch et a/., 1969; Russell et al., 1970; Reynolds et al., 1972; Ohya et al., 1985; Reid et al., 1986; Schenk et al., 1986; Wronski et al., 1989). apparently by a variety of mechanisms. Some bisphosphonates may possess a selective toxicity for phagocytic cells such as macrophages and osteoclasts (Boonekamp et al., 1986; Flanagan and Chambers, 1989), while others may inhibit the proliferation and differentiation of macrophage and osteoclastlike cell precursors from bone marrow (Cecchini et al., 1987; Hughes et af., 1989). The mechanism of action of the newer clinically tested bisphosphonates, however, appears to involve absorption of the compound onto the calcified bone matrix. The bisphosphonate may then either inhibit the action of the mature osteoclast, or, at lower concentrations, prevent attachment of the osteoclast and its precursors to the matrix, thus suppressing resorption and phenotypic expression of the mature osteoclast (Boonekamp et al., 1986; Lowik et ul., 1988; Papapoulos et al., 1989). Inhibition of the osteoclast may not be due simply to cytotoxicity since several bisphosphonates are effective in inhibiting bone resorption by mature osteoclasts at concentrations that are not cytotoxic to the cells (Sato and Grasser, 1990). Because of their selective action on mineralized tissues, their amenity to chemical manipulation and the resultant functional diversity, bisphosphonates


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have been evaluated in a variety of clinical disorders. Their main use is in patients with Paget’s disease (Adami et al., 1986; Thiebaud et al., 1987; Atkins et af., 1987; O’Donoghue and Hosking, 1987; Rico et al., 1988). They have also been evaluated in osteoporosis treatment (Valkema et al., 1989; Mallette et al., 1989; Hodsman, 1989), and in treatment of hypocalcemia associated with malignancy (Thiebaud et al., 1986; Coleman et al., 1988; Morton and Howell, 1988), as well as other disorders (Fleisch, 1989). 2 . Thiazide Diuretics

The primary therapeutic utility of diuretics is to lower blood pressure through the enhanced excretion of sodium and chloride. However, these compounds also effect the renal processing of other ions such as calcium. The effects on calcium metabolism differ depending on the type of compound administered (Stier and Itskovitz, 1986). Among the different diuretics, thiazides have received the greatest attention with respect to therapeutic utility in modifying calcifying processes, being suggested for use in both renal stone disease and osteoporosis. In the urinary tract, thiazides are claimed to prevent the formation of calcium stones (e.g., Yendt and Cohanim, 1978; Brocks et al., 1983) although the positive effects of thiazides on stone formation are not universally accepted (Wolf ef al., 1983; Churchill and Taylor, 1985). In bone, thiazide administration results in higher levels of mineral content in treated compared to untreated individuals (e.g., Wasnich et al., 1983). Estrogen therapy, in combination with thiazides, increases bone mineral content even further (Wasnich et al., 1986). However, not all studies agree with this conclusion (Adland-Davenport et al., 1985). Stier and Itskovitz (1986) have recently reviewed the role of diuretics in calcium metabolism, and point out that different diuretics may either increase or decrease calcium excretion by affecting the physiological mechanisms that influence renal handling of calcium. Long-term administration of thiazides causes a reduction in calcium excretion which results in reducing hypercalcuria, thereby preventing renal stone formation (Stier and Itskovitz, 1986). However, thiazides also increase urinary levels of crystallization inhibitors such as pyrophosphate, magnesium, and zinc, and these also may contribute to thiazide effects on renal stone formation (Brandes et al., 1982). It appears that the primary action of thiazides on skeletal metabolism and as a treatment for osteoporosis is associated with its action on renal tubule calcium readsorption, although their final effects are likely the result of interrelated forces including renal, hormonal, and nonhormonal factors. These aspects of thiazide action need further elucidation (Chestnut, 1983; Stier and Itskovitz, 1986). Other classes of diuretics may likewise affect mineral metabolism in complex ways. Amiloride (a structurally distinct diuretic which appears to block sodium channels), for instance, has an additive effect with thiazides in reducing calcium clearance, but also may have direct effects

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on bone as indicated by its ability to inhibit basal resorption in organ culture (Krieger and Kim, 1988). 3. Anticonvulsants Anticonvulsant drugs, such as diphenylhydantoin (phenytoin) or phenobarbitol, have been reported to have complex effects on bone metabolism, resulting in a clinical instance of osteomalacia (Dziak er al., 1988; Takeshita et at., 1989). While the impact of these compounds in patients can in part be explained by their effect on vitamin D metabolism (Hahn, 1980), the compounds also have direct effects on in virro bone metabolism, including inhibition of bone resorption (Somerman et a f . , 1986) and modulation of osteoblast metabolism (Dziak et al., 1988). In patient populations, osteopenia associated with anticonvulsant therapy is accompanied by high bone turnover ( ie . , increased formation and resorption) e.g., Barden er al., 1982; Takeshita et al., 1989). The interpretation of these data is complicated, however, by the heterogeneous nature of the patient population and accompanying drug therapy regimes the subjects were given, as well as possible differential effects on compact versus trabecular bone (Barden et al., 1982). The effects of these drugs in vivo may involve many indirect effects on bone metabolism. Diphenylhydantoin has, for instance, been shown to inhibit calcium-mediated release of calcitonin from thyroid C cells. This latter effect can be reversed by calcium channel activators (Cooper et a!., 1988). 4 . Flavanoids

Flavanoids have been shown to have a variety of effects on bone related systems. Ipriflavone and several analogs have been shown to inhibit the release of calcium from both unstimulated and PTH-stimulated cultured bones (Tsuda et al., 1986). These compounds can also increase secretory levels of calcitonin in the presence of subeffective doses of estrogen (Yamazaki et al., 1986) and moderate the effect on bone of experimentally induced diabetes, glucocorticoid therapy, and vitamin Dkalcium nutritional deficiency (Shino et al., 1988; Yamazaki et al., 1986; Takenaka et al., 1986). In human studies of relatively short-term (6 month) and limited patient numbers, there appeared to be a positive effect of ipriflavone on bone mineral content in the lumbar spine and distal radius (Agnusdei et al., 1989). Catechin, another flavanoid, can stabilize collagen molecules so that they are less susceptible to collagenase digestion (Kuttan et al., 1981; Pontz et al., 1982) and also can inhibit bone resorption induced in cultured calvaria by PTH, PGE,, or retinoic acid (Delaisse et al., 1986). Other flavanoids have been shown to decrease plasma calcium and calcitonin when injected into rats in short-term (2.5 hr) studies (Saija et al., 1988).


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5. Levamisole

Levamisole is an antihelmentic drug with multiple sites of action in bone. As an antihelmentic agent, levamisole (L-tetramisole) is active while its stereoisorner, dexamisole (D-tetramisole) is inactive. Levamisole and an analog, R823 1 (bromotetramisole), have been reported to inhibit the action of bone-associated alkaline phosphatase in a stereospecific manner (Reynolds and Dew, 1977; Lerner and Granstrom, 1984), though the stereospecificity of this reaction has been questioned with regard to chick bone alkaline phosphatase (Tenenbaum, 1987). Levamisole, dexamisole, and their bromotetramisole analogs have also been shown to inhibit calcium release (bone resorption) and reduce lysosomal enzyme release and lactate production in cultured mouse calvarial bones in a nonstereospecific manner (Lemer and Granstrom, 1984). Levamisole has been shown to affect bone matrix apposition and mineral apposition in vivo, effects which have been attributed to its action on alkaline phosphatase (Garba and Marie, 1986). In that levamisole also has immune-modulatory activity, some of its activity on bone may be mediated through effects on the immune system (Sasagawa et al., 1989).

6 . Imidazole Imidazole and the tetramizoles are structurally related and have similar effects on bone (Lemer and Granstrom, 1984). Imidazole has been shown to inhibit bone resorption both in vitro and in vivo (Wells and Lloyd, 1968; Heersche and Gaillard, 1969; Avery et al., 1971; Heersche and Jez, 1981). It will also increase mineral uptake by cultured calvaria (Heersche and Gaillard, 1969), improve osteomalacia induced by low dietary levels of vitamin D and phosphorus in rats (Roudier and Martin, 1984), and promote calcification in bisphosphonateinduced ricketic growth plates (Eguchi ef al., 1989). Because imidazole has calcium binding activity and its action on ricketic growth plate is reversed by diltiazem, a calcium antagonist, it is possible that its action on skeletal tissue is mediated through an influence on intracellular calcium levels (Lerner and Granstrom, 1984; Eguchi et al., 1989). Alternatively (or in addition), Chambers and Ali (1983) noted that imidazole reduced the effectiveness of prostaglandins and calcitonin in inhibiting motility and shape change in isolated osteoclasts, suggesting that this action was associated with irnidazole stimulation of PDE activity.

7 . Promethazine and Related Compounds Promethazine hydrochloride, a phenothiazine derivative, has been reported to be a depressor of the central nervous system (sedative), an H, (histamine) receptor antagonist, an inhibitor of prostaglandin synthesis, and a potent

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inhibitor of macrophage function (Goldhaber and Rabadjija. 1982). It inhibits PTH-induced bone resorption in calvarial culture at concentrations which do not inhibit osteoid formation (Goldhaber and Rabadjija, 1982). The authors attributed these effects to inhibition of macrophage function. In a subsequent report, Goldhaber and Rabadjija (1983) compared the effects of several H, and H2 receptor antagonists. In this study, the HI antagonists inhibited resorption in calvarial cultures, whereas the H, antagonists did not. Promethazine was found to be the most potent of the H I antagonists tested. However, since histamine agonists (histamine, 2-methyl histamine, 2-pyridylethylamine, and betazole) and a histidine decarboxylase inhibitor [4-(4-imidazolyl)-3-amino-2-butanone] failed to stimulate bone resorption, and histamine was ineffective in reversing the promethazine effect, the authors concluded that promethazine was not acting through H, receptor antagonism. They suggested that the mechanism of action of the H I receptor antagonists was dependent on a membrane stabilizing effect. Tyan (1986) evaluated the effects of promethazine in vivo in aged mice and found that the compound appeared to enhance net bone deposition in femoral shafts. In a second report (Tyan and Blahd, 1986), promethazine was compared to chlorpheneramine (an HI blocker) and trifluoperazine (a phenothiazine without H, blocking activity) and it was found that promethazine, but not the other compounds tested, inhibited bone loss in aging mice. Komoda and co-workers (1985). on the other hand, reported that the phenothiazine derivatives chlorpromazine, trifluoperazine, and perphenazine inhibited aspects of bone formation (e.g., alkaline phosphatase, collagen synthesis, bone cell proliferation) in different in vivo and in vitro models, whereas prornethazine had little effect on these parameters. 8. Thiophene Carbo.qlic Acids Thiophene carboxylic acid and its derivatives have been shown to have antiresorptive effects in several bone models. The hypocalcemic and hypophosphatemic effects of 2-thiophene carboxylic acid in intact, parathyroidectornized and thyroparathyroidectomized rats were initially described by Lloyd ef af. (1969). It was subsequently shown to inhibit both basal and PTH-induced bone resorption in calvarial cultures (Fang et a / . , 1971). Two derivatives, benzo[b]thiophene-2-carboxylic acid (BL5583) and dibenzothiophene-4-carboxylic acid (BL5593) were shown to be effective in reducing heparin-induced osteopenia in mice (Robin at al., 1980). Benzo[b]thiophene-2-carboxylic acid was also evaluated in vitro and found to inhibit basal, PTH-, and A23 187- (a calcium ionophore) induced bone resorption and to decrease calcium uptake by cultured calvarial cell populations, but did not affect CAMP levels in these cells (Robin et al., 1984). Benzo[b]thiophene-2-carboxylic acid (thionapthene-2-car-


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boxylic acid) was found to be the most potent of a series of thiophene carboxylic acid derivatives tested by Raisz et al. (1985)for their effects in inhibiting bone resorption in long bone cultures, and its lysine salt was shown to be effective in lowering serum calcium concentrations in rats bearing hypercalcemic Leydig cell tumors (Decker et al., 1989). 9. WR-2721

WR-2721 [S-2(3-aminopropylamino)ethylphosphorothioic acid] is a thiophosphate analog of cystamine that has been used to protect certain normal tissues against the lethal effects of ionizing radiation and akylating agents (Yuhas et al., 1980).It causes hypocalcemia in animals and humans (Glover et al., 1983;Attie et al., 1985),inhibits PTH secretion (Glover et al., 1983),increases urinary calcium excretion (Hirschel-Scholz et al., 1985), and inhibits osteoclastic bone resorption both in vitro and in vivo (Attie et al.; 1985;Shaker et al., 1989).Attie et al. (1985)concluded that the predominant effect in producing hypocalcemia was associated with the compound’s antiresorptive effect in bone. 10. HeparinlProtamine Heparin is best known therapeutically for its anticoagulant activity. However, it has also been used experimentally to enhance the resorptive activity of agents that stimulate bone resorption (Goldhaber, 1965),and to induce osteopenia in animal models (Thompson, 1973;Ambrus et al., 1978;Matzsch et al., 1986). These models have, in turn, been used to evaluate a variety of compounds for their effects on osteoporosis (e.g., Robin et al., 1980, 1983). Heparin has also been implicated as a causal factor in human osteoporosis (De Swiet et al., 1983;Mazanec and Grisanti, 1989),although the mechanism by which heparin causes osteopenia is not known. It is able to increase both collagenase activity in vitro in mouse bone explants (Sakamoto et a f . , 1973), and resorption of experimentally implanted bone particles (Glowacki, 1983). Crisp et al. (1986) have shown that some heparin preparations could “blunt” the activity of calcitonin in inhibiting bone resorption in vitro, even though heparin alone inhibited resorption in the same model. Recent studies have attempted to better define the activity of heparin based on size characteristics, anticoagulant activity of heparin fragments, and the interactions of heparin with other bone-regulating agents. Cochran (1987)and Cochran and Abernathy (1988)examined glycosaminoglycan effects in mouse calvarial organ culture and found that hyaluronic acid and dermatan sulfate stimulated resorption in the absence of added stimuli, while heparin, heparin fragments with differing anticoagulant activity, dextran, and dextran sulfate potentiated the action of PTH in stimulating resorption but failed to stimulate resorption by themselves. When these glycosaminoglycans were combined with IL-1 p, the effects on resorption differed

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from those seen with PTH. Heparin and hyaluronic acid partially inhibited resorption stimulated by IL- I , and the decreasing inhibition of the heparin fragments corresponded to their decreasing anticoagulant activity. These in vitro results point out the potential complexity associated with heparin effects on the skeletal system. Protamine is a basic polypeptide used therapeutically to antagonize the anticoagulant activity of heparin. Because of this fact, it was evaluated for its effects in patients with hypercalcemia associated with malignancy (Anderson et al., 1967), and has been shown to be a potent hypocalcemic agent in animal models (Johnston et al., 1970 Potts et al., 1984). It is able to inhibit bone resorption both in vitro in cultured calvaria (Johnston et al., 1970) and in vivo in association with subcutaneously implanted bone particles (Glowacki, 1983). The data from both intact and surgically modified animals are consistent with a primary site of protamine’s hypocalcemic action being on bone tissue (Johnston et al., 1970 Potts et al., 1984).

11. Glucocorticoids The effects of glucocorticoids on bone are complex and often contradictory, particularly in comparing in vitro and in vivo data. In vivo, both direct and indirect effects of glucocorticoids on mineral metabolism have been documented. Bone formation in vivo appears to be retarded, whereas bone resorption is reported to be either unaffected or enhanced, with the net effect of glucocorticoid treatment being consistently associated with bone loss both in animal models and in clinical human experience (e.g., Als et al., 1985; Ortoft and Oxlund, 1988; Goulding and Gold, 1988; Lukert and Raisz, 1990; Bockman and Weinerman, 1990). in vitro effects, on the other hand, include impaired bone resorptive activity with isolated osteoclasts (Tobias and Chambers, 1989), inhibition of plasminogen activator activity stimulated by bone resorbing hormones (Hamilton et al., 1985), and stimulation of bone formation in cultured osteoblasts (Bellows et d., 1987). Depending on the experimental conditions, glucocorticoids can either inhibit or enhance bone resorption in organ culture (e.g., Raisz et al., 1972; Reid et al., 1986). One explanation for these conflicting results may be the tendency for glucocorticoids to exhibit biphasic concentration responses in some in vitro systems (e.g., Wong, 1979; Anderson et al., 1984b). The literature on glucocorticoid effects on calcified tissues is far too extensive to review in depth here; however, two very recent reviews that emphasize clinical aspects of the problem are available (Lukert and Raisz, 1990; Bockman and Weineman, 1990). From a therapeutic perspective, one relatively new glucocorticoid, deflazacort, holds promise of reduced adverse effects on the skeletal system (Gennari et al., 1984; Balsan et al., 1987).


F.

INORGANIC

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EFFECTORS OF CALCInCATION

I . Fluoride The use of fluoride in treating metabolic bone diseases such as osteoporosis has been extensively studied, but remains controversial. Fluoride therapy results in increased trabecular bone mass in the axial (vertebral) skeleton of osteopenic patients. This effect is generally found to be associated with decreased vertebral fracture rates (Heaney et al., 1989; Farley et al., 1989). However, concern persists about the quality of the bone laid down and the effect of therapy on fracture rates in the appendicular skeleton. Published data are in disagreement as to the effects of fluoride therapy on appendicular bone. Some studies indicate no significant enhanced risk of fracture with therapy (Riggs er al., 1987; Mamelle et al., 1988; Heaney et al., 1989), whereas others have suggested that fluoride treatment is associated with reduced cortical bone density (e.g., Hodsman and Drost, 1989; Riggs er al., 1990), and increased fracture incidence in the appendicular bones (Schnitzler and Salmon, 1985; Hedlund and Gallagher, 1989; Schnitzler e f al., 1990; Riggs et al., 1990). Some undesirable side effects (e.g., gastrointestinal and rheumatic) associated with fluoride therapy may be related to pharmacokinetic problems associated with the pulsatile administration of fastrelease fluoride preparations that may periodically increase fluoride concentrations to toxic levels (Pak er al., 1986a,b;Turner et aL1989). Slow-release preparations may therefore prove useful (Pak et al., 1986a,b; Pak, 1989). However, significant amounts of fluoride are incorporated into the bone crystalline structure, which can then act as a slow-release reservoir of fluoride, thus effectively increasing the length of treatment (Baud et al., 1988; Grynpas and Cheng, 1988). Since this may mediate some of the adverse skeletal effects, it is not clear that slow-release formulations will eliminate problems of appendicular fracture. Published data consistently demonstrate that fluoride has a dramatic effect on the process of calcification that may be related both to its direct effects on the crystallization processes, as well as to its effects on the cellular aspects of bone formation. Fluoride has been shown to directly stimulate proliferation and alkaline phosphatase activity in bone forming cells (Farley et al., 1983), which may be mediated through the effects of fluoride in inhibiting phosphotyrosyl protein phosphatase and prolonging the stimulatory response of growth factors whose action is mediated through tyrosyl protein phosphorylation (Lundy et af., 1988; Lau et al., 1989). Fluoride also has been shown to modify the crystallinity and reduce the solubility of bone mineral (Baud er al., 1988; Grynpas and Cheng, 1988). These changes in crystalline structure may account for the increases in microhardness and compressive strength associated with fluoridic bone (Baud et al., 1988). High doses of fluoride (60 mg/kg body weight in rats) were

260


demonstrated to be toxic to osteoclasts (Lindskog et al., 1989), but whether this has physiological significance for an osteoclast resorbing fluoridic bone is not clear. Reduced solubility of bone crystal (and/or selective toxicity for osteoclam) could result in decreases in bone resorption (Grynpas and Cheng, 1988) with enhanced bone volume, but reduced repair of microfracture in bone via remodeling processes, and subsequently to stress fracture of bone. Alternatively, Schnitzler et al. ( 1990) suggest that enhanced bone erosion associated with fluoride therapy accompanied by replacement with poorly mineralized osteoid may be the cause of increased incidence of stress fractures.

2. Vanadate Several investigators have demonstrated that vanadate will inhibit bone resorption in mouse calvarial organ culture (e.g., Krieger and Tashjian, 1983), and stimulate DNA, alkaline phosphatase, and collagen synthesis in calvarial cell and organ cultures (Canalis, 1985; Lau et al., 1988b). Moreover, Yamaguchi cr al. (1989b) have recently demonstrated that oral administration of vanadate to weanling rats results in increases in DNA content and alkaline phosphatase activity in femoral diaphyses, while acid phosphatase activity and bone calcium content is not altered. Krieger and Tashjian (1983) conclude that the action of vanadate in stimulating bone resorption is through its effect on Na/K transport and the inhibition of Na,K-ATPase, whereas, Lau et a f .(1988b) suggest that the effects of vanadate in the calvaria may be due to its ability to prolong and/or potentiate the action of mitogenic agents by inhibiting the activity of phosphotyrosy1 protein phosphatase. 3. Aluminrtm

Aluminum accumulation in the skeleton has been connected with the development of a vitamin D-resistant form of osteomalacia, and may be associated with impaired osteoblast numbers and/or rates of matrix synthesis and mineralization (Sedman e t a / . , 1987; Hodsman et al., 1988). Alternatively, aluminum binding to hydroxyapatite crystals may interfere with nucleation and crystal growth (Blumenthal, 1985). The factors causing the development of this syndrome have not been completely defined, but, in animal models, appear to be linked with the vitamin D/PTH status of the subject (Hodsman et al., 1988). Aluminum toxicity has also been implicated in the development of aplastic bone disease in dialysis patients. This disease differs from pure osteomalacia in the absence of a marked osteoid excess. While aluminum-associated aplastic bone disease has been less studied than osteomalacia associated with aluminum toxicity, it does appear to involve impaired osteoblast function (Parisien et al., 1988).


26 1

4. Gallium

Gallium salts have been used therapeutically as an antitumor agent and to reduce hypercalcemia and bone turnover associated with certain malignant tumors (e.g., Warrell et al., 1984, 1987; Warrell and Bockman, 1989). Gallium inhibits bone resorption in vitro in organ culture (Warrell et al., 1984), as well as in cultures of isolated osteoclasts (Hall and Chambers, 1990a), and short term treatment of rats in vivo results in increases in bone calcium and phosphorus content (Repo et al., 1988). In chronically treated rats, both acid phosphatase and alkaline phosphatase levels were increased in calvaria (Coumot-Witmer et al., 1987). Like aluminum, gallium interacts with the mineral as well as the cellular components of bone (Bockman et al., 1986; Blumenthal et al., 1989; Hall and Chambers, 1990a), and this interaction may partially explain the in vivo effects of gallium. 5 . Gold

Complexes of gold are used as treatment for rheumatoid arthritis (Klaushofer et al., 1989). Several groups have reported that these complexes will also inhibit bone resorption in vitro (Goldhaber et al., 1978; Katz and Gray, 1986; Vargas et al., 1987; Klaushofer et al., 1989). These studies that might help to explain the possible mechanism of gold action on bone resorption differ in the models used (e.g., mouse vs. rat, long bone vs. calvaria), and in their results. Klaushofer et al. (1989) reported that, depending on concentration, gold salts can either stimulate or inhibit bone resorption in calvarial organ cultures, with stimulation at lower concentrations (3 x 10-'-3 x M) mediated through effects on prostaglandin production. They suggest that the inhibitory effect of high concentrations of auranofin, the gold compound used in their studies, may be due to cytotoxic effects. Katz and Gray (1986), however, found that while auranofin inhibited collagen synthesis at doses comparable to those inhibiting bone resorption in mouse calvaria, DNA and protein synthesis and lysosomal enzyme release were not significantly affected. Vargas et al., using fetal rat long bone cultures, found that both auranofin and gold sodium thiomalate (GST) inhibited resorption. The effects of auranofin were irreversable and accompanied by inhibition of ['Hlthymidine and ['Hlproline incorporation into bone, whereas GST effects on resorption were reversible and it did not effect DNA or protein synthesis. Both auranofin and GST decreased lysosomal (P-glucuronidase) release from bones.

6. Zinc Zinc is an essential trace element. It is a cofactor for many enzymes, including a number that participate in the processes of transcription and translation (Prasad, 1983). Several investigators have shown a correlation between tissue and body fluid levels of zinc and osteoporosis (e.g., Atik, 1983; Herzberg et al.,

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1990). Zinc deficiency has also been shown to be associated with retardation of ectopic bone formation and calcification in response to implantation of demineralized bone matrix. Evidence indicates that zinc may play an active role in bone metabolism (Calhoun er a/., 1975). However, an alternative explanation for the zinc effects in the ectopic bone implant model relate to impaired vascularization at the site of implantation (Belanger et al., 1977). Yamaguchi and co-workers have studied the effects of zinc on bone metabolism using both in vitro and in v i w animal models (e.g., Yamaguchi and Inamoto, 1986; Yamaguchi and Matsui, 1989; Yamaguchi er af., 1982a,b, 1987, 1988, 1989a,b). In these studies, short termbow dose administration of zinc in weanling rats increased parameters associated with bone formation (increased femur dry weight, DNA content, alkaline phosphatase activity), whereas longer term administration or higher doses decreased formation parameters and increased resorption parameters (femur acid phosphatase activity) (Yamaguchi et al., 1982a,b). Zinc was found to potentiate the bone formation effects of 1,25-(OH),-D,, but not the effects of PTH or calcitonin (Yamaguchi and Inamoto, 1986; Yamaguchi et al., 1989a). The actions of zinc in bone were attributed to its effects in stimulating protein synthesis and aminoacyl-tRNA synthetase (Yamaguchi et al., 1987, 1988). In tissue culture, the positive effects of zinc could be reversed by chelation with dipicolinate, which Yamaguchi and Matsui (1989) suggest supports a role for endogenous zinc in regulating bone protein synthesis.

V. Summary and Conclusions Biological calcification is a highly regulated process which occurs in diverse species of microorganisms, plants, and animals. Calcification provides tissues with structural rigidity to function in support and protection, supplies the organism with a reservoir for physiologically important ions, and also serves in a variety of specialized functions. In the vertebrate skeleton, hydroxyapatite crystals are laid down on a backbone of type I collagen, with the process being controlled by a wide range of noncollagenous proteins present in the local surroundings. In bone, cells of the osteoblast lineage are responsible for the synthesis of the bone matrix and many of these regulatory proteins. Osteoclasts, on the other hand, are continually resorbing bone to both produce changes in bone shape and maintain skeletal integrity, and to establish the ionic environment needed by the organism. The proliferation, differentiation, and activity of these cells is regulated by a number of growth factors and hormones. While much has already been discovered over the past few years about the involvement of various regulators in the process of mineralization, the identification and functional characterization of these factors remains an area of intense investigation.


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As with any complex, biological system that is in a finely tuned equilibrium under normal conditions, problems can occur. An imbalance in the processes of formation and resorption can lead to calcification disorders, and the resultant diseases of the skeletal system have a major impact on human health. A number of pharmacological agents have been, and are being, investigated for their therapeutic potential to correct these defects. While much has been learned recently concerning mineralization and methods of therapeutic intervention in the process, research in this area is only in its infancy. The next few years should bring exciting advancements in this field that will most likely give a better insight into how calcification is initiated and controlled and how to best deal with abnormalities in the process.

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Index

A Abscisic acid cereal aleurone and, 49 a-amylase genes, 54,59,61-62 calcium, 81-82 mechanism of hormone action, 6 3 4 4 , 6647 uptake mechanisms in plants and, I10 Abscisic acid-responsive element, cereal aleurone and, 64-65.67 N-Acetylgalactosamine, intestinal epithelium and, 141, 157 Golgi apparatus, 174-175 oligosaccharides, 143, 145-147 N-Acetylglucosamine, intestinal epithelium and, 139,141,143,156 Acid phosphatase, calcification and, 215,261 Acidification, calcification and, 2 15-2 17 Actin, calcification and, 213 Adaptation, melanin-concentrating hormone and, 7,33,35 Adenyl cyclase, calcification and, 221,226228, 240,242-243 Adrenocorticotropin (ACTH), melaninconcentrating hormone and, 13.35-36, 3940 Affinity calcification and, 219,223,234,241 glycosylation in intestinal epithelium and, 176177 uptake mechanisms in plants and, 91-92, 119, 125 Aleurone, regulation of secreted proteins in, see Cereal aleurone 293

Alkaline phosphatase, calcification and biochemistry, 2 0 6 2 0 7 cells of bone, 209-210 endocrine factors, 226228,231 pharmacological regulation, 249,255, 259-26 1 physiological regulation, 237,240-241, 243-244 Aluminum, calcification and, 260-261 Amiloride, calcification and, 253 Amino acids calcification and, 204,22&222,240 cereal aleurone and a-amylase genes, 5&57 protein transport, 70.73.78-79 glycosylation in intestinal epithelium and, 139,141,143 melanin-concentrating hormone and, 14-1 8, 21,23 uptake mechanisms in plants and, 89, 105-109, 113,123 a-Aminoisobutyric acid, uptake mechanisms in plants and, 106 Amphibians, melanin-concentrating hormone and, 1-3 anatomical distribution, 9-1 1, 13 physiology, 33-34 structure-activity studies, 23 Amrinone, calcification and, 248 a-Amylase, cereal aleurone and, 49,83 calcium, 79-83 genes, 54-62 mechanism of hormone action, 6 3 4 8 protein transport, 70-74.7677 Androgen, calcification and, 230-23 I , 233,249

294

INDEX

Anguilla, melanin-concentrating hormone and,

3, 14 Anolis, melanin-concentrating hormone and, 23-24.34 Anti-idiotypic antibodies, cereal aleurone and. 63-64 Antibodies calcification and, 206,220,235,240 cereal aleurone and, 63-64,7 1,75 glycosylation in intestinal epithelium and. 146, 149. 154, 168, 176-178 melanin-concentrating hormone and, 3 Anticonvulsants, calcification and, 254 Antiestrogens, calcification and. 249 Antigens calcification and, 209 cereal aleurone and. 63 intestinal epithelium and, 149, 176-178 Apical region, calcification and, 2 16-2 17 A p ~ w i umelanin-concentrating . hormone and, 41-43 Arginine. uptake mechanisms in plants and. 107 ATP. melanin-concenaating hormone and, 3 1 ATPase calcification and, 216-217,224,260 cereal aleurone and. 8 1-82 uptake mechanisms in plants and, 89, 123, 125, 131 multiphasic model, 114-116. I18 Atrial natriuretic factor. melaninconcenaating hormone and, 4 2 4 3 Autoantagonism, melanin-concentrating hormone and, 24 Autoradiography calcification and, 227,237 glycosylation in intestinal epithelium and, 137, 154. 175-176, 184 Auxins, uptake mechanisms in plants and, 110

B Bacteria, uptake mechanisms in plants and, 90, 116-118, 192 Barley cereal aleurone and, 83-84 aleurone tissue, 49-52 a-amylase genes, 54-57,59,61 calcium. 79-83

mechanism of hormone action, 6 3 , 6 5 4 8 protein transport, 7&7 I , 73-79 uptake mechanisms in plants and, 124.13 I dual model, 9 1-92 multiphasic model, 93-98. 102-107. 111-1 13 Basement membrane, melanin-concentrating hormone and, 7 Bean, uptake mechanisms in plants and, 104 Benzyladenine, uptake mechanisms in plants and, 110 Binding protein, cereal aleurone and, 75 Biological calcification, see Calcification Bisphosphonates, calcification and, 252-253 Bone, see Calcification Bone inductive proteins, calcification and, 239-24 I Bone lining cells, calcification and, 21 2 Bone marrow, calcification and, 213.215.223, 242,245,252 Bone morphogenetic proteins, calcification and, 239-24 1 Bone sialoprotein, calcification and. 202-203 Boron, uptake mechanisms in plants and, 104 Brain, melanin-concentrating hormone and, 1,3, 4243 anatomical distribution, 3-5,7-8, 11, 13 physiology, 30.37.39-40

C Calcification, 195-196,262-263 biochemistry, 199 r-carboxyglutamic acid, 2 W 2 0 5 collagens, 200-201 enzymes, 206-207 mineral phase, 200 noncollagenous proteins, 201-203 proteolipids, 205 cells ofbone, 208 bone lining cells, 21 2 osteoblasts, 209-2 I2 osteoclasts, 2 13-2 I7 osteocytes, 2 12-2 13 diversity, 198-199 function, 196 matrix vesicle theory. 197 modeling, 207

INDEX nucleation theory, 196-197 pharmacologicalregulation, 246 enzyme inhibitors, 247-249 estrogen, 249-250 immune modulators, 250 inorganic effectors, 259-262 ion flux effectors, 251 organic agents, 252-258 physiological regulation, 2 17-2 19 bone inductive proteins, 239-24 I calcitonin, 222-223 cytokines, 244-246 endocrine factors, 2 18 epidermal growth factor, 24 1-242 fibroblast growth factor, 242-243 growth hormone, 232-233 insulinlike growth factors, 233-236 parathyroid hormone, 217,219-222 platelet-derived growth factor, 243 steroid hormones, 227-232 transforminggrowth factors, 236-239 vitamin A, 226-227 vitamin D, 223-226 Calcitonin, calcification and cells of bone, 215-216 pharmacological regulation, 247-248,257,261 physiological regulation, 222-223.229, 231-232.238 Calcium cereal aleurone and, 49.84 a-amylase genes, 54,56,61 metal loproteins, 79-80 protein transport, 71-72,74,76-77 transport, 81-83 melanin-concentratinghormone and, 2 4 2 5 Calcium phosphate, calcification and, 197,200, 205-206,252 Calmodulin calcification and, 203 cereal aleurone and, 82 Cancer cells calcification and, 210 glycosylation in intestinal epithelium and, 135,186 Carbohydrate calcification and, 203 glycosylation in intestinal epithelium and, 137,147-148,186-188 distribution, 153,155-158, 161

295

post-Golgi apparatus distribution, 177-178 Carbonate, calcification and, 198,200 Carbonic anhydrase, calcification and, 216 inhibition, 247 T-Carboxyglutamic acid, calcification and, 204-205 Carriers, uptake mechanisms in plants and, 126-128 Cartilage, calcification and biochemistry, 199-200,205-206 endocrine factors, 222,224-227 physiological regulation, 235-236,238-24 1, 244-245 Catalysis, glycosylation in intestinal epithelium and, 182 Cathespin H, cereal aleurone and, 78.84 cDNA, see ComplementaryDNA Cellulose, cereal aleurone and, 72 Central nervous system, melanin-concentrating hormone and, 10,39 Cereal aleurone, regulation of secreted proteins in, 49,83-84 aleurone tissue, 49-53 a-amylase genes expression, 60-62 multigene family, 55-59 synthesis, 54-55 calcium metalloproteins, 79-80 transport, 8 1-83 hormone action genes, 64-68 perception, 63-64 steroid model, 68-70 protein transport, 7 4 7 9 ER lumen, 73-74 route, 70-72 Channels, uptake mechanisms in plants and, 126-128,131 Chloride calcification and, 253 uptake mechanisms in plants and, 91,102, 113,124 CHO cells, glycosylation in intestinal epithelium and, 173,181 Choline sulfate, uptake mechanisms in plants and, 92 Chondrocytes,calcification and, 217,220, 224-225

296

INDEX

cytokines. 245-246 endocrine factors. 217,220,224-225.232 physiological regulation, 233-236.238-239, 243 steroid hormones. 227-228.23 I Chorellu, uptake mechanisms in plants and, 99-101, 107. 110, 116 Chromatin. cereal aleurone and. 68 Chromatophores. melanin-concentrating hormone and, 30,43 Chromosomes,cereal aleurone and, 57.59 Chymotrypsin,cereal aleurone and, 80 Cis-acting elements, cereal aleurone and, 59.62, 64-67 Citrate. calcification and, 198.200 Clones calcification and. 240 cereal aleurone and, 83 a-amylase genes, 5657.59 mechanism of hormone action, 63.6-7 protein transport. 73,77 glycosylation in intestinal epithelium and, 177. 181 Colchicine glycosylation in intestinal epithelium and, I84185 melanin-concentratinghormone and. 8 Collagen. calcification and, 197,262 biochemistry, 200-203 cells of bone, 209-210 endocrine factors, 220, 222-226,231 pharmacological regulation, 248.25 1,254, 260-261 physiological regulation, 2 17. 235-237, 239-245 Collagenase.calcification and, 21 2,238, 243-244,254,251 Collagenase inhibition. calcification and, 247 Colloidal gold, glycosylation in intestinal epitheliumand, 148. 159. 161 Colon, glycosylation and. 158-159. 163, 168, 171, 178 Color change, melanin-concentratinghormone and, 1-2.30.39.43 Competition. uptake mechanisms in plants and, 92.95. 107 Complementary DNA cereal aleurone and a-amylase genes. 56-57.59 protein transpon, 73.77-78

glycosylation in intestinal epithelium and, 173, 188 melanin-concentratinghormone and, 14-17, 40.42 Concanavalin A, glycosylation in intestinal epithelium and, 158-159 Cooperative kinetics, uptake mechanisms in plants and. 114, 127-128 Cooperative model, uptake mechanisms in plants and, 123 Corn, uptake mechanisms in plants and, 102. 114, 120, 124-126 Corticosteroids, melanin-concentratinghormone and, 39 Corticotropin-releasingfactor anatomical distribution, 13-14, 18 physiology, 35-37.39-40 Conisol calcification and, 236, 25 I melanin-concentratinghormone and, 35-36, 38-39,43 Cortisone, glycosylation in intestinal epithelium and, 183-184 Creatine kinase, calcification and, 228 Crystallization, calcification and, 197-198.262 biochemistry,201,206 cells of bone, 212 pharmacological regulation, 252-253,259 physiological regulation, 2 17,223-224 Crenophupngodon, melanin-concentrating hormone and, 3,5,25,30 Cyclic AMP,calcification and cells of bone, 2 I 1.215 endocrine factors, 220-222,231 pharmacologicalregulation, 247,256 Cycloheximide,glycosylation in intestinal epithelium and, 185 Cyclooxygenase,calcification and, 249 Cyclosporin, calcification and, 250 Cyclostomes, melanin-concentratinghormone and, 8-9 Cysteine, uptake mechanisms in plants and, 106 Cytochemistry,glycosylation in intestinal epithelium and, 138. 148, 157, 165 Cytochrome c reductase, cereal aleurone and, 82 Cytokines, calcification and, 230,24&241, 244-246.250 Cytokinin, uptake mechanisms in plants and, I10 Cytoplasm. cereal aleurone and, 63. 68

INDEX Cytosol, cereal aleurone and, 70.81-83

D Daruru srramonium lectin (DSL), intestinal

epithelium and, 159 Depletion, uptake mechanisms in plants and, 104 Dexamethasone, glycosylation in intestinal epithelium and, 183 Diacylglycerol, calcification and, 221 Dicalcium phosphate dihydrate, calcification and, 200 2,4-Dichlorophenoxyaceticacid, uptake mechanisms in plants and, 110 Differentiation, glycosylation in intestinal epithelium and, 185-187 Diffusion, uptake mechanisms in plants and, 89, 101,124-125 Dihydrotachysterol, calcification and, 224 Dihydrotestosterone, calcification and, 22 I , 230-23 1 2,4-Dinitrophenol, uptake mechanisms in plants and, 1 1 1 Discontinuous models, uptake mechanisms in plants and, 125-126 Dixon plot, uptake mechanisms in plants and, 99 DNA calcification and cells of bone, 2 11 endocrine factors, 220,222.23 1 pharmacological regulation, 249,260-261 physiological regulation, 234,236, 238-239,241-243 cereal aleurone and, 57,63-65,68,74 DNase, cereal aleurone and, 62 Dolichos biflorus lectin (DBL), intestinal epithelium and, 158 Dual and diffusion model, uptake mechanisms in plantsand, 124-125

E Eadie-Hofstee plot, uptake mechanisms in plants and, 93-95 EDTA, cereal aleurone and, 79 Elasmobranchs, melanin-concentrating hormone and, 9.20 Electron microscopy

297

cereal aleurone and, 72 glycosylation in intestinal epithelium and, 137, 148, 151,159,168, 176 Electrophoresis cereal aleurone and, 54 melanin-concentrating hormone and, 20 Emetine, glycosylation in intestinal epithelium and, 185 Endocrine factors, calcification and, 217-219 calcitonin, 222-223 growth hormone, 232-233 parathyroid hormone, 217,219-222 steroid hormones, 227-232 vitamin A, 226-227 vitamin D, 223-226 Endocytosis, calcification and, 217 Endoplasmic reticulum calcification and, 217 cereal aleurone and, 70.73-75.8042.84 glycosylation in intestinal epithelium and, 143, 145, 147 melanin-concentrating hormone and, 42 Endosperm, cereal aleurone and, 79.82 aleurone tissue, 49,5 1 a-amylase genes, 54-55 Energy, uptake mechanisms in plants and, 89,111 Enterocytes, glycosylation in intestinal epithelium and, 137, 157 Environment melanin-concentrating hormone and, 30 uptake mechanisms in plants and, 89 Enzymes calcification and, 197 biochemistry, 206-207 cells of bone, 212,217 inhibitiors, 247-249 pharmacological regulation, 252,255,261 physiological regulation, 241-242 cereal aleurone and, 49.80.83 aleurone tissue, 5 1.53 a-amylase genes, 54,61 protein transport, 70-72.74-75.77-78 melanin-concentrating hormone and, 20 uptake mechanisms in plants and, 123 Epidermal cells, uptake mechanisms in plants and, 110 Epidermal growth factor, calcification and, 221, 226,236-239-242.245 Epithelium calcification and, 237

298

INDEX

glycosylation in, see Glycosylation in intestinal epithelium Epitopes glycosylation in intestinal epithelium and. 177-1 78 melanin-concentratinghormone and, 8, 14, 18-19 Ervthrrno cristugalli lectin (ECL), intestinal epithelium and, 159 Escherichia coli

cereal aleurone and, 74 glycosylation in intestinal epithelium and, I78 uptake mechanisms in plants and, 117 Estradiol, calcification and, 228,230 Estrogen. calcification and cells of bone. 212 endocrine factors, 223.225.227-231,233 pharmacological regulation, 249-250,254 physiological regulation. 236.245 Evolution melanin-concentratinghormone and. I , 14, 29,34.41-43 uptake mechanisms in plants and, 89 Extracellular matrix. calcification and endocrine factors, 2 17,224 physiological regulation, 235,237,239, 242-243

F Fibroblast growth factor, calcification and, 236, 238-240,242-245 Fibroblasts,calcification and, 203.2 10,220, 224.236.240 Fibronectin. calcification and, 237 Fish, see also Teleosts melanin-concentratinghormone and, 1-2.43 biosynthesis. 20-2 1 color-change hormone, 3 6 3 4 stress respone, 35-40 structure, 14, 18 Flavanoids. calcification and, 254 Fluorescence cereal aleurone and. 8 1 glycosylation in intestinal epithelium and, 158- I59 melanin-concentratinghormone and, 5 Fluoride, calcification and, 200.251, 259-260 Fucose, intestinal epithelium and, 143, 175, 188 distribution, 155-157. 161, 163 exogenous agents, I X3-I85

Fucosyltransferase, intestinal epithelium and, 151, 157, 161,165. 185 G G-binding protein, calcification and, 219 Galactose, intestinal epithelium and, 143, 147 distribution of glycosyltransferases. 1 5 6 157 Golgi apparatus, 174-176 investigation methods, 153-154 Gdactosyltransferase. intestinal epithelium and, I45 distribution, 151-152, 154 exogenous agents, 183, 185 Golgi apparatus, 165, 168 post-Golgi apparatus distribution, 175-177, 182 Gallium, calcification and, 261 Genes, cereal aleurone and differential expression, 6 0 6 2 mechanism of hormone action, 64-68 multigene family, 55-59 synthesis. 54-55 Genetics, calcification and, 198-199 Genomes, cereal aleurone and, 56-57.59.66 Genotype, cereal aleurone and, 54 Gibberellic acid, cereal aleurone and, 49.5 I , 83 a-amylase genes, 5 4 5 5 , 5 9 4 2 calcium, 79.81-83 mechanism of hormone action, 6 3 4 , 6 6 7 0 protein transport, 7 1-72 Gibberellic acid-responsive element, cereal aleurone and. 64-65.67 Glucagon, melanin-concentratinghormone and. 8 Glucocorticoids, calcification and, 225,227, 235-236.254.258 Glucose intestinal epithelium and, 142-143, 174-175, 186 uptake mechanisms in plants and, 94-95, 108 Glucose-&phosphate dehydrogenase, calcification and, 21 3 Glutamic acid, calcification and, 202 Glycogen, calcification and, 209,228 Glycolipids, intestinal epithelium and, 148, 157, 185

Glycopeptides, intestinal epithelium and, 156, 186 Glycoprotein calcification and, 201-203,206,219,237

INDEX cereal aleurone and, 78 glycosylation in intestinal epithelium and, 135,139,141 distribution, 151, 153, 155-157 exogenous agents, 185 Golgi apparatus, 173 investigation methods, 148 oligosaccharides, 143, 147 post-Golgi apparatus distribution, 177, 181 Glycosaminoglycans,calcification and, 257 Glycosylation calcification and, 240 cereal aleurone and, 78 Glycosylation in intestinal epithelium, 135-141, 187-1 88 differentiation, 185-187 distribution of gl ycosyltransferases adult animals, 151-154 development, 154-155 lectin binding, 157-166 sugar content, 155-157 drugs, 184-185 Golgi apparatus, 165 lectin-binding sites, 173-175 localization, 168-173 subcompartmentalization,165, 167-1 68 hormones, 183-184 investigation methods, 147 biochemical, 148 morphological, 148-15 1 oligosaccharides N-glycosidically linked, 141-143 0-glycosidically linked, 143-147 post-Golgi apparatus distribution, 175-182 Glyoxsomes, cereal aleurone and, 78 Goblet cells, glycosylation and, 137-138, 182 distribution, 154-155,158-159, 161 Golgi apparatus, 168-169,17&175 ol igosaccharides, 145- 146 Gold, calcification and, 261 Gold sodium thiomalate, calcification and, 261 Golgi apparatus calcification and, 198,209,217 cereal aleurone and, 7&71 glycosylation in intestinal epithelium and, 137 distribution, 155, 158-159 exogenous agents, 183-1 85 investigation methods, 147-148.15 1 lectin-binding sites, 173-2 75 localization, 168-173 oligosaccharides, 141, 143, 145-147 post-Golgi apparatus, 175-182

299

subcompartmentalization,165,167-1 68 Granculocyte colony-formingcells, calcification and, 215 Granculocyte-macrophage-colonyforming cells, calcification and, 215 Granculocyte-macrophagecolony-stimulating factor, calcification and, 21 1,221,245-246 Growth factors, calcification and, 262 pharmacologicalregulation, 259 physiological regulation, 217,220,222,226 steroid hormones, 229-230 Growth hormone, calcification and, 227,230, 232-233,235

H Helixpomatia, intestinal epithelium and, 145-146 Helixpomatia lectin (HPL), intestinal epithelium and, 159 Hemopoietic cells, calcificationand, 213,215 Heparin, calcificationand, 242,257-258 Histamine, calcification and, 256 Homology cereal aleurone and, 56,59,73 melanin-concentratinghormone and, 1, 18, 20,4142 Hormone-responsiveelements,cereal aleurone and, 59.62 Hormones, see also specific hormone calcification and, 199,217,253,258,262 cereal aleurone and, 49,5 1,82-83 genes, 64-68 perception, 63-64 steroid model, 68-70 glycosylation in intestinal epithelium and, 183-184 Human growth hormone-releasingfactor, melanin-concentratinghormone and, 13-14,18-19 Hydroxyapatite,calcification and, 197-198.262 biochemistry, 199-200,202-205 pharmacologicalregulation, 260 Hypercalcemia,calcification and, 221,257-258, 26 1 Hypertrophy,calcification and, 234,239 Hypocalcemia,calcification and, 223,253, 256-258 Hypothalamo-pituitary-interrenalaxis, melaninconcentrating hormone and, 35-37 Hypothalamus,melanin-concentratinghormone

300

INDEX

and, 2-3,40 anatomical distribution. 3-5.7-9, biosynthesis, 2 C 2 1 structure, 15, 20

1I , 13

I Imidazole. calcification and, 255 Immune modulators, calcification and, 250 Immune system, calcification and, 255 Immunocytochemical localization, glycosylation in intestinal epithelium and, 147, 151, 175, 177, 182 Golgi apparatus, 167-173 Immunocytochemistry cereal aleurone and. 70 glycosylation in intestinal epithelium and. 148, 1.53, 165, 175, 181 melanin-concentrating hormone and, 3-4.8, I1 Immunofluorescence, gl ycosylation in intestinal epithelium and, 168, 177 Immunoglobulin, glycosylation in intestinal epithelium and, 176 Immunoreactive melanin-concentrating hormone, 3.9. 1 1. 14, 39 Immunoreactive a-melonocyte-stimulating hormone, 8 Immunoreactivity glycosylation in intestinal epithelium and. 146,167, 176 melanin-concentrating hormone and, 8.21 lndoleacetic acid. uptake mechanisms in plants and, 110 Inflammatory diseases, calcification and, 244-246 Inhibition calcification and, I97 biochemistry, 201,204-206,210-212,215 cytokines, 245-246 endocrine factors, 217,219,221-226 enzymes, 247-249 pharmacological regulation, 250-261 physiological regulation. 235-239, 241-243 steroid hormones, 228-229,23 I cereal aleurone and, 53,55.61,64,71 glycosylation in intestinal epithelium and. 177, 185 melanin-concentrating hormone and, 7,34

uptake mechanisms in plants and, 96-99, 107, 111, 124 Insulin, calcification and, 240 Insulinlke growth factors, calcification and endocrine factors, 220,225-226,229-230, 232 physiological regulation, 233-236.243 Integral membrane proteins, intestinal epithelium and, 175 Interleukin- 1, calcification and endocrine factors, 221,230,238-239 physiological regulation, 240,242,244-248 Intestinal epithelium, glycosylation in, see Glycosylation in intestinal epithelium Invertebrates, melanin-concentrating hormone and, 14 Ion-carrier complex, uptake mechanisms in plants and, 125 Ion channels, uptake mechanisms in plants and, 125 Ion flux effectors, calcification and, 25 1 Ion transport, calcification and, 199 3-Isobutyl-methylxanthine(IBMX), calcification and, 248 Isoelectric focusing, cereal aleurone and, 55 Isoelectric point (PI), cereal aleurone and a-amylase genes, 55,57,59-60,62 mechanism of hormone action, 65-66 protein transport, 76-77 Isoforms, cereal aleurone and a-amylase genes, 55-56,58,6&62 calcium, 79-82 protein transport, 7 1,7&77 Isotherm, uptake mechanisms in plants and, 122-127, 130-131 dual model, 91-92 multipha.icmodel.93, 113-114, 116-121 amino acids, 105-106 benzyladenine, 110 inorganic ions, 102, 104

K Kidney, calcification and, 217.221-222.227

K,, uptake mechanisms in plants and, 91,93, 111-114, 126-127

L Lacuna. calcification and, 212,215-217

NDEX Lectin, glycosylation in intestinal epithelium and, 145 distribution of glycosyltransferases. 155-1 63 exogenous agents, 183-184 Golgi apparatus, 165,167,172-175 investigation methods, 148, 150, 153 Leucine, uptake mechanisms in plants and, 106-107 Levamisole, calcification and, 255 Leydig cells, calcification and, 227,257 Light microscopy, glycosylation in intestinal epithelium and, 159, 176 Limaxflavus lectin (LFL), intestinal epithelium and, 161,163, 165, 167, 172-173 Lineweaver-Burk plot, uptake mechanisms in plants and, 93-95,99, ilU, 128 amino acids, 107 inorganic ions, 102-104 Lipid intestinal epithelium and, 135, 139, 141, 143 uptake mechanisms in plants and, 127 Localization, glycosylation in intestinal epitheliumand, 147, 151, 175, 177, 182 Golgi apparatus, 167-173 Lotus rerragonolobus lectin (LTL), intestinal epithelium and, 158-159, 161 Lupinus polyphyllus, uptake mechanisms in plants and, 110 Luteinizing hormone, melanin-concentrating hormone and, 40 Lysine, uptake mechanisms in plants and, 105-107 Lysosomes, cereal aleurone and, 84

M Maackia amurensis lectin (MAL), intestinal epithelium and, 161 Macrophage colony-forming cells, calcification and, 215,252 Macrophage-colony stimulating factor, calcification and, 244 Macrophages, calcification and, 21 1,215,221, 245-246.256 Mammals, melanin-concentrating hormone and, 3,23,35,42 anatomical distribution, 8, 11-14 structure, 14-15, 18-20 Mannose glycosylation in intestinal epithelium and,

30 1

157, 165 intestinal epithelium and, 139, 141-143, 155, 186-1 87 Mannose-&phosphate receptor, calcification and, 216 Mapping, melanin-concentrating hormone and, 11

Matrix vesicle, calcification and, 197,205-206, 224-225 Melanin-concentrating hormone, 1,4@43 anatomical distribution, 3.8 in amphibians, 9-1 I in cyclostomes, 8-9 in elasmobranchs, 9 in invertebrates, 14 in mammals, 11-14 in teleost fish, 3-8 biosynthesis, 20-21 discovery, 1-3 physiology color-change hormone in fish, 30-34 higher vertebrates, 3 9 4 0 stress-response in fish, 35-39 precursor molecule of mammalian MCH, 18-19 of teleost MCH, 15-18 structure, 1 4 1 5 , 2 0 structure-activity studies, 2 1-23 melanin-concentrating activity, 25-30 aMSH-like activity, 23-24 Melanin-concentrating hormone-gene-related peptide, 15, 18,21 Melanocyte-stimulating hormone, 2-3 a-Melanocyte-stimulating hormone, 43 distribution, 8-9, 13-14 physiology, 3 I-36,40 structure, 18-20 structure-activity studies, 23-24,30 Melanophores, 3, 15 physiology, 30-33,40 structure-activity studies, 23-26 Mesenchyme, calcification and, 209.238-239, 24 1 Messenger RNA calcification and biochemistry, 202,204-205 endocrine factors, 220,225-230 physiological regulation, 235,238 cereal aleurone and, 73,83 a-amylase genes, 5455,57,60-62 mechanism of hormone action, 68-69

302

INDEX

melanin-concentratinghormone and, 15,20

MET.melanin-concentratinghormone and, 15 Metalloproteins. cereal aleurone and, 79-80 Michaelis-Menten kinetics, uptake mechanisms in plants and, 90. I3 1 dual model, 92 multiphasic model, 93.95,99. 101, 104 Microtubules, glycosylation in intestinal epithelium and. 184-185 Milrinone. calcification and, 248 Mineralization. calcification and. 198-199, 262-263 biochemistry. 200-207 cells of bone, 209-21 2 epidermal growth factor, 241-242 pharmacological regulation, 25 I. 260 physiological regulation. 223-224.226-228, 230-233 transforming growth factors. 237-238 Mitogen, calcification and, 220,237,243 Monensin, cereal aleurone and, 70-7 I Monocytes,calcification and, 215,230,244245 Morphogenesis,calcification and. 200,236 Morphology calcification and, 209,237 glycosylation in intestinal epithelium and, 147-151, I85 melanin-concentratinghormone and, 13 Mucin. intestinal epithelium and, 146, 153. 182 Multienzyme system. intestinal epithelium and. 165 Multiphasic uptake mechanisms in plants, see Uptake mechanisms in plants Mutagenesis. cereal aleurone and, 65 Mutation, melanin-concentratinghormone and, 14. 18

N NEI. melanin-concentratinghormone and, 18-20,4 1 Neurohypophysis,melaninconcentrating hormone and, 7-9.20.39 Neuromodulators,melanin-concentrating hormone and, 5.7.37.39 Neuropeptides,see Melaninconcentrating hormone Neurotransmitters,melanin-concentrating hormone and, 5.3 I , 39

NGE, melanin-concentratinghormone and, 18-19.41 Nitrate, uptake mechanisms in plants and, 1w105 Nocodazoie, glycosylation in intestinal epithelium and, I85 Noncollagenousproteins, calcification and, 197, 201-207.262 Nonsteroidal antiinflammatorydrugs (NSAIDS), calcification and, 249 Nucleation theory. calcification and, 196-197 Nucleotide pyrophosphate, intestinal epithelium and, 151-152, 176-177 Nucleotides cereal aleurone and, 57 glycosylation in intestinal epithelium and, 141, 182 Nucleus lateralis tuberis, melanin-concentrating hormone and, 4-5.9 Nutrition, cereal aleurone and, 5 I

0 Oats. uptake mechanisms in plants and, 114, 116 Oligosaccharides cereal aleurone and, 78 glycosylation in intestinal epithelium and differentiation, 186-1 87 Golgi apparatus. 167, 171 OnchorhJnchus.melanin-concentrating hormone and, 14-15.20 Osteoblasts, calcification and, 209-2 12,262 biochemistry, 199,204 endocrine factors, 2 17,220-228.230-232 pharmacologicalregulation, 247-25 1.258, 260 physiological regulation, 234,236-239, 24 1-246 Osteocalcin, calcification and biochemistry. 204-205 cells of bone, 2 I2 endocrine factors, 217,220,224,227 pharmacologicalregulation, 250-25 I physiological regulation, 237,243,250-251 Osteocalcium phosphate, calcification and, 197, 200 Osteoclasts. calcification and, 2 10-2 17,262 biochemistry, 199,204

INDEX endocrine factors, 220-221.225-226 organic agents, 252,255,257-258 pharmacologicalregulation, 247-248,250,

260 physiological regulation, 237.241-242,

244-246

303

Peptides calcificationand, 203-204 cereal aleurone and, 73,78 glycosylation in intestinal epithelium and,

139,141,145-146,181 melanin-concentratinghormone and, 15, 18,

Osteocytes, calcification and, 209.212-213.223 Osteogenesis, calcification and, 197.211,241 Osteogenin, calcification and, 239-240 Osteoinductive factor, calcification and,

239-240 Osteomalacia, calcification and, 260 Osteonectin, calcification and, 203,217,237 Osteopenia, calcification and, 210,250,254,

257,259 Osteopetrosis, calcification and, 213,215 Osteopontin, calcification and, 201,203,237,

243,249 Osteoporosis, calcification and, 195 pharmacologicalregulation, 253,257,259,

261 physiological regulation, 222,227,230-231 Osteoprogenitors,calcification and, 209,

223-224.242 Osteosarcomacells, calcification and, 210 biochemistry,203,205 endocrine factors, 221,225-231 pharmacologicalregulation, 248,251 physiological regulation, 233-234.238, 240-24 I , 243 Oxalates, calcification and, 198

P Palindromes, cereal aleurone and, 654% Pancreas, cereal aleurone and, 73 Pancreatica-amylase, cereal aleurone and, 77 Parathyroid hormone, calcification and biochemistry, 204 cellsofbone,2lO,212,215-216 endocrine factors, 217-222.226-229.231 inorganic effectors, 260,262 pharmacologicalregulation, 247-248.251,

21.40-43 Perichondrium,calcification and, 227 Periosteal cells, calcification and, 21 1,

227.237 pH, uptake mechanisms in plants and, 95-96, 110

Phagocytes, calcification and, 215 Pharmacologicalregulation, calcification and,

246-25 1,263 inorganic effectors,259-262 organic agents, 252-258 Phenotype, calcification and, 210,212 pharmacologicalregulation, 252 physiological regulation, 238,240,243,246 Phosphate calcification and, 197-198 biochemistry,205,207 pharmacologicalregulation, 248,252 physiological regulation, 217,223 uptake mechanisms in plants and, 124-126 multiphasic model, 95-97.99,102,113,122 Phosphatidylserine,calcification and, 205,223 Phosphodiesterase,calcification and, 21 1,255 inhibitors, 247-248 PhospholipaseA,, calcification and, 225,244 Phospholipase C, calcification and, 221 Phospholipids,calcification and, 205,223 Phosphoprotein,calcification and, 201-202,217 Phosphorus calcification and, 224,255,261 uptake mechanisms in plants and, 122 Phosphorylation,calcification and, 202-203,

206,259

Pituitary calcification and, 227,235-236 melanin-concentratinghormone and, 1-3,43 anatomical dismbution, 3-5,7-9,I I , 13 biosynthesis, 20 physiology, 30,32-37,3940 254,256-258 structure, 14 physiological regulation,238,240,242-243.245 Plants, uptake mechanisms in, see Uptake Parathyroid hormone-relatedpeptide, mechanisms in plants calcification and, 221-222,239,245 Plasma, melanin-concentratinghormone and, Peanut lectin (PNL), intestinal epithelium and,

156,159,184

31-32,36,3840

304

INDEX

Plasma membrane calcificationand. 197. 213. 219. 224 cereal aleurone and, 68.70-7 I. 82-83 glycosylation and, 135. 137. 147-148 differentiation, I86 distribution of glycosyltransferases. 15 I. 155-156 exogenous agents, 184-1 85 Golgi apparatus. 173 lectin binding, 158-159, 161. 163, 165 post-Golgi apparatus distribution. 175-176. 178-179, 182 uptake mechanisms in plants and, 89 Plasmalemma,uptake mechanisms in plants and, 89-90. 130- 1 3 1 molecular basis, 127 multiphasicmodel. 110, 114. 116, 118-120 Plasmids. cereal aleurone and. 77 Plasminogen activator, calcification and. 2 12. 238.24X. 258 Plastids, uptake mechanisms in plants and, 90, 99. 118. 125 Platelet-derivedgrowth factor. calcification and, 240,243 foecilia. melanin-concentratinghormone and, 2. 5.7.33 Polyamine, glycosylation in intestinal epithelium and. 185 Polypeptides calcification and. 200.2 19 cereal aleurone and, 54.56.78-79 glycosylation in intestinal epithelium and, 143. 145. 147. 165, 177-178 POMC. melanin-concentratinghormone and, 8. 13.20,35 Porcine pancreatic amylase. cereal aleurone and. 79 Posttranslational modifications. cereal aleurone and, 55. 7 6 7 1 Potassium calcification and, 2 16.25 I. 260 uptake mechanisms in plants and, 90, 124, 130-131 dual model, 9 I multiphasicmodel. 102, I l l . 114-116. 118-119,121 Pre-prohormonemelanin-concentrating hormone. IS, 18-19.41 Preosteoblasts. calcification and, 209 Procollagen. calcification and, 226-228

Proenzymes, cereal aleurone and, 78-79 Progesterone,calcification and, 228,230 Prohormone melanin-concentratinghormone, 4 1 anatomical distribution, 8 biosynthesis, 2 I structure, 20 Prohormones, melanin-concentratinghormone and, 15, 18.4W1 Prolactin, melaninconcentratinghormone and, 40 Promethazine, calcification and, 255-256 Prostaglandin,calcification and cellsofbone,211-213 endocrine factors, 220,229 pharmacological regulation, 247.25 I , 254 physiological regulation, 238,241-245 synthesis inhibitors, 248-249,255 Proteases, cereal aleurone and. 74.78,80,84 Protein, see also specific protein calcification and, 197,262 biochemistry, 200-207 endocrine factors, 226,23@-232 pharmacological regulation, 259-262 physiological regulation, 234,236,243 glycosylation in intestinal epithelium and, 135, 139. 147-148, 185 distribution of glycosyltransferases, 154, 156157, 172 post-Golgi apparatus distribution, 175, 177-178, 182 melanin-concentratinghormone and, 20-21, 4142 regulation in cereal aleurone. see Cereal aleurone uptake mechanisms in plants and, 1 10, 126127 Protein kinase C , calcification and. 221,241 Protein-A gold technique. glycosylation in intestinal epithelium and. 149, 178 Proteoglycans,calcification and biochemistry, 201-202 endocrine factors, 2 17,223,226,228.23 I physiological regulation, 235-236.238-242, 245-246 Proteolipids,calcification and, 199,205 Proteolysis cereal aleurone and, 74,80 melanin-concentratinghormone and, 18 Prothrombin,calcification and, 204 Protoplasts cereal aleurone and. 6345,72,76,81

305

INDEX uptake mechanisms in plants and, 89-90, 124-125 multiphasic model, 107, 110, 118 Pseudo-dual model, uptake mechanisms in plants and, 125 Pyrimidine, cereal aleurone and, 65-68 Pyrophosphate calcification and, 217,252-253 uptake mechanisms in plants and, 96

R Radioimmunoassays,melanin-concentrating hormone and, 3.1 1,39 Rana, melanin-concentratinghormone and, 24 Rana pipiens. melanin-concentratinghormone and, 23 Rana ridibunda, melanin-concentrating hormone and, 9.20.39 Rana temporaria, melanin-concentrating hormone and, 11 Rat growth hormone-releasingfactor, melaninconcentrating hormone and, 19 Rat melanin-concentratinghormone, 11 Rat osteosarcoma cells, calcification and, 226-221,229,238 Recombination,cereal aleurone and, 77 Retinoic acid, calcification and, 225-227 Retinoids, calcification and, 226-227.232 Retinol, calcification and, 226-227 Rheumatoid arthritis, calcification and, 195, 244,26 1 Rice, uptake mechanisms in plants and, 122 Ricinus communis lectin I (RCL I), glycosylation in intestinal epithelium and, 156,158-159,161,168,172 Ricinus communis lectin I1 (RCL 11). glycosylation in intestinal epithelium and, 159 RNA calcification and, 213 cereal aleurone and, 60,63 melanin-concentratinghormone and, 42 RNA polymerase, cereal aleurone and, 64.68 Rough endoplasmic reticulum, glycosylation in intestinal epithelium and, 141, 143, 145-146 Rubidium, uptake mechanisms in plants and, 90, 124

S Saccharide, glycosylation in intestinal epithelium and, 140 distribution, 151-157 Golgi apparatus, 165-175 lectin binding, 157-166 post-Golgi apparatus distribution, 175-182 Salivary a-amylase, cereal aleurone and, 77 Salmon, melanin-concentratinghormone and, 3-4, 14-15.18 Salmonid melanin-concentratinghormone, 40, 42 anatomical distribution,4, 11 physiology, 3 9 4 0 structure, 14-15 structure-activity studies, 23.25-28 Sambucus nigra L. lectin (SNL I), intestinal epitheliumand, 161, 163,173 Scutellum,cereal aleurone and, 49.5 1,54,71, 78 Second messengers, cereal aleurone and, 83 Secreted proteins in cereal aleurone, see Cereal aleurone Selective pressures, melanin-concentrating hormone and, 4 1 4 3 Sequences calcification and, 204 cereal aleurone and a-amylase genes, 56-57,59,62 mechanism of hormone action, 64-68 melanin-concentratinghormone and, 3.4 1 4 2 structure, 14-20 structure-activity studies, 23,26,29 Sialic acid calcification and, 202 intestinal epithelium and, 143, 147, 176, 183 distribution, 156-157, 159, 161, 163, 165 Golgi apparatus, 168-169, 171 Sialyltransferase,intestinal epithelium and distribution, 151-154, 161 exogenous agents, 183-184 Golgi apparatus, 165,167-169,171,173 post-Golgi apparatus distribution, 175-178, 181-182 Signal transduction cereal aleurone and, 83 melanin-concentratinghormone and, 25 Skeleton, vertebrate, calcification in, see Calcification

306

LNDEX

Sodium calcification and, 216,251,253,260 uptake mechanisms in plants and, 91, 102. 113. 119 Soybean lectin (SBL),intestinal epithelium and, 156,158-159 Spinal cord, melanin-concentratinghormone and, 9. I I Spleen, calcification and, 2 I3,2 15 Starch, cereal aleurone and, 5 I , 54,79,82 Steroids calcification and, 221.225,227-232 cereal aleurone and, 65,68-70 Stress, melanin-concentratinghormone and, 3540 Structure-activity studies, melaninconcentrating hormone and, 2 1-30 Subcompanmentalization,glycosylation in intestinal epithelium and. 165, 167-168, 171-172, 177 Sucrose. uptake mechanisms in plants and, 110 sugar glycosylation in intestinal epithelium and, 135. 137,139. 141,143, 148 distribution of glycosyltransferases, 153, 155-157, 163 Golgi apparatus, 167, 173 uptake mechanisms in plants and, 108, I10 Sulfate calcificationand, 198,201.226 uptake mechanisms in plants and, 93,95-98, 112-113,121 aminoacids, 1 W 1 0 7 inorganic ions, 99-100 Synbranchrcr, melanin-concentratinghormone and. 23-24.26.30

T Tamoxifen, calcification and, 249 Teleosts. melanin-concentratinghormone and, 1-2.42-43 anatomical distribution, 3-8 physiology. 30.33-34.39-40 structure. 14-18 structure-activity studies, 23-25.30 Testosterone calcification and. 221,223,227.230-232 glycosylation in intestinal epithelium and, 183 Theophylline,calcification and, 247-248

Thiamine pyrophosphatase,intestinal epithelium and, 167 Thiazide diuretics, calcification and, 253-254 Thiophene carboxylic acids, calcification and, 256-257 TIMP, calcification and, 238 Tonoplasts, uptake mechanisms in plants and, 119-120,131 Transcription calcification and. 224,261 cereal aleurone and a-amylase genes, 55.61-62 mechanism of hormone action, 63-65, 67-69 Transforming growth factor-a, calcification and, 241-242,245 Transforming growth factor+, calcification and biochemistry, 204 cells of bone, 21 I endocrine factors, 220,222,226,230 physiological regulation, 236241,245 Transforming growth factors, calcification and, 236239 Trans-Golgi network, intestinal epithelium and, 145, 168 Translation, melanin-concentratinghormone and, 42 Translocation cereal aleurone and, 73-74 melanin-concentratinghormone and, 2 1 Tropocollagen,calcification and, 200 Tumor necrosis factor, calcification and, 241, 245-246

U Ulex europaeus lectin I (UEL I), intestinal epitheliumand. 157-159, 163, 184 Uptake mechanisms in plants, 89-91. 122-123. 128, 130-131 cooperative model, 123 discontinuous models, 125-1 26 dual and diffusion model, 124-125 dual model, 9 1-92 molecular basis, 126-129 carriers, 126-1 27 channels, 126-127 transition site, 127 multiphasic model. 1 18-1 22, 128, 130 amino acids, 105-109

INDEX initial experiments, 93-95 inorganic ions, 99-105 K,, 112-1 15 potassium, 114-1 16 solute concentrations, 110-1 12 sugars, 108,110 transition sites, 95-99 transport in bacteria, 116-117 VmaX,112-115 pseudo-dual models, 125 single and diffusion model, 124

V Vacuoles calcification and, 2 13 cereal aleurone and, 5 I , 70,82 uptake mechanisms in plants and, 90, 120, 131 Vanadate, calcification and, 260 Vertebrate skeleton, calcification in, see Calcification Vertebrates, melanin-concentrating hormone and, see Melanin-concentrating hormone Vesicles calcification and, 197-198.205.217 glycosylationin intestinal epithelium and, 143, 145,159,179 Vitamin A, calcification and, 226-227 Vitamin D, calcification and biochemistry, 204

307

endocrine factors, 221,223-227 pharmacological regulation, 25 1,254,260, 262 physiological regulation, 236,238-240.245 Vitronectin, calcification and, 203,215 V,,,,,, uptake mechanisms in plants and, 101, 111-114,121,126-127

W Warfarin, calcificationand, 204 Wheat, cereal aleurone and, 49,5 1 a-amylase genes, 54 mechanism of hormone action, 6 3 , 6 5 4 6 Wheat germ agglutinin (WGA), glycosylation in intestinal epithelium and, 156,158-159, 161, 184 WR-2721,calcification and, 257

X Xenopus, melanin-concentrating hormone and, 2 Xenopus laevis cereal aleurone and, 57,74,77 melanin-concentrating hormone and, 11

Z

Zinc, calcificationand, 206,261-262


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