Vitamins and Hormones, Volume 62

Preface This volume of Vitamins and Hormones begins with a lengthy and timely review of the health benefits of green tea...

Author: Gerald Litwack

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Preface This volume of Vitamins and Hormones begins with a lengthy and timely review of the health benefits of green tea, entitled, "Green Tea: Biochemical and Biological Basis for Health Benefits," by S. Liao, Y.-H. Kao, and R. A. Hiipakka. A relatively new field is covered in the next contribution: "Proteins Regulating the Biosynthesis and Inactivation of Neuromodulatory Fatty Acid Amides" by M. P. Patricelli and B. F. Cravatt. This is followed by the review: "Three-Dimensional Organization of the Aquaporin Water Channel: What Can Structure Tell Us About Function?" by A. K. Mitra. Jolinda Traugh's laboratory then describes a protein kinase with cytostatic activity: "Cytostatic p21 G Protein-Activated Protein Kinase ~-PAK" by J. Roig and J. A. Traugh. The remainder of the volume is devoted to steroid hormones. D. J. Lamb, N. L. Weigel, and M. Marcelli discuss androgen receptors and their biology. S. Safe describes transcriptional activation of genes by 17~-estradiol through estrogen receptor-Spl interactions. Finally, in a new approach to make drugs more specific, J. N. Miner and C. M. Tyree review drug discovery and the intracellular receptor family. The Editorial Board provided some of the suggestions for authors in this volume and I appreciate their support. Academic Press continues to make the progress in this serial smooth, for which I express my appreciation. GERALDLITWACK

xi

VITAMINSANDHORMONES,VOL.62

Green Tea: Biochemical and Biological Basis for Health Benefits SHUTSUNG LIAO, YUNG-HSI KAO, AND RICHARD A. HIIPAKKA Tang Center for Herbal Medicine Research, Ben May Institute for Cancer Research, and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 I. Introduction II. Catechins and Other Constituents of Green Tea III. Structure, Pharmacology, and Metabolism of Catechins A. Structures, Isolation, and Analysis B. Pharmacodynamics of Green Tea Catechins IV. Chemical and Biochemical Properties of Catechins A. Antioxidant Activity B. Prooxidant Activity C. Protein-Binding Activity D. Ion-Chelating Activity V. Biological Activity of Green Tea Catechins A. Endocrine Effects B. Cancer C. Cardiovascular Disease and Hypertension D. Allergy, Asthma, Arthritis, and the Immune System E. Diabetes F. Obesity G. Oral Health H. Nervous System and Memory I. Osteoporosis J. Antibacterial Activity K. Antiviral Activity L. Dermatological Therapy VI. Molecular and Cellular Bases for Biological Effects of Catechins A. Importance of Catechin Structure and Bioavailability B. Modulation of Enzyme Activity C. Antimutagenic Activities of Tea Catechins D. Control of Cellular Activity by Catechins VII. Epilog References

Copyright© 2001by AcademicPress. All rights ofreproductionin any formreserved. 0083-6729/01 $35.00

2

S. LIAO, Y-H. KAO, AND R. A. HIIPAKKA I. INTRODUCTION

According to Zen Buddhist legend, the first patriarch, Bodhidharma, around the 6th century A.D., trying to keep himself awake, cut off his eyelids, which fell to the ground and grew as tea plants. Ever since, tea has been used to fend off sleep and clear the soul. Historians believe that tea became part of h u m a n culture about 5000 years ago in an area around the Yunnan plateau of southwestern China, in the state of Assam in northeastern India, as well as in Tibet. Tea trees more than 1000 years old and 100 feet tall can be found in these areas. At one time two species of tea plants, Camellia sinensis and Camellia assamica, were recognized by botanists. However, these plants are now considered different strains of Camellia sinensis (L) O. Kuntze. Commercial tea trees are trimmed often, and so they are small bushy plants about 3 to 4 feet high. Tea leaves are picked three to four times between spring and fall of each year. Green tea is produced from leaves that are picked and heated quickly, either in a pan or with hot steam, to stop enzymatic action and to prevent fermentation. Fermentation involves air oxidation and polymerization of tea components including polyphenolic catechins that are major constituents of tea leaves. Some tea products are fermented to enhance taste and flavor. Oolong tea, often served in Chinese restaurants, is partially fermented, whereas black or red teas are extensively fermented and are most often consumed in Western societies. In oriental cultures it has been widely believed for a long time that tea has medicinal efficacy in the prevention and treatment of many diseases, and so longevity is often associated with drinking tea. According to Chinese history, about 47 centuries ago, Emperor Sheng-Nong found that a daily cup of tea could dissolve many poisons in the body (Committee, 1991). Scientific and medical evaluation of tea, however, started only very recently. A literature survey based on PubMed shows a dramatic increase in the number of publications on green tea and catechins since 1995 (Fig. 1), reflecting increased research on the possible health benefits of green tea beverage. In recognition of their possible importance in vascular health, polyphenolic flavonoids, like tea catechins, were once called vitamin P (Rusznyak and Szent Gyorgyi, 1936). Green tea consumption may be linked to a lower incidence of various pathologies, including cancer, cardiovascular disease, diabetes, and obesity. The major green tea catechin, (-)-epigallocatechin 3-gallate (EGCG), has been the focus of much of the research by the scientific community because of its ability to mimic some of the biological effects of green tea. To determine possible molecular mechanisms for the

GREEN TEA: BIOCHEMICAL AND BIOLOGICAL BASIS FOR HEALTH BENEFITS

3

Green ~o_100" ~

:~

50-

EGGG 01960 1970 1980 1990 2(300 Year

FIG. 1. The yearly n u m b e r of publications related to green tea, catechins, and EGCG. The data were obtained from PubMed. The data for the year 2000 are projected from the n u m b e r available for the first 3 m o n t h s of 2000.

putative health benefits of green tea, pure green tea catechins have been used in numerous studies examining catechin effects on isolated enzyme systems and cells in culture. Unfortunately, the relevance of these in vitro studies to the possible in vivo effects of green tea consumption is very difficult to determine. In many of these studies, the predominantly inhibitory effects are seen only at high catechin concentrations that would be difficult to achieve in vivo. Many studies using whole cells also lack demonstration of a specific interaction ofcatechins with a defined cellular component that would explain possible biological effects in vivo. Many of the reported findings may not have relevance to the effect of tea in vivo. In this chapter, we focus on green tea and green tea catechins rather than on fermented teas or catechin by-products that are produced during fermentation. Theaflavins (Fig. 2) and thearubigins, a complex mixture of catechin condensation products with a heterogeneous molecular weight distribution, are major fermentation products in black tea, which, like catechins, have antioxidant and antitumorigenic activities (Shiraki et al., 1994; Yoshino et al., 1994). In addition to catechins, green tea contains vitamins, caffeine, and other phytochemicals that are medically important, but are not discussed in this chapter. Experimental studies on the physiological effects of some polyphenolic tannins from other plants indicate that they also may be beneficial for decreasing serum lipids, reducing blood pressure, and modulating immune responses and for use as antitumorigenic and antibacterial agents and use in food preservation (Chung et al., 1998). This chapter, however, does not cover health benefits of these tannins. Tea polyphenols are also widely used as natural antioxidants for prevention of oxidation of

4

s. LIAO,Y-H. KAO,AND R. A. HIIPAKK Gallyl:

.OH

OH

o2 :: ~"~,~OR

2

OH

R1

R2

(-)Epicatechln (EC) H H (.)Epigallocatechin (EGC) OH H (.)Epicatechin-3-gallate (ECG) H G (-)Epigallocatechin-3-gallate (EGCG) OH G (-)Gallocatechin-3-gallate (GCG)* OH G * Ring-B is attached to C-2 in the tran$ position In respect to the attachmentof the gallatesubstitutionat C-3. OH

oR2

HO~ / L " ~ O / ~

HO

.-,. ~O,

"F

.///

~

"'oR, OH

o OH

"OH

C'NH'C2H5 ~H2 I

CH2 cooH

OH Theaflavins (R1 , and R2 , can be H or a galloyl group)

Theanine (7-glutamylethylamine)

FIG. 2. Structures of catechins and theanine in green tea and theaflavins in fermented tea.

edible oils or discoloring of foods (Chen and Ho, 1995), but we do not cover this aspect of tea catechin usage.

II. CATECHINS AND OTHER CONSTITUENTS OF GREEN TEA

Green tea leaves are unique in t h a t they are very rich in catechins, caffeine, and theanine. These constituents are soluble in hot water and impart flavor and taste to green tea beverages. The amounts of these compounds in tea vary considerably depending on the strain of tea plant, light and soil conditions for cultivation of tea (Hilton, 1974;

GREEN TEA:BIOCHEMICALAND BIOLOGICALBASISFOR HEALTHBENEFITS

5

Graham, 1992; Ruan et al., 1999), and the processing method used for making tea products (Lin et al., 1996). Fermentation often moderates tea bitterness due to catechins by oxidative conversion or polymerization of catechins, thereby improving tea taste and flavor. Dry green tea contains as much as 15 to 20% catechins, 2 to 3% caffeine, and about 1 to 6% amino acids. Green tea has about 600 mg vitamin C and 80 mg vitamin E per 100 g of freshly dried leaves. Ordinary green tea beverage is made with about 2 to 4 g of tea leaves and about 100 ml to 200 ml of hot water. Most of these green tea constituents are soluble in hot water and are major components in tea beverage. The concentration of caffeine in tea beverage is about one-third of that in ordinary brewed coffee. The aromatic flavor of green tea has been attributed to a number of alcohols (pentenols, hexenols, amyl alcohol, and benzylalcohol), theaspirane and hexenoate derivatives, and sulfur compounds (dimethylsulfide, methylmercaptan, sulfonic acid ester, and sulfonium salts). Some of these compounds are present in green tea as glycosides that are known as aroma precursors, which are enzymatically hydrolyzed during fermentation to alter tea aroma and flavor (Guo et al., 1995; Nishitani et al., 1996). Inorganic substances that are relatively high in tea leaves are potassium, aluminum, manganese, and fluorine. The fluorine content of tea is 100 to 300 ppm, which is higher than the level found in most other plants. Each cup of tea beverage can provide 0.5 to 0.8 mg of fluorine, approximately one-fifth of the daily flourine requirement for humans. About 50% of the amino acid content in green tea is theanine (T-glutamylethylamine; Fig. 2), which imparts a pleasantly sweet taste to tea. Theanine is degraded to glutamic acid (Terashima et al., 1999) and has been shown to have a relaxation effect in humans (Juneja et al., 1999). Theanine can elicit a neurochemical effect; it can alter the metabolism of brain monoamines or the striatal release of dopamine (Yokogoshi et al., 1998a). Theanine and its derivative, T-glutamylmethyl amide, can also reduce blood pressure in spontaneously hypertensive rats (Yokogoshi et al., 1998b).

III.

STRUCTURE,PHARMACOLOGY,AND METABOLISMOF CATECHINS

A. STRUCTURES, ISOLATION,AND ANALYSIS

More than 80% of green tea polyphenols are catechins, which are derivatives of flavan-3-ol. Catechins (3,3',4',5,7-pentahydroxyflavan)

6

s. LIAO, Y-H. KAO, AND R. A. HIIPAKKA

contain two asymmetric carbon atoms at C2 and C3 (Fig. 2). The major catechins in green tea are (+)-catechin (CA), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG), and (-)-epigallocatechin gallate (EGCG). GCG may be formed by epimerization of EGCG during extractions with solvents at high temperatures (Roberts and Wood, 1953). EGCG is the most abundant catechin in green tea and constitutes 25 to 40% of the total catechin content. Catechins were isolated from green tea and their structures were determined during the 1930s (reviewed in Bradfield and Bate-Smith, 1950; Roberts, 1952; Tsujimura and Takasu, 1955). Catechins are the major solid matter extracted by hot water from tea leaves and constitute 70 to 80% of the solid matter of dried aqueous tea extracts. Caffeine also extracted by hot water from tea can be removed from catechins by chloroform extraction. Catechins in aqueous solution can be extracted by ethyl acetate and then separated by high-porosity polystyrene or Sephadex LH-20 chromatography (Liao and Hiipakka, 1995). A supercritical carbon dioxide extraction method can also be used to isolate catechins from green tea (Chang et al., 2000). The identity and purity of isolated catechins can be assessed by NMR, mass spectrum analysis, or high-performance liquid chromatography (HPLC) (Goto and Yoshida, 1999; Hollman et al., 1999). In tea leaves, EC is synthesized from flavanone, which is derived from phenylalanine and acetyl-CoA, while gallic acid is synthesized from shikimic acid. The biological function of catechins in tea plants is not clear and why tea leaves accumulate high concentrations of catechins is intriguing. The fact that shaded tea leaves have less catechins may indicate that catechins are used to protect leaves from excessive sun light. The bitter taste as well as antimicrobial and antioxidant properties ofcatechins may also protect tea leaves from insects, infections, and other environmental damage. Ironically, many of the health benefits of green tea for humans, as described in the following sections, are based on similar properties. Colorimetric methods based on phenolic structures can be used for catechin analysis (Das, 1971; He and Kies, 1994). A simple colorimetric method for quantitation of catechins that was developed recently is useful for measurement of total catechins levels in biological fluids. In this method, catechins are adsorbed onto alumina and treated with 4-methylaminocinnamaldehydeunder strong acidic conditions to give a colored product (Kivits et al., 1997). The detection sensitivity in human plasma is 50 nM. An improved HPLC method that uses coulometric array detection is suitable for quantitative analysis of various catechins (Lee et al., 2000). Using this technique, the detection limits for

GREEN TEA: BIOCHEMICALAND BIOLOGICAL BASIS FOR HEALTH BENEFITS

7

catechins in saliva, plasma, or urine are 10 to 20 nM. A very sensitive chemiluminescense detection method, when combined with HPLC, has a detection limit of 2 nM and is suitable for measuring plasma EGCG (Nakagawa and Miyazawa, 1997). The stability of green tea in aqueous solutions depends on a variety of factors, including pH, oxygen concentration, temperature, and ionic strength (Yoshino et al., 1999). All of the green tea catechins are stable in acidic solution at a pH from 1.8 to 6.4. EGC and EGCG are rapidly degraded at pH levels above 7.4, which is the pH of many body fluids. ECG is degraded at pH 11.2, but EC is stable between pH 1.8 and 11.2. When EGCG is incubated in plasma (pH 7.8) or mouse intestinal fluid (pH 8.5), 50% is degraded in less than 10 min. EGCG degradation follows a pseudo-first-order kinetic model (Zimeri and Tong, 1999). The rate constant for degradation increases linearly with both pH and oxygen concentration. The instability of EGCG may be related to the aromatic B ring of EGCG, since the ability to donate a hydrogen from the B ring is elevated in alkaline solutions and forms radicals easily (Yoshioka et al., 1991; Guo et al., 1999). The instability of EGCG in alkaline solution may mean that some of the effects of EGCG in vivo may be due to oxidized forms of EGCG (Yoshino et al., 1999). B.

PHARMACODYNAMICS OF GREEN TEA CATECHINS

Green tea catechins have numerous biological effects in vitro and generally effects are observed in the range of 10 to 100 ~tM; however, what is not clear at this time is whether pharmacologically effective doses of catechins can be attained in blood or tissues simply by consuming tea infusions. Green tea polyphenols taken orally at dosages equivalent to 5 to 10 cups of tea have few, if any, serious side effects. The median lethal dose of a green tea extract containing 85% EGCG and given orally to rats is about 3 to 5 g/kg (Yamane et al., 1996). No significant changes are observed in body weight or blood hematological and biochemical parameters when 15 or 75 mg/kg of a green tea extract is administered to rats orally for 28 days. Rats injected ip with EGCG at a dosage of 75 mg/kg, however, experience rapid weight loss due to decrease in food intake (Kao et al., 2000a). The bioavailability of green tea catechins depends on several factors, including catechin structure, purity, and dosage. For example, after consumption of 1.5 g of decaffinated green tea solids, the catechins in human plasma reach peak levels in 1.5 to 2.5 h. At this time, the plasma levels (free and conjugated) of EGCG, EGC, and EC levels are 0.26, 0.48, and 0.19 ~xM, respectively, while ECG is not detected

8

S. LIAO, Y-H. KAO, AND R. A. HIIPAKKA

(Yang et al., 1998a). The half-life in plasma is 5 h for EGCG and 3 h for EC and EGC. Most catechins in plasma are in the form of glucuronide and sulfate conjugates (Lee et al., 1995). Drinking one cup of tea every 2 h maintains the plasma level of free and conjugated catechin at about 0.8 to 1.0 ~M (Unno et al., 1996; van het Hof et al., 1999). Consumption of a single high dose of green tea, equivalent to six cups of tea, can raise plasma levels of catechins to 2 to 4 ~M in 60 min (Unno et al., 1996). After oral ingestion of an infusion of green tea containing 400 mg of a mixture of green tea catechins, the human plasma EGCG and ECG concentration reaches a maximum of approximately 2 ~M at 2 h (Pietta et al., 1998). A few minutes after two to three cups of green tea are consumed, the saliva levels of various catechins reach a peak at 39 to 144 ~M EGC, 11 to 48 ~M EGCG, and 7 to 28 ~M EC. The half-life of these catechins in saliva is about 10 to 20 min. The absorption and distribution of green tea catechins in laboratory animals depend on the route of administration and the particular catechin. Sixty minutes after intragastric administration of EGCG at a dose of 500 mg/kg body wt to rats, the level of EGCG is 12 ~M in plasma, 48 }~M in liver, 0.5 ~M in brain, 565 ~M in small intestinal mucosa, and 68 ~M in colon mucosa (Nakagawa and Miyazawa, 1997). EGCG administered intragastrically has a lower bioavailability than EGC and EC in term of fraction of absorption (Chen et al., 1997). When [3H]EGCG is administered directly into the stomach of mice, radioactivity is found in the digestive tract, liver, lung, pancreas, mammary gland and skin, as well as in brain, kidney, uterus, ovary, and testes (Suganuma et al., 1998). Since the chemical nature of the radioactivity in these tissues was not determined, whether the radioactivity represents EGCG or metabolites is not known. When green tea catechins are given by intravenous injection to rats, EGC and EC are removed from plasma faster than EGCG (Chen et al., 1997). Five hours after administration less than 1% of EGC and EC and 12% of EGCG remain in plasma. The highest amount of EGCG is found in the intestine, whereas low levels of catechins are found in liver. When EGCG is administered at a dose of 100 mg/kg body wt to rats by intraperitoneal injection, the plasma concentrations of unmetabolized EGCG, determined by HPLC, are 24, 2, 1, and 1 ~M at 0.5, 1, 2, and 24 h, respectively (Kao et al., 2000a). A plasma concentration of unconjugated and conjugated EGCG of i ~M would be similar to levels in a 70-kg human 1 h after drinking 6 to 12 cups (200 ml/cup) of tea (Lee et al., 1995). EGCG is also absorbed when it is topically applied to mouse and human skin. Intradermal uptake of catechins can be substantial, reaching

GREEN TEA: BIOCHEMICAL A N D BIOIX)GICALBASIS FOR HEALTH BENEFITS

9

i to 20% of the dose of EGCG when applied in a hydrophilic ointment (Dvorakova et al., 1999). The metabolism of catechins, including green tea catechins, has been studied in various animals, including humans (Das, 1971; Hackett and Griffiths, 1983; Hackett et al., 1985; Mesely et al., 1997; Pietta et al., 1998; Okushio et al., 1999; Yang et al., 1999; Li et al., 2000). Carechins orally administered to humans are absorbed, metabolized, and rapidly excreted largely within 24 h. EGCG appears to be converted by a salivary esterase to EGC (Yang et al., 1999). Catechins are detected in rat plasma and bile as free or sulfate and glucuronide conjugates (Harada et al., 1999), which are excreted within 6 to 10 h in urine and feces. More than a dozen urinary and plasma metabolites of green tea catechins are found in rats and humans, including m-hydroxyphenyl propionic acid, 3,4-dihydroxybenzoic acid, 3-methyl4-hipppuric acid, 3-methoxy-4-hydroxybenzoic acid (vanillic acid), 1-(dior trihydroxyphenyl)-3-(di- or trihydroxyphenyl)propan-2-ols, and ~/-valerolactones ((-)-5-(3',4'-dihydroxyphenyl or 3',4',5',trihydroxyphennyl)-~/-valerolactone). These products are formed by the cleavage ofcatechin rings (Das, 1971; Pietta et al., 1998; Li et al., 2000). Some of these catechin metabolites are apparently formed by microbial transformation in the intestine (Meselhy et al., 1997). Metabolism of CA has been studied in various animals, including humans. Urinary metabolites of CA and 3-methyl-CA orally administered to humans are predominantly glucuronides of 3,3'-dimethyl-CA and glucuronide and sulfate conjugates of CA (Hackett and Griffiths, 1983; Hackett et al., 1985). Incubation of catechins with rat liver homogenates and S-adenosyl-Lmethionine also produces 4'-O-methyl derivatives of EGC, ECG, and EGCG (Okushio et al., 1999).

IV. CHEMICAL AND BIOCHEMICAL PROPERTIES OF CATECHINS

A. ANTIOXIDANTACTMTY Reactive chemical species such as superoxide radical ('02) , hydroxyl radical ('OH), hydrogen peroxide, peroxynitrite, or alkoxyl radical (RO') cause cellular injury and cellular dysfunction by destruction and alteration of lipids, lipoproteins, enzymes, nucleic acids, and other cellular biochemicals as well as of cellular components such as ion channels, membranes, and chromatin. These radicals can originate from both exogenous and endogenous sources, such as pollutants, radiation, and metabolic activity. Radical damage contributes to the etiology of many

10


/

chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. In addition to free radicals, many carcinogenic agents can be converted into electron-pair-deficient electrophiles that can react with and damage DNA (Miller and Miller, 1971). If DNA damage by such agents is biologically fixed through cell division, cancer may be initiated. The radical scavenging activity of catechins can be studied using hydroxyl radicals, azide radicals, superoxide anions, and larger radicals such as, 1,1-diphenyl-2-picrylhydrazyl (DPPH), using pulse-radiolysis combined with kinetic spectroscopy, electron spin resonance (ESR), LC/MS spectroscopy, and semiempirical molecular orbital calculations (Bors and Michel, 1999; Kondo et al., 1999; Nanjo et al., 1999; Senba et al., 1999; Bors et al., 2000). When ESR and radicals generated from DPPH and 2,2'-azobis(2-amidinopropane)hydrochloride are used, the radical scavenging activity is stronger for gallated catechins (EGCG and GCG) than for nongallated catechins (EGC, GC, EC, and CA). Larger differences are seen especially at low concentrations ofcatechins. Steric effects may play a more important role in the ability to scavenge larger free radicals generated from DPPH and AAPH than in the ability to scavenge small free radicals, such as superoxide, especially in the case with EGCG and GCG, which have more bulky groups to potentially contribute to steric hindrance (Guo et al., 1999). Various antioxidants, including catechins, can act as scavengers of radicals caused by reactive oxygen species (ROS) and prevent radical damage (Sichel et al., 1991; Rice-Evans and Diplock, 1993; Wei et al., 1999). For example, tea polyphenols inhibit oxidant-induced DNA strand breakage in cultured lung cells (Leanderson et al., 1997). Pretreatment of cultured lung cells with green tea extract causes a significant decrease in both cigarette smoke and H202-induced DNA strand breakage. Tea polyphenols appear to reduce ROS that can reach and damage DNA molecules. In vitro, EGCG at 0.1 to 1.0 mM inhibits a-, 6-, ~/- and X-ray-induced scission of DNA in tritiated water (Hasegawa et al., 1997; Yoshioka et al., 1997, 1999). EGCG may also influence cellular mechanisms that are related to induction of mutagenesis, such as DNA synthesis and repair processes (Hayatsu et al., 1992). Superoxide and nitric oxide react to form peroxynitrite, which is a potent oxidant. Peroxynitrite enhances formation of 8-oxodeoxyguanosine, an oxidation product of deoxygnanosine, which is representative of oxidative damage to DNA. It also produces 3-nitrotyrosine from L-tyrosine in proteins. These oxidant activities of peroxynitrite are inhibited by EGCG with an IC5o of 250 and 110 ~M, respectively, for the two processes (Fiala


11

et al., 1996). These concentrations are much higher than can be expected in plasma of individuals consuming tea. The antioxidant and radical scavenger activities of catechins are apparently mainly due to its phenolic structures. Several important features of the radical scavenging mechanism are delocalization of electrons, formation ofintra- and intermolecular hydrogen bonds, ability to undergo molecular rearrangements, and ability to chelate metals that may be involved in oxidation (van Acker et al., 1996a,b; Morel et al., 1993; Balentine et al., 1997). The reduction potential is a measure of the energy required to donate an electron and usually is representative of a compound's antioxidant activity. However, the redox potentials of catechins and related compounds do not predict their antioxidant activities in biological systems (Balentine et al., 1997). Usually EGCG has the highest radical scavenging activity, while ECG and EGC are clearly more active than EC, CA, or other antioxidants, such as ~-tocopherol, L-ascorbic acid, or butylated hydroxytoluene. The ortho-trihydroxy group in catechins (Fig. 2) appears to play an important role in antioxidant activity, radical scavenging, and preventing oxidative destruction of biological compounds. Both free catechins and their glucosides are active radical scavengers. In general, the 3-gallate ester appears to play a very important role in free radical scavenging activity. The hydroxyl groups in the B ring also contribute to radical scavenging activity. Green tea extracts are more potent antioxidants than extracts from fermented teas, such as oolong and black teas (Yokozawa et al., 1998a), perhaps due to destruction of the o-trihydroxy structure during fermentation. In addition to ring B and the gallate ester (ring C), ring A of catechins (Fig. 2) may play a role in antioxidant activity. When EGCG or EGC reacts with H202, ring A is oxidized, cleaved, and decarboxylated to yield acids without any change in the trihydroxyl groups on ring B (Zhu et al., 2000). B. PROOXIDANTACTMTY While catechins and other plant polyphenols are excellent antioxidants that act as radical scavengers and protect cell components from radical damage, these antioxidants can also be prooxidants under certain conditions and generate hydroxyl radicals especially in the presence of Fe(II or III), Ag(I), or Cu(II) (Hiramoto et al., 1996; Hayakawa et al., 1997). In the presence of these metal ions and under aerobic conditions, tea catechins (EC, EGC, and EGCG) generate radicals that can cleave DNA, deoxyribose, and chromatin and accelerate the

12

s. LIAO,Y-H.KAO,ANDR.A.HIIPAKKA

peroxidation of unsaturated fatty acid (Yen et al., 1997; Hayakawa et al., 1999). In vitro, DNA breakage is not significant with nitric oxide, peroxynitrite, or nitroxyl anion alone, but DNA damage is enhanced by 10 to 500 ~M concentrations of different flavonoids, including EC, EGC, ECG, and EGCG (Shirahata et al., 1989; Ohshima et al., 1998). These studies suggest that catechins may have adverse effects in biological systems (see Section VI,B,10). However, direct evidence that biological damage is caused by drinking green tea is lacking. C. PROTEIN-BINDING ACTIVITY

Tannins, includingtea catechinsand their derivatives,have longbeen known to have activity necessaryfor the conversionof animal hides to leathers becausethey can bind proteins nonspecifica]]y(Haslam, 1989). This process is dependent on the multiple phenolic groups present in tannins, which can form hydrogen bonds and on flexible hydrophobic structures that form surfaces that can interact with other mo]ectfles (Fersht, 1987; Cai et al., 1990). Nonhydrolyzableor condensedtannins are po]ymerizedforms of flavonoids, whereas hydrolyzab]etannins are gallic acidesters of simple sugars like glucose.Proline-richproteins and synthetic polymers,such as po]yvinylpyrrolidine,have a high affinity for natural po]yphenols(Hagerman and Butler, 1981). Catechins can precipitate enzymes and other proteins in vitro or in vivo and inhibit enzymesor other activities(Sekiyaet al., 1984). By interacting with catechins and other tannins, proteins or enzymes change their chemical and physicalnature, their ability to be degraded, and even their nutritional value. The numerous reports of the effectsof green tea catechins on biologicallyimportant enzymesor proteins in vitro, therefore, need very careful assessment of whether these observations are relevant to possible in vivo effectsof green tea. Human serum proteins that bind to catechins have been identifiedusing affinitychromatography(Sazuka et al., 1996). Fibrinogen, fibronectin, and a histidine-richgiycoprotein all bound to an EGCG-agaroseaffinitycolumn.These proteins may act as carrier proteins for ga]]ated catechins absorbed into blood and may influence the biological and pharmacologicalactivity and metabolism of green tea catechins. Nonspecific catechin-protein interactions, however, can be biologically important. For example, po]ypheno]shave a harsh and stringent taste and produce in the mouth a feelingof constriction, dryness, and roughness. These effectsmay be, in part, due to binding of catechinsto giycoproteinson the epithelium of mouth (Haslam, 1989). The reduced

GREEN TEA: BIOCHEMICALAND BIOLOGICAL BASIS FOR HEALTH BENEFITS

13

digestibility of tannin-rich food may be explained, in part, on the basis of the inhibition of digestive enzymes (Honda et al., 1994). Catechinbound protein substrates in food may also have reduced reactivity with digesting enzymes. Herbal medicines often contain plant tannins as essential components. These tannins may be important because they can protect pharmacological ingredients from enzymatic degradation, provide a slow release of active components, and moderate medicinal activities. For example, angiotensin-converting enzyme plays an i m portant role in the regulation of blood pressure. This enzyme is inhibited by tannins in the crude medicines used in China and Japan for control of symptoms related to hypertension (Inokuchi et al., 1984, 1985). D. IoN-CHELATING ACTIVITY

Some of the effects of green tea polyphenols may be due to chelation of metal ions. Tea polyphenols, from green and black tea, stoichiometrically bind ferric iron to form a redox-inactive Fe-polyphenol complex (Grinberg et al., 1997). EGCG and ECG have greater chelating activity than EGC and EC, suggesting that the gallate ester is important for Fe2+-chelating activity (Yoshino et al., 1999). CA and other flavonoids protect iron-loaded hepatocytes from lipid peroxidation by removing iron from these cells (Morel et al., 1993). The chelating activity of tea catechin apparently is active in vivo, since consumption of both green and black tea lowers absorption of dietary iron in controlled feeding studies and decreases iron balance (Prystai et al., 1999). Green tea polyphenols also chelate copper ions and this is one of the suggested mechanisms by which polyphenols protect LDL from peroxidation (Yokozawa and Dong, 1997). EGCG inhibition of angiotensin-converting enzyme is modulated by zinc ion, suggesting that EGCG may also chelate zinc ions (Hara and Matsuzaki, 1987). The iron-chelating activity of green tea catechins may affect the function of various heme or metalloproteins that are dependent on metal ions for activity and, therefore, may have adverse effects in some situations. Although tea catechins appear to chelate a variety of metals, consumption of green and black tea extracts by humans does not affect the body's calcium, copper, magnesium, and zinc balance (Prystai et al., 1999). Also, injection of rats with EGCG does not alter serum levels of Na +, K +, Ca 2+, Cl-, and PO43- (Kao et al., 2000a). Rats given EGCG-containing green tea extracts orally accumulate less cadmium in the liver, kidney, and bone than rats not given green tea extracts (Kim and Rhee, 1994). Therefore, consumption of green tea

14

S. LIAO,Y-H.KAO,AND R. A. HIIPAKKA

may reduce toxicity due to heavy metals. In addition to chelating metals, green tea catechins react chemically with certain metal ions. For example, EGCG reduces Cr 6+, Cu 2+, and Fe 3+ to Cr 3+, Cu +, and Fe 2+ (Okuda et al., 1982).

V. BIOLOGICALACTIVITYOF GREEN TEA CATECHINS

Green tea polyphenols modulate many of the risk factors that contribute to common medical problems when examined in experimental models. The mechanisms responsible for the chemopreventative activities of tea catechins may be very complex (Yang and Wang, 1993; Dreosti, 1996; Katiyar et al., 1998). While antioxidant, radical scavenging, and metal-chelating activities of green tea catechins (see Section IV) may play an important role in the mechanisms by which catechins produce biological effects, some catechin effects may involve modulation of the activity of enzyme and other cellular components (see Section VI) that are involved in the disease process. Catechin effects may involve alterations in signal transduction pathways that are critical for cell transformation. Since catechins can modulate many endocrine systems as well as appetite in vivo (Kao et al., 2000a), catechins are expected to alter hormones and other physiological properties in vivo. A. ENDOCRINE EFFECTS

Although many of the possible health benefits of tea consumption have been investigated, until recently the effect of tea catechins on endocrine function was not well studied. Since green tea and other tea products that are orally consumed as beverages have very undefined compositions and since their absorption, organ distribution, metabolism, and cellular interactions with biological compounds are not well defined, it is helpful to use purified catechins in experiments designed to determine possible mechanisms for the health effects of tea. As a first step in studying the effects of green tea on endocrine systems, we injected pure EGCG ip into Sprague-Dawley (SD) and Zucker rats and studied the effects of EGCG on many endocrine systems (Table I; Figs. 3 and 4) in these animals (Kao et al., 2000a).

1. Sex Hormones and Accessory Sex Organs Rats treated with EGCG have significant changes in various endocrine parameters. After 7 days of ip treatment with EGCG at a


15

TABLE I EFFECTS OF (--)-EPIGALLOCATECHINGALLATEON HORMONES, NEUROPEPTIDES, GROWTHFACTORS, CYTOKINES, IMMUNOGLOBULINS,AND SERUM NUTRIENTSa Mediators

Effect

Dose/route

Models

References

C DHT GHb Insulin Leptin LH T

(0) (-) (-) (-) (-) (-) (-)

Endocrine systems 70-92 mg/kg bw, ip SDR or ZR >100 ~M, in vitro Rat 1 cells 70-92 mg/kg bw, ip SDR or ZR 70-92 mg/kg bw, ip SDR or ZR 70-92 mg/kg bw, ip, p.o. SDR or ZR 70-92 mg/kg bw, ip, p.o. SDR or ZR 70-92 mg/kg bw, ip SDR or ZR

Kao et al. (2000) Liao et al. (1995) Kao et al. (2000) Kao et al. (2000) Kao et al. (2000) Kao et al. (2000) Kao et al. (2000)

Histamine IGF-I IL-1 IL-10 IL-12 LTB4 PGE2 TNFa c

(-) (-) (+) (-) (+) (-) (+) (÷) (+) (-) (-) (-)

Growth factors and cytokines 100 btM, in vitro RBL-2H3 cells 70 ~ 92 mg/kg bw, ip SDR, ZR 109 ~M, in vitro Monocytes 3 mg/mouse, t.o. Mice w/UVB 3 mg/mouse, t.o. Mice w/UVB 100 ~M, in vitro Peritoneal cells 1-25 ~M, in vitro Macrophage 70-92 mg/kg bw, ip SDR ECso < 200 ~M, in vitro Macrophages ICs0 = 100 ~M, in vitro Macrophages ICs0 = 20 ~M, in vitro 3T3 cells ICso = 100 }~M, in vitro 3T3 cells

Matsuo et al. (1997) Kao et al. (2000) Sakagami et al. (1992) Katiyar et al. (1999) Katiyar et al. (1999) Matsuo et al. (1996) Liang et al. (1999) Kao et al. (unpubl.) Yang et al. (1998) Yang et al. (1998) August et al. (1999) Suganuma (1996)

IgA

(-) (+) (-) (+)

Yamada et Yamada et Yamada et Yamada et Yamada et Yamada et Yamada et

IgG

(-) (0)

IgM

(0)

Immunoglobulins (Ig) ICso = 100 }~M, in vitro Lymphocytes 0.1 ~M, in vitro Lymphocytes 0.1-100 ~M, in vitro Lymphocytes 1000 ~M, in vitro Lymphocytes ICs0 = 200 }xM, in vitro Lymphocytes 0.1-10 ~M, in vitro Lymphocytes 0.1 }LM,in vitro Lymphocytes

Protein Glucose Lipid Triglyceride Cholesterol

(0) (-) (-) (- ) (-)

70-92 70-92 70-92 70-92 70-92

IgE

Serum nutrientsd mg/kg bw, ip SDR or ZR mg/kg bw, ip SDR or ZR mg/kg bw, ip SDR or ZR mg/kg bw, ip SDR or ZR mg/kg bw, ip SDR or ZR

Kao Kao Kao Kao Kao

et et et et et

al. al. al. al. al.

al. al. al. al. al. al. al.

(1997) (1997) (1997) (1997) (1997) (1997) (1997)

(2000) (2000) (2000) '(2000) (2000)

aPlasma levels of neuropeptides such as adrenocorticotrophic hormone, corticotrophin-releasing factor, galanin, neuropeptide Y, and urocortin are not affected by EGCG (Kao et al., 2000a). Plasma levels of cholecystokinin in rats are increased when 0.5--2.5% green tea polyphenols are added to the diet (Yang et al., 1992). Abbreviations: C, corticosterone; DHT, 5a-dihydrotestosterone; GH, growth hormone; LH, luteinizing hormone; T, testosterone; IGF-I, insulinlike growth factor I; IL, interleukin; LTB, leukotriene B; PGE2, prostaglandin E2; TNF~, tumor necrosis factor ¢x; SDR, Sprague-Dawley rat; ZR, Zucker rat; ip, intraperitoneal; p.o., oral; t.o., topical; (0), no effect; (+), increased; (-), decreased. bEGCG increases serum GH levels in males, but decreases serum GH levels in females. CEGCG reduces LPS- and OA-induced TNFcx secretion and gene expression in 3T3 cells, but increases serum TNFa levels in SDR and TNF~ secretion and gene expression in the macrophages not treated with inducers. dEGCG reduces blood cholesterol levels in rats fed a high cholesterol diet (Fukuyo et al., 1986).


16

A

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FIG. 3. Differential effects of EGCG a n d t h r e e related green tea catechins on body weight (A), serum testosterone (B), weight of the ventral prostate (C) in male S p r a g u e Dawley r a t s a n d body weight (D), serum 171~-estradiol (E), a n d weight of the uterus (F) in female Sprague-Dawley rats. Symbols in A a n d D correspond to control (o), EC (), EGC (H), ECG, (A), and EGCG (e) groups. Values are the m e a n s 4- SEM from five animals (Kao et al., 2000a).

dosage of 85 mg/kg body wt, circulating levels of testosterone (Fig. 3B) are reduced by about 75% in male rats and 17~-estradiol levels by 34% (Fig. 3E) in female rats. The weights of androgen-sensitive organs, such as ventral (Fig. 3C) and dorsolateral prostates, seminal vesicles, and A

Male

~1.2

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Male

Female C

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FIG. 4. The effects of different catechins on hormone levels of Sprague-Dawley rats. Male and female rats were injected ip with indicated catechins (85 mg/kg body wt for male a n d 92 mg/kg body wt for female) for 7 days, and serum levels of leptin (A), insulin (B), a n d IGF-1 (C) were measured. Values are t h e m e a n s 4- SEM from five animals (Kao et al., 2000a).


17

coagulating and preputial glands are reduced by 50 to 70% after EGCG treatment. Similarly, the weights of estrogen-sensitive organs, such as the uterus (Fig. 3F) and ovary of female SD rats, are reduced by about 50% after EGCG treatment. These changes in the weights of sexual organs are catechin-specific, with EGCG showing the largest effect. The effect of EGCG on prostate or uterine weight loss appears to be due to reduced sex hormone levels and is not a direct effect of EGCG on organs, since organ weight loss is completely blocked, if rats are treated with sex hormones when given EGCG. With male and female rats treated with EGCG for 7 days, the serum levels of LH are significantly reduced by 40 to 50%, suggesting that low LH production led to the reduced blood levels of sex hormones.

2. Leptin, IGF-I, and Insulin In both male and female rats, 7 days of EGCG t r e a t m e n t causes significant reduction in blood levels of leptin, IGF-I, and insulin (Fig. 4). Dose-dependent effects of EGCG on levels of serum leptin, IGF-I, and insulin in male rats are also observed. The effects of EGCG on various peptide hormones are not mimicked by structurally similar catechins, EC, EGC, or ECG at an equivalent dose.

3. Food Intake and Body Weight Male rats injected ip with EGCG at a dosage of 70 to 92 mg/kg body wt consume about 50 to 60% less food than control rats. Apparently for this reason, EGCG causes an acute body weight loss (30% decreased compared to the body weight of control rats) in male (Figs. 3A, 5A, and 5C) and female (Figs. 3D, 5B, and 5D) rats within 2 to 7 days of treatment. EC, ECG, and EGC t r e a t m e n t did not reduce food intake or body weight loss at this dose. The effects of EGCG on various endocrine parameters that we observed may be explained as secondary effects of EGCG on food intake. For example, the large decrease in circulating leptin in EGCG-treated rats could have been caused by diminished fat stores due to low food intake in these rats. Both glucose and insulin stimulate leptin gene expression (Saladin et al., 1995; Friedman and Halaas, 1998), and so low circulating levels of glucose and insulin possibly resulting from low food intake m a y also contribute to the effect of EGCG on the leptin level. However, other mechanisms for the effects of EGCG, besides lowering food intake, may be operating and should be considered.

18

S. LIAO, Y-H. KAO,AND R. A. HIIPAKKA A

15

B

375 t

15

30

240"

.rTTr~

.

o lHll 2

5 0 ~ 0 5 10 15 20 25 30 35 Days

160

c

D

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0 0 ~ 0 5 10 15 20 25 30 Days

. . . . . . 0 5 10 15 20 25 30 Days

3oollo'~' 2

do

5 0 ~ 0 5 10 15 20 25 30 Days =

i

=

i

i

i

FIG. 5. Body weight loss in lean, leptin receptor-functional and obese, leptin receptordefective Zucker rats treated with EGCG. Lean male (A) and female (B) and obese male (C) and female (D) Zucker rats were injected ip daily with the indicated (number under arrow is milligrams injected) amount of EGCG. Changes in the amount of EGCG injected are shown with arrows. Obese rats were injected with vehicle (o) or EGCG (o). Values are the means -t- SEM from five animals.

4. Serum Nutrients and Proximate Body Composition In EGCG-treated male SD rats, the serum levels of protein, fatty acids, and glycerol are not altered, but significant reductions in serum glucose (-32%), lipids (-15%), triglycerides (-46%), and cholesterol (-20%) are observed (Table I). Based on proximate body composition analysis, rats treated daily with EGCG for 7 days have no change in percentage water and protein content, a moderate decrease in carbohydrate content, but a very large reduction in fat content, decreasing from 4.1% in control to 1.4% in EGCG-treated group. Within 7 to 8 days, EGCG treatment decreases subcutaneous fat by 40 to 70% and abdominal fat by 20 to 35%, but not epididymal fat, in male SD and lean (leptin receptor normal) Zucker rats. A 20% loss of


19

abdominal fat is seen in obese (leptin receptor deficient) male Zucker rats (Phillips et al., 1996) within 4 days of EGCG t r e a t m e n t (Kao et al., 2000a). 5. Blood Constituents Rats treated with EGCG have a 20% increase in red blood cell and hemoglobin concentrations, whereas the concentrations of white blood cells, lymphocytes, and monocytes decrease about 10, 31, and 24% respectively. Both eosinophil and platelet concentrations increase by 100%. EGCG does not appear to be toxic to the liver and kidney since (a) EGCG does not cause significant changes in the serum level of total protein, albumin, blood urea nitrogen, creatine, PO 3-, Na +, K +, Ca 2+, el-, and enzymes that are indicative of severe damage to liver and other organs; (b) EGCG has no effect on male SD rat liver ornithine decarboxylase activity, an indicator of cell proliferation that increases upon liver damage; and (c) in lean and obese Zucker rats, no visible differences are observed between the microscopic histology of the liver and kidney of EGCG-treated rats and those from the control (Kao et al., 2000a). Although detailed toxicological studies of EGCG have not been reported, a condensed polyphenol structurally related to EGCG, procyanidin B-2, has a lethal dose greater t h a n 2 g/kg body wt when subcutaneously injected into rats (Takahashi et al., 1999). 6. Route of E G C G Administration and Health Benefits The effects of EGCG on body weight loss, hormone level changes, and food intake depend on the route of administration. The effects of EGCG observed when EGCG is administered by ip injection are not present when the same amount of EGCG is given to rats orally for 7 days (Kao et al., 2000a). This m a y be due to inefficient absorption of EGCG or metabolism in the digestive tract (Yang, 1997; Suganuma et al., 1998) and suggests t h a t the effects ofip administration of EGCG are not caused by interaction of EGCG with food or by EGCG action inside the gastrointestinal tract. Although oral administration of EGCG is not effective within 7 to 14 days, long-term oral consumption of green tea or EGCG-containing extracts m a y mimic some of the acute EGCG effects caused by ip administration of EGCG and may be beneficial to health. Based on oral and ip effects of EGCG on serum hormones and nutrients, long-term consumption of green tea m a y influence the incidence and provide therapies for obesity, diabetes, and cardiovascular disease.

20

S. LIAO, Y.H. KAO, AND R. A. HIIPAKKA A. PC-3

EGCG r ~ l

600, o ~, 400. '~ 2OO. ,,~

eB 250 B. LNCaP 104-R "~ ~ 200

Be

--~ 150 "E ~ 100

-~ 100.

C. MCF-7

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"r "r " ,

0.

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,

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14 21 2 8 Days

50

EGCG

"5 50o~

0 7 1421283542 Days

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EGCG

I

Days

l~G. 6. The effect of EGCG on t u m o r growth in nude mice. Intact and castrated male mice were used for h u m a n prostate PC-3 (A) a n d LNCaP 104-R (B) t u m o r growth studies, respectively, whereas intact female nude mice were used for h u m a n b r e a s t MCF-7 (C) t u m o r growth studies. Two weeks after inoculation of cancer cells, the t u m o r size was m e a s u r e d a n d designated as 100% on this day (day zero). When used, 1 mg EGCG was injected ip daffy for the period indicated (Liao et al., 1995).

By lowering plasma levels of sex steroids and other endocrine factors, such as IGF-I, long-term use of EGCG or green tea may be effective in prevention and suppression of the growth of hormone-dependent and hormone-independent prostate and breast cancers (Fig. 6) (Liao et al., 1995; C h a n e t al., 1998; Hiipakka and Liao, 1998). This may relate to the low occurrence of breast and prostate cancer metastasis and mortality in some Asian countries where green tea is consumed regularly (Giovannucci, 1995; Liao et al., 1995). B. CANCER

1. Epidemiological Observations Numerous reports of the ability of green tea and its associated catechins to act as antioxidants and radical scavengers and to inhibit the growth of cancer cells in culture, as well as to inhibit the induction of carcinogenesis in experimental animals, raise the possibility that consumption of green tea and its associated catechins may lower cancer risk in humans. However, epidemiological studies, most of them based on fermented tea users, have not produced conclusive evidence for health benefits of tea consumption (Blot et al., 1996, 1997; Kohlmeier et al., 1997; Chung et al., 1998). Some studies have suggested lower risks, while others demonstrated no association or even an adverse relationship. Several possible confounding factors may be responsible for these equivocal results, including population sampling errors, the diversity o~tea products used and the amounts consumed, possible interference


21

by tea adulterants (for example, addition of milk to tea m a y affect the antioxidant activity of catechins), overriding effects of lifestyle (e.g., smoking), and selectivity of health benefits for certain types of cancer. Several studies based on green tea users appear to provide more convincing data that green tea m a y have health benefits in terms of cancer incidence or mortality. In a study in China (Gao et al., 1994), green tea drinkers who consumed more than two cups of tea a day had their risk of esophageal cancer reduced by 50%. Another large study from Shanghai, China, that controlled for socioeconomic, lifestyle, and other factors found that green tea users had a nearly 50% reduction in stomach cancer incidence (Yu et al., 1995). In Japan, inhabitants of tea producing districts have a lower mortality due to stomach cancer, perhaps due to consumption of green tea (Kono et al., 1988; Oguni et al., 1989). In contrast to these studies, an early case-control study in England found a positive correlation between tea consumption and the occurrence of stomach, kidney, and lung cancers (Kinlen et al., 1988). An increase in the risk of esophageal cancer with ingestion of tea has been attributed to the hot temperature of the tea rather than due to the tea itself (De Jong et al., 1974; Victoria et al., 1987). Although many reports have described a lack of association between breast cancer risk and tea consumption, a Japanese study involving 472 breast cancer patients found that increased consumption of green tea was closely associated with a decreased number of axillary lymph node metastases among premenopausal patients with stage I and II breast cancer (Nakachi et al., 1998). Increased expression of progesterone and estrogen receptors was observed in biopsies from these patients, which is perhaps indicative of less tumor progression. Increased consumption of green tea also correlated with decreased recurrence of stage I and II breast cancer. The recurrence rate was 17% for individuals drinking more than five cups and 24% for those drinking less than four cups per day, suggesting an increased benefit for heavy green tea drinkers. No improvement in prognosis was observed in stage III breast cancer (Nakachi et al., 1998). Certain epidemiological studies have not found an association between tea drinking and incidence of colorectal, lung, bladder, and pancreatic cancer. However, in a large population-based case-control study, conducted in Shanghai, China that adjusted for socioeconomic and other individual factors, like cigarette smoking, an inverse association with green tea consumption (>200-300 g per month) and risk of pancreatic and colorectal cancers, in both men and women, was found

22


(Ji et al., 1997). Studies on urinary tract and kidney cancer also have shown little association between cancer risk and tea drinking. In a case control study in Taiwan, however, oolong tea consumption was found to be associated with an increased risk of bladder cancer (Lu et al., 1999). Whether this increased risk is directly associated with the tea is not clear. Overall, green tea consumption appears to be associated with a lower risk of upper gastrointestinal cancer, and since this part of the body is exposed to high levels of green tea catechins, this finding may be reliable. In other tissues, the bioavailability of green tea components may be limited, and so, in these tissues, the effects of tea consumption on cancer initiation and progression may be harder to discern. Also, even though moderate consumption of green or black tea (one to two cups a day) does not have consequence that are apparent in epidemiological studies, the potential health benefits of long-term green tea consumption may be significant. 2. S k i n Cancer The effect of green tea on skin cancer initiation and promotion has been thoroughly investigated in experimental animals (Conney et al., 1992; H u a n g et al., 1992; M u k h t a r et al., 1994). In these studies, skin tumors are induced by ultraviolet (UV) irradiation or by applying carcinogenic polycyclic aromatic hydrocarbons, such as 7,12-dimethylbenz[a]anthracene (DMBA), to study the initiation process of skin cancer. A tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), is then used to study promotion of tumor growth. By giving green tea extracts as the sole source of drinking water or topical application of isolated catechins at different stages, before, during, or after the application of carcinogenic agents, it is possible to determine at which stage the catechin exerts its antitumorigenic effects. Analyses of the changes in the activities of different enzymes that are known to play key roles in the tumor initiation, promotion, and progression during carcinogenesis are helpful in understanding the mode of action of catechins. EGCG topically applied to skin inhibits teleocidin-induced tumor promotion in mice previously initiated with DMBA (Yoshizawa et al., 1987). Oral (Wang et al., 1989, 1994) or topically applied (Huang et al., 1992) green tea polyphenols inhibit the tumor-initiating activities of benzo[a]pyrene (BP) and DMBA in mouse skin. Skin carcinogenesis has two stages (Slaga et al., 1980; DiGiovanni, 1992), and the tumor promoters TPA and mezerein can be used to analyze the effects of green tea at different stages. Green tea polyphenols protect against stage I skin papilloma formation in terms of both tumor multiplicity by 42 to 50% and


23

stage II tumor growth by 43 to 54%. With topical application, green tea polyphenols inhibit TPA-induced events in stage I such as increased activities of cyclooxygenase, lipoxygenase, arachidonic acid metabolism, hyperplasia, inflammation, and hydrogen peroxide production, as well as stage II events such as elevated levels of ornithine decarboxylase (ODC), polyamines, protein kinase C, and cell proliferation in the epidermis of mice (Agarwal et al., 1992; Huang et al., 1992; Katiyar et al., 1992). Catechin inhibition of skin tumor formation is also observed when mezerein is used (Katiyar et al., 1993). These observations indicate that both tumor initiation and tumor promotion are inhibited by green tea polyphenols. Catechin effects may also involve inhibition of the production of other growth-promoting factors. Pretreatment of mouse skin with green tea extract or individual catechins 30 min before application of TPA results in a significant inhibition of TPA-induction of epidermal interleukin-1 (Katiyar et al., 1995). EGCG and ECG are more effective than EGC and EC, suggesting that the gallate ester is important for the effects. Therefore, catechins may lower agents associated with growth, such as polyamines, arachidonic acid, and prostaglandins, which can promote hyperplasia, inflammation, and tumor growth. Green tea administered in drinking water also inhibits skin tumor formation induced by UV irradiation when given prior to and during irradiation. This treatment also decreases the number and the size of skin tumors (Wang et al., 1991, 1992a,b). The effect of green tea can be mimicked by EGCG. The incidence of skin tumors induced by UV exposure is reduced from 96 to 62 and 29%, when 10 and 50 mg of EGCG in acetone are applied to the skin of mice three times weekly before and throughout a 25-week experimental period. Oral EGCG is not effective (Gensler et al., 1996). EGCG inhibits photocarcinogenesis in these mice with no visible toxicity. 3. Digestive Tract Cancer

Induction of cancer by DMBA in the buccal pouch of the golden Syrian hamster is an animal model of carcinogenesis that closely resembles events involved in the development of precancerous lesions and cancer in the human oral cavity (Gimenez-Conti and Slaga, 1993). This model has proven to be valuable for screening for chemopreventative agents. When a mixed tea preparation containing 1.5% green tea extract, tea pigments, and tea polyphenols is given as the sole source of drinking water, before and during topical DMBA treatment of buccal pouches of hamsters, the mean tumor burden and the incidence of dysplasia and oral carcinoma are significantly reduced (Li et al., 1999a). The mixed tea preparation is more effective than green tea or tea pigment

24


alone. The advantages of a tea mixture for cancer prevention is similar to reports on the prevention of liver precancerous lesions induced by N-nitrosodiethylamine in rats (Qi et al., 1997). N-Nitroso compounds and their precursors are possible etiological factors for esophageal cancer (Yang, 1980). In rats, several asymmetric nitrosamines are potent inducers of esophageal tumors (Druckrey et al., 1963). The induction of esophageal tumors by N-nitrosomethylbenzylamine (NMBA) is a valuable model for screening for the chemopreventative efficacy of various compounds. Using this model, green tea extracts inhibit esophageal tumorigenesis induced by NMBA and its precursors, sodium nitrite and methylbenzylamine (Chen, 1992; Wang et al., 1995). Theaflavins and EGCG are also effective in reducing esophageal tumorigenesis induced by NMBA in rats (Morse et al., 1997). Epidemiological studies (Section V,B,1) have found that green tea consumption may reduce stomach cancer incidence. Carcinogenesis in the glandular stomach of rats can be induced by N-methyl-N'-nitrosoguanidine (MNNG). EGCG treatment at a dose of 1 mM in drinking water reduces the percentage of tumor-bearing rats in this model from 62% in the control without EGCG to 31% in EGCG-treated rats. EGCG treatment decreases ODC activity and tissue polyamine levels, suggesting that EGCG inhibits cellular proliferation of gastric mucosa during the promotion stage of MNNG-induced gastric carcinogenesis (Yamane et al., 1995b). In a multiple-organ model of carcinogenesis induced by nitrosoamines, green tea extract administered at a dosage of 0.1-1% in drinking water, and given during and after carcinogen exposure, reduces the number of small intestinal tumors (adenomas and carcinomas) per rat by 60 to 85%. However, in the same animals, a slight but significant increase in precarcinogenic foci in liver is observed (Hirose et al., 1993). Green tea extracts and EGCG given orally to mice or rats, after oral dosing with carcinogen for 4 to 28 weeks, also inhibits N-ethyl-N'-nitroN-nitrosoguanidine-induced duodenumal carcinogenesis, MNNGinduced gastric carcinogenesis, and azoxymethane-induced colon carcinogenesis (Yamane et al., 1995a, 1996). In a clinical study, 10 patients with esophageal dysplasia and familial adenomatous polyposis after subtotal colectomy were administered 1 g of green tea polyphenols (85% EGCG and 20 mg polyphenol/kg body wt) per day for 2 to 32 months. This treatment resulted in the disappearance of polyps and a decrease in rectal mucosal ODC activity (Yamane et al., 1995). However, a more comprehensive study with proper controls is needed to assess the clinical efficacy of this treatment (Yamane et al., 1996).


25

Green tea catechins at a dosage of i to 3 mg per mouse and EGCG at a dosage of 2 mg per mouse, administered five times per week for 20 to 23 weeks by stomach perfusion, reduced the incidence of large intestinal cancers induced by 1,2-dimethylhydrazine and metastasis to lung (Yin et al., 1994). How catechins inhibit tumor formation in this model is unknown but the mechanism may be related to the observation that t r e a t m e n t with tea catechins enhances the activity of superoxide dismutase in red blood cells and in large intestinal cancers. When a green tea extract at a dosage of 0.01 or 0.1% in drinking water is given to rats after t r e a t m e n t with the carcinogen azoxymethane, colon carcinogenesis is inhibited. EGCG also inhibits proliferation of the h u m a n colon carcinoma cell line SW620, which is resistant to doxorubicin (Stammler et al., 1997). 4. Liver a n d L u n g Cancer Green tea extracts at dosages of 1 to 50 ~g/ml prevent the cytotoxic effects on isolated mouse hepatocytes induced by oxygen freeradical-generating enzymes, such as xanthine oxidase, and glucose oxidase, in a dose-dependent manner and decrease the DNA labeling index in hepatic preneoplastic foci of mice treated with the tumor promoter phenobarbital (Klaunig, 1992). Mice given the carcinogen diethylnitrosoamine (DENA) and administered green tea and black tea at a dosage of 1.25% in drinking water have a 50% decrease in the number of lung and liver tumors (Cao et al., 1996). Green, oolong, and black teas, as well as EC, EGC, ECG, and EGCG, when given in the diet at a dosage of 0.05 or 0.1% significantly reduce the number and area of preneoplastic foci in liver of rats treated with DENA and phenobarbital (Matsumoto et al., 1996). Since catechins are effective when they were given during or after carcinogenic treatment, they appear to act both during the tumor initiation or promotion stages. Both green and black tea extracts in drinking water at dosages of 1 to 2% inhibit spontaneous formation of lung tumors in A/J mice (Landau et al., 1998). 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), which is one of the most potent carcinogenic nitrosamines in tobacco smoke, is highly specific for lung cancer induction in laboratory animals. Green tea at a dosage of 2% and EGCG at 1.2 mM in drinking water inhibit lung tumorigenesis in mice treated with N N K (Xu et al., 1992; Chung et al., 1999a). Both green and black teas are effective in this system (Shi et al., 1994). Green tea or EGCG administration suppresses increases in the level of 8-OH-dG in mouse lung DNA due to

26

S. LIAO, YoH. KAO, AND R. A. HIIPAKKA

NNK treatment. Since 8-OH-dG is a DNA lesion caused by oxidative damage, the EGCG effect may be in part due to its antioxidant activity (Xu et al., 1992). At a concentration of 0.4 mg/ml, green and black tea extracts inhibit NNK oxidation and NNK-induced DNA methylation in the presence of lung microsomes. The concentration of EGCG needed for 50% inhibition is 0.12 ~M. At this concentration, EGCG also inhibits the catalytic activities of various cytochrome P450 enzymes and perhaps bioactivation of NNK. However, in vivo, a statistically significant inhibition of lung DNA methylation is not found, while a significant reduction in lung tumor multiplicity is observed. Therefore, inhibition of the metabolic activation of NNK and DNA methylation by high concentrations of tea compounds in vitro may not be related to the inhibition of tumorigenesis by tea catechins (Shi et al., 1994). Oral administration of green tea infusion and EGCG inhibits metastasis of Lewis lung carcinoma LL2 cells in mice (Taniguchi et al., 1992; Sazuka et al., 1995). In vitro, LL2-Lu3 cell invasion of an artifically reconstituted basement membrane (Matrigel) is inhibited by EGCG, but not by C,a_, EC, or methyl gallate. Since superoxide can enhance invasiveness of tumor cells (Taniguchi et al., 1992), the radical scavenging activity of EGCG may be related to its inhibition of cancer cell invasion and metastasis (Sazuka et al., 1995). The antimutagenic activity of EGCG against BP-induced mutations has been assessed by using transgenic mice carrying the rpsL (streptomycin sensitivity) gene as a monitor of mutations. EGCG given in drinking water during a 3-week experiment reduces BP-induced mutations in the lung by 60% (Muto et al., 1999). The BP-induced mutations occur mainly (52%) at G:C base pairs. BP-diolepoxide, which is the metabolically activated form of BP, binds to DNA and forms adducts predominantly at the 2-amino group of guanine (Phillips, 1983). The BP-induced mutations in the rpsL gene have motif similarities to mutational hotspots in human lung cancer, namely TGG and GGT for codon 12 of the tG-ras gene and CGT for codons 157 and 273 and CGC for codon 248 of the human p53 gene. The activation of Ki-ras and the inactivation of the p53 gene by mutation are common genetic lesions found in both human and rodent lung tumors. The Ki-ras mutation occurs in 30 to 50% of all small cell lung cancers, and approximately 60 to 70% of human cancers have been reported to contain mutations in the p53 gene (Gao et al., 1997). EGCG may reduce mutations in these genes, leading to the reduction of lung tumors (Muto et al., 1999).


27

Individuals with a p53 germ-line mutation (Li-Fraumeni syndrome) have a 50% risk of developing lung cancer by age 60. To determine if green tea can modulate the risk of cancer from germ-line mutations, p53 heterozygous knockout mice and p53 transgenic mice carrying a dominant negative m u t a n t were crossed with A/J mice, which are highly susceptible to lung tumor induction. Green tea at a dosage of 0.6%, as the sole drinking water, reduces tumor multiplicity in these transgenic mice treated with N N K (Zhang et al., 2000). Drinking tea for 4 or 8 weeks reduces NNK-induced expression of mouse lung oncogenes, such as c-myc, c-raf, and c-H-ras, suggesting a possible mechanism of green tea action through modulation ofoncogene expression (Hu et al., 1995). 5. Breast a n d Prostate Cancer M a m m a r y gland carcinogenesis is very effectively induced by a single dose of DMBA in female SD rats (Huggins, 1965). When female rats are given one intragastric dose of 50 mg/kg of DMBA, and then 1 week later start a diet containing 1% of a green tea catechin extract, the incidence and multiplicity of DMBA-induced m a m m a r y tumors are not significantly affected. However, starting at week 10 and toward the end of the 18th week, when all animals are still alive, the average size of palpable m a m m a r y tumors is significantly smaller in the catechintreated group. The survival rate is 94% for the green tea catechin group and 33% for the control group not receiving the catechins during the 36-week experiment (Hirose et al., 1994). However, when rats are given a diet containing 0.5% of an EGCG-rich tea extract 13 weeks after DMBA treatment, there is no significant effect on tumor incidence (Hirose et al., 1997; Tanaka et al., 1997). Based on these studies, green tea catechins appear to be more effective at early postinitiation stages, b u t not at later stages of tumorigenesis. In both of these experiments, catechins were not given to animals during carcinogen treatment. It is possible that catechins also act at the initiation stage of DMBA induction of m a m m a r y cancer, since rats given black tea in their drinking water during treatment with DMBA, have decreased m a m m a r y tumor multiplicity and volume. Milk appears to potentiate this inhibitory effect of black tea (Weisburger et al., 1997). A green tea catechin extract at a dosage of 0.5% in drinking water prevents spontaneous m a m m a r y tumor incidence and burden in C3H (Jax) mice and inhibits DMBA-induced m a m m a r y tumors in rats (Bhide et al., 1994). H u m a n prostate and breast cancer cell lines injected subcutaneously into nude mice produce tumors. Injection of mice ip with EGCG at

28

S. LIAO, Y-H. KAO,AND R. A. HIIPAKKA

a dosage of 50 mg/kg per day inhibits the growth of tumors derived from human cancer cell lines (Fig. 6), including (a) an androgen receptor (AR)-positive and androgen-dependent prostate cell line (LNCaP 104S), (b) an AR-positive and androgen-suppressed prostate cell line (LNCaP 104R), (c) an AR-negative and androgen-independent prostate cell line (PC3), and (d) an estrogen receptor (ER)-positive and estrogendependent breast tumor cell line (MCF-7) (Liao et al., 1995). The effect of EGCG on prostate and breast tumor growth and regression is very rapid and is observed within 1 to 2 weeks (Fig. 6). The EGCG effect is reversible; when EGCG treatment is stopped, the tumors resume their growth. While EGCG is effective, the structurally very similar compound ECG is not active. ECG lacks only one of the eight hydroxyl group on EGCG and this is on the B ring. Since EGC, which lacks a gallate group, also is not active, both the B and C aromatic rings that contain o-trihydroxy groups (Fig. 2) are important for activity. EGCG in vitro may control prostate cell growth by inducing apoptosis (Paschka et al., 1998); however, in vivo studies suggest that complex endocrine changes (Kao et al., 2000a) may be responsible for EGCG-induced human prostate cancer regression in mice (Liao et al., 1995). Green tea polyphenols also down-regulate the androgen receptor in LNCaP cells (Ren et al., 2000), and EGCG affects cell growth and cell cycle regulation and induces apoptosis in androgen-sensitive and androgen-insensitive human prostate carcinoma cells (Gupta et al., 2000). 6. Angiogenesis

Angiogenesis is required for many important physiological processes, such as maturation of the corpus luteum, embryogenesis, endometrial regeneration, and wound healing. The growth of tumors also requires new blood vessel growth, which provides nutrients to tumors and also permits tumor metastasis (Folkman, 1985). The growing tips of the developing blood vessels produce urokinase plasminogen activator, which creates proteolytic activity in the proximity of the migrating vessel tip, degrades stromal structures, produces space for vessel expansion, and allows local tumor invasion and metastasis (Goldfarb and Liotta, 1986). Inhibitors of angiogenesis, such as angiostatin, may restrict formation of new blood vessels by inhibiting proliferation of endothelial cells (Folkman, 1990). Angiogenesis inhibitors, therefore, can suppress tumor growth and metastasis without a direct effect on tumors. EGCG inhibits urokinase in vitro with an IC5o of about 4 mM (Jankun et al., 1997). When EGCG and other urokinase inhibitors are analyzed for their effect on angiogenesis using a chicken embryo


29

chorioallantoic membrane assay, all cause a significant reduction in angiogenesis (Swiercz et al., 1999). The ICso for EGCG is about 50 ~g/embryo. EGCG also inhibits endothelial cell proliferation at concentrations in the range of 22 to 110 ~M (Cao and Cao, 1999). It has been pointed out that these high EGCG concentrations are very difficult to achieve by drinking green tea extracts (Yang, 1997). However, green tea given to mice in their drinking water prevents corneal neovascularization induced by vascular endothelial growth factor, a potent angiogenic factor (Cao and Cao, 1999). Since high concentrations of EGCG are required to block endothelial cell proliferation in vitro, it is not clear by what mechanism tea blocks angiogenesis in vivo, where high blood levels of EGCG are unlikely (see also Section V,B).

C. CARDIOVASCULAR DISEASE AND HYPERTENSION

1. Epidemiological Observations a n d Clinical Studies Although some epidemiological studies have not provided clear-cut evidence for a link between tea consumption and cardiovascular disease (Kark et al., 1985; Green and Jucha, 1986; Hollman et al., 1999; van het Hof et al., 1999), several studies have shown that tea intake is associated with a lower risk of cardiovascular disease (Hertog et al., 1993, 1995; Keli et al., 1996; Knekt et al., 1996). A large epidemiological study carried out in Norway compared 9856 men and 10,233 women, 35 to 49 years of age, without a history of cardiovascular diseases or diabetes (Stensvold et al., 1992). These researchers found that mean serum cholesterol and systolic blood pressure are somewhat lower and inversely related to increasing tea (probably black tea) consumption when individuals consuming less than one cup per day are compared to individuals consuming more than one cup or more than five cups per day. Several studies in J a p a n have related green tea consumption to serum cholesterol and lipoprotein cholesterol levels (Kono et al., 1992; Imai and Nakachi, 1995). In a study involving 2062 men, ages 49 to 55 years, the level of green tea consumption was inversely associated with serum levels of total cholesterol and low-density lipoprotein cholesterol (LDL), b u t not with either high-density lipoprotein cholesterol or triglycerides (Kono et al., 1996). In another Japanese study with 1371 men over 40 years of age, higher levels of green tea consumption were associated with decreased serum concentrations of total cholesterol and triglyceride and an increased proportion of high-density lipoprotein

30

s. LIAO, Y-H. KAO, AND R. A. HIIPAKKA

cholesterol together with a decreased proportion of low and very low density lipoprotein cholesterol. Increased consumption of green tea, especially more than 10 cups a day, was related to decreased concentrations of hepatological markers in serum, such as aspartate aminotransferase, alanine transferase, and ferritin, suggesting a protective role for tea in disorders of the liver (Imai and Nakachi, 1995). In a clinical study, 18 healthy Japanese male volunteers were orally given a green tea extract containing 254 mg catechins. Their plasma level of EGCG reached 0.27 nM. The plasma phosphatidylcholine hydroperoxide level, a marker of oxidized lipoproteins, decreased from 74 pM in controls to 45 pM in EGCG-treated subjects, suggesting that tea catechins are effective as antioxidants, in vivo, to prevent lipid oxidation and, therefore, may reduce the risk of cardiovascular disease (Nakagawa et al., 1999). However, another Japanese study using 14 men who consumed black tea at a dosage of 750 ml daily for 4 weeks found no significant changes in LDL oxidation (Ishikawa et al., 1997). Another report also concluded that there was no effect of consumption of green and black teas on plasma lipid and antioxidant level and on LDL oxidation in smokers (Princen et al., 1998). 2. L i p i d Peroxidation Analysis Lipid-laden foam cells are characteristic of atherogenesis. Native LDL does not induce foam cell formation. Oxidative modification of LDL is necessary for the cellular uptake of lipid and foam cell formation. Modified LDL is recognized by macrophage scavenger receptors and massive uptake of LDL and associated cholesterol converts the macrophages into foam cells. Modified LDL is present in atherosclerotic lesions, but not in normal arterial walls. For this reason, LDL oxidation is an important step in the formation of atherosclerotic plaques and subsequent cardiovascular diseases (Haberland et al., 1988; Steinberg et al., 1989; Stanton et al., 1992). Therefore, various studies have examine inhibition of LDL oxidation by green tea catechins. However, green tea inhibition of atherosclerotic lesion formation may not be fully explained by the ability of catechins to inhibit LDL peroxidation (Tijburg et al., 1997). The antioxidant activities of tea catechins have been assessed in vitro and ex vivo after ingestion of tea. LDL isolated from blood can be oxidized by various cells, including endothelial cells and macrophages, as well as by metal ions, such as Cu 2+, in vitro. The extent of lipid peroxidation and protection by catechins can be determined by measuring the quantity of thiobarbituric acid-reactive substances (TBARS) generated in the culture medium using colorimetric analysis (Yoshida et al., 1999).


31

The electrophoretic mobilities of native and modified LDL can also be determined by agarose gel electrophoresis for comparison. In addition, LDL oxidation can also be analyzed with a spectrophotometric method in which the amount of lipid peroxides or conjugated dienes formed are measured. 3. In Vitro Inhibition of Lipid Peroxidation

Green tea catechins, at 5 and 10 ~g/ml (about 10-20 ~M), dosedependently inhibit LDL oxidation and also lower the cholesterol level in endothelial cells. LDL oxidation by reactive oxygen species increases the negative charge on LDL, increasing its electrophoretic mobility (Esterbauer et al., 1990). Green tea catechins prevent this increase in the electrophoretic mobility of LDL induced by endothelial cells, which prevents the uptake of LDL by macrophage scavenger receptor and formation of foam cells (Yang and Koo, 2000). LDL oxidation, as monitored using in vitro assays, has a lag phase perhaps due to endogenous antioxidants in LDL particles. Green tea polyphenols at a concentration of 0.5 ~M increase the lag phase for LDL oxidation from 79 to 211 min. At the same concentration, vitamin C increases the lag phase to 95 min and vitamin E to 213 rain (Luo et al., 1997). Therefore, in vitro, the antioxidant activity of green tea is greater than that of vitamin C, but equivalent to vitamin E on a molar basis. EGCG is the most potent inhibitor of lipid peroxidation among 28 beverages or compounds tested using an in vitro assay. The ICao for EGCG is 80 nM, whereas those for vitamin C (1.45 ~M), tocopherol (2.4 ~M), and ~-carotene (4.3 ~M) are much higher (Vinson et al., 1995). Copper sulfate also catalyzes LDL oxidation and an in vitro assay of LDL oxidation using Cu 2+ also has a lag phase before oxidation begins. An increase in the lag phase is observed when tea catechins are present in the assay. EGCG and ECG are clearly more active than EC and EGC when present at 5 to 40 ~M (Zhang et al., 1997a), At 40 ~M vitamin C does not prevent Cu2+-mediated LDL oxidation. EGCG also is more active than other catechins based on other in vitro studies (Miura et al., 1994, 1995). The ICso for inhibition" of Cu 2+ -mediated hydroperoxide2+ formation was about 1 ~M for ECG and EGCG, and Cu -mediated cholesterol ester degradation in LDL was almost completely inhibited by 5 ~M EGCG. The activity of EGCG and ECG may be dependent on the gallate and hydroxyl groups that may make these compounds better radical scavengers. Alternatively, EGCG and ECG may function as better metal chelators than EC and EGC, sequestering Cu 2+ and other ions involved in initiation of free radicals. EGCG and ECG are

32

s. LIAO, Y-H. KAO,AND R. A. HIIPAKKA

more hydrophobic t h a n EC and EGC based on their elution times from C-18 reverse-phase chromatography columns, and so these catechins may more readily partition into LDL particles and prevent oxidation t h a n EC and EGC. ~-Tocopherol functions as a major antioxidant in h u m a n LDL. However, ~-tocopherol can be depleted by oxidants very rapidly. Since, in vitro, various catechins at 2 to 20 ~M protect ~-tocopherol from oxidation, one of the functions of green tea catechins may be their ability to assist, in vivo, in the regeneration or protection of ~-tocopherol or other antioxidants from oxidation (Nanjo et al., 1993; Zhu et al.,

2OOO). The effective dose of a tea catechin for protection of LDL from oxidation in vitro varies in different studies. While in some studies catechins are effective at less t h a n 5 ~M, which is probably the m a x i m u m concentration achievable with very heavy tea drinking, other studies show t h a t much higher doses are required. For example, in one report (Yoshida et al., 1999) protection of LDL from cell-mediated oxidation requires as much as 100 to 400 ~tM EGCG or theaflavin digallate for 25 to 40% inhibition. Blood concentrations of catechins as high as 100 ~M are very difficult to achieve by drinking tea. The reason t h a t effective doses vary in different studies could be due to the use of different assay methodologies. The cell-induced LDL oxidation assay requires higher concentrations of catechins for inhibition t h a n Cu2+-mediated LDL oxidation assays. This may be due to the high amount of protein in the cell-induced oxidation assay, which may nonspecifically bind catechins making them inactive. In the Cu2+-induced oxidation system no cells are present, and so lower concentrations of catechins may be required to chelate metal ions t h a t are required for oxidation.

4. Ex Vivo Inhibition of L i p i d Peroxidation The ability of green tea catechins to inhibit in vitro lipoprotein oxidation does not necessarily mean t h a t these catechins function in a similar m a n n e r in vivo. Polyphenols may block oxidants before they interact with lipoproteins or they may bind in vivo to LDL and VLDL and provide protection within the lipoprotein particle. Lipoprotein-bound antioxidant effectiveness can be evaluated using an ex vivo assay method. Although such an approach often shows significant increases in the total antioxidant capacity of serum after tea ingestion, findings are not always consistent (Serafini et al., 1996; van het Hofet al., 1997; McAnlis et al., 1998). When LDL is isolated from plasma for use in in vitro assays of lipid peroxidation, most of the catechins are associated with the water-soluble


33

protein-rich fraction of the plasma and in HDL. The concentration of catechins in LDL is low and not sufficient to enhance the resistance of LDL to oxidation ex vivo (van het Hof et al., 1999). An alternative method is to study the acute effects of ingested tea on ex vivo lipoprotein oxidation without prior isolation of lipoproteins from serum. For example, in a study using this approach, 20 men who were 35 to 73 years of age, healthy, and nonsmokers were given four cups of green or black tea or caffeine. Blood was taken 60 min later and the oxidation of LDL, induced by exogenous Cu 2+, was analyzed and compared. There is some indication that tea consumption increased antioxidant activity, b u t there are no highly significant differences in assay variables, such as oxidation lag time and total antioxidant activity related to peroxidation of serum components (Hodgson et al., 2000). Therefore, whether green tea catechins affect lipid peroxidation in vivo is still inconclusive at present. It may be necessary to use tests and biomarkers that are more representative of oxidative stress in vivo. 5. L i p i d Peroxidation in Heart and Brain

The heart is a target organ for biological damage caused by oxygen free radicals formed under oxidative stress (Antonius, 1988). Cardiac mitochondria are constantly susceptible to oxidative stress (Hruszkewycz, 1988) and lipid peroxidation in cardiac tissue plays an important role in the pathogenesis of cardiac dysfunction (Fukuchi et al., 1991). High B-carotene levels in adipose tissue are also associated with reduced risk of myocardial infarction (Kardinaal et al., 1993). When lipid peroxidation in rat heart mitochondria is induced with FeSO4, and the inhibitory effect of various catechins on peroxidation is evaluated, EGCG, ECG, and GCG are more active inhibitors than catechins without a gallate group. The IC5o for these gallated catechins is 5 to 16 ~M. Brain tissue contains a high content of oxidizable substrates, such as u n s a t u r a t e d fatty acids and catalytically active metals such as iron and copper, and so this tissue is sensitive to free radical assault. Therefore, catechins can act as radical scavengers and chelate iron to protect brain tissue from oxidative damage. Catechins inhibit the formation of TBARS in rat synaptosomes and EGCG is the most active among green tea catechins, with an IC5o of 100 ~M. The radical scavenging activity of different catechins can be measured with a spin trap probe. ECG and EGCG are the most effective, with ICs0 values of 7 and 15 ~M, respectively. EC and EGC are not as active, suggesting that the gallate ester plays an important role in radical scavenging activity and inhibition of lipid peroxidation in synaptosomes (Guo et al., 1996).

34


6. Cholesterol When male weanling rats are fed a diet containing 1% cholesterol for 28 days, the addition of 1 to 2% green tea catechins to the diet significantly lowers the levels of plasma total cholesterol, cholesterol ester, very low density lipoprotein, and LDL-cholesterol. Decreases in free cholesterol are not statistically significant. HDL levels tend to increase with higher doses of green tea catechins. Green tea catechins also increase fecal total lipids and cholesterol (Muramatsu et al., 1986). EGCG or ECG, at a dosage of 0.5% in diet or 300 mg/kg given orally, is as effective as crude green tea catechins (Fukuyo et al., 1986; Matsuda et al., 1986; Ando et al., 1989), indicating that the green tea catechins EGCG and ECG have a hypocholesterolemic effect and may have a protective effect against the atherosclerotic process. The antihypercholesterolemic effect of EGCG may be due to inhibition of cholesterol absorption from the digestive tract (Chisaka et al., 1988). Green tea does not affect incorporation of[ 14C]acetate into cholesterol (Chisaka et al., 1988) or the activity of 3-hydroxy-3-methylglutarylCoA reductase or cholesterol 7a-hydroxylase (Yang and Koo, 2000). Inhibition of cholesterol absorption may be due to decreased micellar solubility of cholesterol in the presence of EGCG (Ikeda et al., 1992). Male rats fed a basal diet supplemented with 1% cholesterol and 0.5% cholic acid develop a high atherogenic index. However, if the diet is supplemented with a black tea extract at a dosage of 200 mg/kg body wt/day in the drinking water for 20 days, these rats have a smaller atherogenic index than control rats not fed black tea extracts (Yokozawa et al., 1998b). Black tea extracts reduce the plasma level of free and LDL-bound cholesterol in the rat and inhibit in vitro the proliferation of smooth muscle cells and suppress the production of oxidized LDL. The effective agents have not been identified, but they may be metabolites or derivatives of catechins. 7. Hypertension Green tea extracts and catechins have vasodilator effects in vitro (Fitzpatrick et al., 1995). However, green tea and black tea, when consumed at the rate of four to five cups per day and containing about 3% caffeine, cause acute increases in blood pressure that are larger than the increases caused by similar doses of caffeine alone (Hodgson et al., 1999). The tea constituent that causes this increase has not been identified. The acute effect, however, does not translate into significant alterations in 24-h ambulatory blood pressure over longer periods of tea consumption.


35

Tea polyphenols at a dosage of 0.5% in the diet lower blood pressure in rats (Taniguchi et al., 1988) and increase the life span of hypertensive rats (Uchida et al., 1995). When 0.5% EGCG or persimmon tannin is given in drinking water to stroke-prone spontaneously hypertensive rats (SHRSP) during weeks 5 to 51 of life, the incidence of hemorrhage or infarction is 20% for control rats, but 0% for EGCG or persimmon tannin-treated rats. The survival rate was 40% in the controls, but 80 and 100% respectively for EGCG and persimmon tannin-treated rats. It is possible that EGCG, by acting as a radical scavenger, inhibits lipid peroxidation and lessens the incidence of stroke, prolonging the life span of SHRSP. Angiotensin-converting enzyme, which converts angiotensinogen to angiotensin, a key factor in controlling blood osmolarity and blood pressure, is specifically inhibited by EGCG (Hara and Matsuzaki, 1987; Uchida et al., 1987). 8. Platelets a n d Thrombosis

Platelets play an important role in normal hemostasis, as well as in thrombosis when blood vessels are damaged. Thrombus formation occurs through the activation and aggregation of platelets (Di Minno and Silver, 1983). Therefore, inhibition of platelet function may provide a promising approach for prevention of thrombosis. Green tea, but not black tea, consumption significantly reduces thromboxane levels in rats (Ali et al., 1990). Green tea catechins affect vascular smooth muscle tension and aSca2+ uptake in rat aorta (Ahn et al., 1996) and selectively inhibit the intracellular signal transduction pathway of platelet-derived growth factor-BB in vascular smooth muscle cells (Ahn et al., 1999). EGCG given orally to mice 90 min before intravenous injection of a combination of epinephrine and collagen protects mice from paralysis or death due to pulmonary thrombosis (Di Minno and Silver, 1983). Green tea or EGCG at 10 and 50 mg/kg provides 40 and 70% protection from thrombosis (Kang et al., 1999). When mouse tail bleeding time is used as an index for platelet aggregation, green tea or EGCG administered ip at dosages of 4 and 10 mg/kg prolongs tail bleeding time from 64 to about 140 to 200 s. In contrast to this acute effect of EGCG, treatment of rats for i week with EGCG at a dosage of 75 mg/kg causes platelet numbers to double (Kao et al., 2000a). Green tea catechins inhibit in vitro the collagen-induced aggregation of rabbit platelets and the potency of EGCG is similar to aspirin (Sagesaka-Mitane et al., 1990). EGCG also inhibits thrombin- and platelet activating factor-induced aggregation ofplatelets. ECG inhibits ADP-induced human platelet aggregation (Chang and Hsu, 1991). EGCG in vitro inhibits human platelet aggregation induced by many

36

S. LIAO,Y-H.KAO,AND R. A. HIIPAKKA

agents, such as ADP, collagen, epinephrine, and calcium. ICso values are in the range of i to 2 mM. Green tea given orally to rats inhibits ADP or collagen-induced platelet aggregation by 40 to 50% at a dose of 100 mg/kg (Kang et al., 1999). Bovine thrombin amidolysis activity is inhibited by EGCG and ECG with an IC5o of 1 mM (Kinoshita and Horie, 1993). 9. Red Blood Cell a n d Arterial Function

Low vitamin E in the red blood cell (RBC) membrane is associated with increased susceptibility to hemolysis (Delmas-Beauvieus et al., 1995), which can be prevented by antioxidants such as ~-tocopherol. When RBCs are incubated with 2.5 to 40 ~tM EC, ECG, EGC, and EGCG, cells are protected from hemolysis induced by a free radical initiator. Ingestion of green tea extract also causes a significant decrease in the susceptibility of rat RBC to hemolysis (Zhang et al., 1997b). Vibrio cholerae toxin or Staphylococcus aureus s-toxin induces hemolysis of cells and this is also inhibited by 10 to 100 ~M ECG and EGCG (Ikigai et al., 1990). ECG has been shown to stimulate h u m a n blood monocyte production ofinterleukin-1 (IL-1), which enhances host resistance to bacterial infection. EGCG and ECG dimer, trimer, and t e t r a m e r are about five times more effective t h a n ECG at 50 ~g/ml (Sakagami et al., 1992a,b). Catechins noncompetitively reduce the contractile response of rat mesentric arteries in vitro to phenylephrine in a concentration-dependent m a n n e r (Huang et al., 1998). EGCG is the most active catechin and effective doses are about 30 to 100 ~M. Caffeine-induced transient contraction is not affected. D. ALLERGY, ASTHMA, ARTHRITIS, AND THE IMMUNE SYSTEM 1. Allergy

Incidences of allergic disorders, in particular, hypersensitivity to food or environmental allergens, appear to be increasing. Among the four types of allergic reactions, type I allergy plays an important role in the incidence of all allergies against food components and airborne antigens (Metcalfe, 1991). The events involved in the development of type I allergy include production of antigen-specific IgE, which binds to a specific receptor on mast cells or basophils, interaction of IgE with absorbed allergens, and release of chemical mediators, such as histamine and leukotrienes from cells, followed by the elevation ofintracellular C a 2+ concentration (Plaut and Zimmerman, 1993). Interference


37

with any of these processes can lead to an attenuation of allergic symptoms. Agents that are effective include corticosteroids, epinephrine, antihistamines, and inhibitors of leukotriene synthesis (Bochner and Lichtenstein, 1991). Tea catechins have been reported to inhibit chemical mediator release from mast cells (Kakegawa et al., 1985; Ohmori et al., 1995). Some catechins inhibit the calcium ionophore-stimulated release of histamine from rat peritoneal exudate cells in vitro (Table I). EGCG is a potent inhibitor, while EGC and ECG have moderate effects, and CA and EC are not active (Matsuo et al., 1996). EGCG is a dose-dependent inhibitor of histamine release from rat basophilic leukemia cells stimulated with a calcium ionophore. At 10 to 100 ~M, EGCG inhibition is 13 to 59%. GC and ECG moderately inhibit histamine release, whereas CA and EC are not active, suggesting that an o-trihydroxyl group is important for the activity. Triphenols, such as pyrogallol and gallic acid, are active, but diphenols, such as pyrocatechol and resorcinol, are not. EGCG inhibits histamine release induced by IgE-antigen complex formation, but does not inhibit the increase of intracellular C a 2+ in cells due to binding of antigen to IgE. Therefore, EGCG seems to exert its effects on signaling pathways through events occurring after elevation ofintracellular C a 2+ concentrations (Yamada et al., 1997). 2. A s t h m a Green tea-induced asthma is an occupational disease among workers in tea factories. EGCG has been identified as the causative agent (Shirai et al., 1994). In a study involving four men and four women with green tea-induced asthma, EGCG evoked a dose-dependent histamine release in heparinized whole-blood samples taken from five of these individuals. A significant correlation was noted between the maximum percentage histamine release and the threshold EGCG concentration producing a positive intradermal skin test. Green tea-induced asthma appears to be based on a series of physiological events including binding of EGCG-specific IgE on the basophile membrane and then antigenantibody reactions that cause the release of histamine (Shirai et al., 1997). Since clustering of IgE receptors may be required for signaling, a small molecule like EGCG may act as a hapten and bind nonspecifically to multiple sites on proteins and this complex induces a response on binding to IgE. 3. Arthritis Collagen-induced arthritis (CIA) in mice is a widely studied animal model of inflammatory polyarthritis. CIA is induced by immunization

38


of susceptible strains of mice with articular chicken type II collagen (Myers et al., 1997). Using this experimental model, mice fed green tea have a 50% reduction in the incidence of CIA. Green tea significantly lowers the arthritic index; the levels of cyclooxygenase 2, interferon-~/, and tumor necrosis factor-~ (TNF) in arthritic joints are markedly reduced (Table I). In addition, mice fed green tea have lower total and type II collagen-specific IgG levels in serum and in the arthritic joints. Therefore, polyphenols in green tea may be useful in the prevention and treatment of arthritis (Haqqi et al., 1999). 4. Immune Responses Allergen-specific IgA inhibits the allergic reaction through the inhibition of allergen absorption in the gut. Inhibition of IgE production and the stimulation of IgA production may alleviate some allergic reactions. Therefore, studies of the effects of tea catechins on immunoglobulin production are important for finding agents that may control certain aspects of the immune system and allergy. When the effect of green tea catechins on Ig production by mesenteric lymph node lymphocytes of male rats is examined, tea polyphenols having a triphenolic group such as EGCG, ECG, and EGC enhance IgE production at 1 mM, but inhibit it at 100 ~M or below (Table I) (Yamada et al., 1997). EC does not affect IgE production. All catechins tested exert inhibitory effects on the production of IgA and IgG at 10 }zM. EGCG, gallic acid, and pyrogallol enhance IgA production at 0.1 ~M. Catechins appear to exert bifunctional effects on IgE production, stimulatory at high concentrations and inhibitory at low concentrations. Since various tea products contain different concentrations of diphenols and triphenols, their effects on the immune system and allergies may not be easily predicted (Yamada et al., 1997). However, regular consumption of green tea, which is rich in EGCG and provides a blood level of 0.1 ~M EGCG, may be effective in raising the blood level of IgA, while lowering IgE, which may provide a benefit in the control of allergies. 5. Complement, Monocytes, and Macrophages In vivo anticomplement activity of 19 phenolic compounds has been evaluated (Nakagami et al., 1995). Two flavonoids, EGCG and myricetin, exhibit marked anticomplement activities with ICso values of about 10 ~M. The two phenolic compounds appear to interact with both antibody-sensitized erythrocytes and complement components. They may function as natural biological response modifiers by multiple mechanisms and affect the body's autoimmune response.


39

Tea polyphenols, such as EGCG, have been found to stimulate interleukin-1 (IL-1) production by human peripheral blood monocytes in vitro (Table I) (Sakagami et al., 1992b). Since IL-1 enhances host resistance to bacterial infection, EGCG may assist antibacterial activity in blood. EGCG at high concentrations also may be a strong stimulant of human phagocytes (Sakagami et al., 1992a). UV exposure of the skin can cause adverse biological effects, including alterations in cutaneous immune cells. Topical application of EGCG at a dosage of 3 mg/mouse, given before UV irradiation, prevents l_W-induced monocyte/macrophage infiltration into skin inflammatory lesions, which is responsible for the UV-induced immunosuppressive state. EGCG also decreases UV-induced production of the immunomodulatory cytokine IL-10, but markedly increases IL-12 (Table I), which is a mediator involved in contact sensitivity (Katiyar et al., 1999). E. DIABETES Bai-Yu-Cha (BYC) is prepared from the catechin-rich tender leaves of old tea trees growing in certain areas in China. It is used as a Chinese medicine for treatment of diabetes. The aqueous extract of BYC (10 g/kg), orally administered to mice, protects pancreatic islets from necrosis and cell degranulation, experimentally induced by alloxan (Zhu et al., 1990). Orally administered, BYC at a dosage of 1.5 g/kg also decreases blood glucose concentration in normal rabbits. Other Chinese teas, such as Fujing woolong tea or Zhejiang Long-Jing green tea, do not show any antidiabetic action in animal tests. EC, EGC, GC, and caffeine individually do not have any antidiabetic activity. However, mixtures reconstructed from the isolated compounds according to the relative levels of these four compounds, as determined by HPLC, reproduce the protective action against diabetes induced by alloxan in mice. The blood-lowering effect of the prescription mixture is comparable to that of clinically used antidiabetic drugs such as Daonil and Glimicron (Zhu et al., 1990). EC was once identified as the active component of an Indian folk medicine for treatment of diabetes (Chadrawarthy et al., 1980); however, in a later study (Bone et al., 1985), EC was not active in reversing established diabetes in drug-induced or spontaneously diabetic rats. In a case-control study in Finland, the risk for type 1 (insulindependent) diabetes in 600 newly diagnosed children (14 years or younger) was higher in those who consumed at least two cups of coffee or one to two cups (110 ml/cup) of tea daily (Virtanen et al., 1994). About 60% of diabetic children consumed more than one cup of tea, while only

40


33% of nondiabetic children consumed tea. The mother's consumption of coffee or tea during pregnancy did not affect the risk for diabetes in their children. The nonspecific interaction of catechins with proteins and various food components probably will reduce digestibility and absorption of food components. Many reports have also shown that catechins can, in vitro, inhibit digestive enzymes, such as salivary s-amylase (Hara and Honda, 1990; Hara, 1997), intestinal sucrase, and (~-glucosidase (Honda et al., 1993; Honda et al., 1994), which suggests that the reduced digestibility may be responsible for lowering blood glucose and insulin levels (Goldstein and Swain, 1965; Matsumoto et al., 1993; Zhang et al., 1998). The ability of EGCG to lower blood levels of glucose, insulin, and lipids in rats may in some cases be dependent on other mechanisms, such as alterations in appetite (Kao et al., 2000a). Tea extracts (Kreydiyyeh et al., 1994; Murata et al., 1994) and gallated catechins (Shimizu, 1999) inhibit intestinal glucose transporters, indicating that they may be useful as functional foods for diabetic patients. Aldose reductase catalyzes the conversion of aldoses to sugar alcohols, and it is a key enzyme in the synthesis of polyol sugars that cause diabetic complications, such as cataracts, retinopathy, neuropathy, and nephropathy (Kador et al., 1985). The enzyme is considered to be a target enzyme for the pharmacological control of diabetes-related pathologies. Several natural and synthetic compounds have been shown to be inhibitors of aldose reductase. Some of the green tea catechins have been shown to be active inhibitors. EC and ECG are active and EGCG is weakly active, while EGC is not active. Catechins with a catechol (dihydroxylated) structure in ring B are more active than catechins with a trihydroxylated ring B, and gallated catechins have increased inhibitory activity. The IC50 for ECG is 38 ~M (Murata et al., 1994). F. OBESITY

In oriental countries, long-term use of green tea beverage is considered to be beneficial for keeping a healthy body weight. However, clear scientific evidence for an effect of green tea on body weight has not been available until recently. As discussed in Section V,A,3, EGCG given to rats by ip injection at a dosage of 50 to 90 mg EGCG/kg body wt daily could within 2 to 7 days reduce body weight by about 20 to 30% (Liao and Liang, 1997; Kao et al., 2000a). Other structurally related catechins, such as EC, EGC, and ECG, are not effective at the same dose. Reduction of body weight appears to be due to EGCG-induced reduction in food intake. The loss of appetite might involve neuropeptide(s)


41

other than leptin, since EGCG is effective in reducing body weight of lean and obese (leptin-receptor negative) female and male rats (Fig. 5). The effective dose of EGCG is, at first, 30 to 50 mg EGCG/kg body wt. However, rats gradually adapt and within 1 week higher doses of EGCG (100 mg/kg body wt) are needed to reduce or prevent body weight increases. The body weight loss is reversible; when EGCG administration is stopped, animals regain body weight (Kao et al., 2000b). The EGCG effect on food intake is apparently not dependent on an intact leptin receptor. Lean (leptin-receptor positive) and obese (leptinreceptor deficient) male (Figs. 5A and 5C) and female (Figs. 5B and 5D) Zucker rats treated with EGCG lose body weight and have lower serum levels of sex hormones, leptin, IGF-I, and insulin (Kao et al., 2000a,b). EGCG may interact specifically with a component of a leptin receptor-independent appetite control pathway and reduce food intake. Various hormones, including cholecystokinin, glucagonlike peptide-1, glucagon, substance P, somatostatin, and bombesin, have been reported to inhibit food intake and plasma cholecystokinin levels are elevated in rats given a diet supplemented with tea polyphenols (Yang et al., 1992). Further study is required to determine whether the expression of other hypothalamic or gastrointestinal neuropeptide genes that control appetite are altered by EGCG and perhaps responsible for the effect of EGCG on food intake. Although orally administered EGCG is not as effective as ip injected EGCG, probably due to the difficulty in absorption of EGCG from the intestine, long-term oral use of green tea beverage (two to four cups per day) or EGCG-containing drinks may mimic the effects of ip injected EGCG. Since EGCG can also selectively reduce body fat accumulation (Sections V,A,3 and V,A,4), EGCG may be useful for treatment of obesity. EGCG-containing green tea extracts that contain caffeine are more potent than caffeine alone for stimulating 24-h energy expenditure in humans (Dulloo et al., 1999) and for stimulating in vitro the respiration rate of rat brown adipose tissue (Dulloo et al., 2000). The in vitro thermogenic effect of a green tea extract on brown adipose tissue could be mimicked by EGCG. EGCG also reduces total triglyceride accumulation of murine 3T3-LI preadipocytes during their differentiation to adipocytes (Kao et al., 2000b) or in differentiated 3T3-L1 adipocytes (Watanabe et al., 1998). Reduced triglyceride accumulation may be due to EGCG inhibition of the activity of the key enzyme in lipid synthesis, acetyl-CoA carboxylase (Watanabe et al., 1998). EGCG, however, also inhibits the proliferation of 3T3-L1 preadipocytes (Kao et al., 2000b). The ICso for EGCG is about 10 ~M, and at this concentration EGCG, but not EC, EGC, or ECG, inhibits insulin-induced increase in cell

42


number and triglyceride content during fat cell differentiation. At 10 to 100 ~M EGCG also reduces cell number and trigylceride content of differentiating preadipocytes treated with dexamethasone and 1-methyl3-isobutylxanthine in the presence or absence of insulin. Therefore, the in vitro effect of EGCG on fat tissues may be mediated by modulation of hormone-stimulated cell proliferation or by inhibition of fat cell functions, and this effect may be related to the effect of EGCG on obese animals (Fig. 5) (Kao et al., 2000a). G. ORALHEALTH In oriental countries, tea drinking is often taken after meals for "keeping the mouth clean." Tea leaves are rich in fluoride, which is known to enhance dental health. However, the possible dental health benefits of tea are not solely due to fluoride, but involve other tea components (Onisi et al., 1981a,b). Dental carries and periodontal diseases are induced by oral microflora. Among hundreds of microorganisms in the oral cavity, only the cariogenic streptococci, especially Streptococcus mutans, play key roles in causing dental caries (Hamada and Slade, 1980). Several green tea polyphenols have preventative effects on dental caries (Sakanaka et al., 1989, 1990, 1992). Among the catechins, GC and EGC are most active, inhibiting the growth of 10 strains of cariogenic bacteria. ECG and EGCG are less active, whereas CA and EC are not active at these concentrations (Sakanaka, 1989). Cariogenic bacteria synthesize water-soluble and -insoluble glucans using glucosyltransferase (GTase). Highly branched glucans are responsible for bacterial cell adherence to the tooth surface (Hamada and Slade, 1980). ECG, GCG, and EGCG strongly inhibit GTase at 20 to 50 ~g/ml and inhibit adherence of the bacteria to glass surfaces (Sakanaka et al., 1990; Otake et al., 1991). Inhibition of the adherence of bacteria is apparently related to the gallate moiety, but gallic acid is not inhibitory. Porphyromonas gingivalis has been identified as the bacterium that most frequently causes inflammatory and destructive lesions in periodontal tissue. The bacteria adhere to host tissue cells, colonize, and produce virulent factors, including collagenase, that injure host tissue cells (Slot and Gibbsons, 1978). Tea catechins inhibit the activity of collagenase (Makimura et al., 1993) and bacterial adherence to human buccal epithelial cells. At 20 to 50 ~g/ml, gallated catechins, such as EGCG, ECG, and GCG, almost completely inhibit bacterial adherence (Sakanaka et al., 1996). In mice, bacteria-induced periodontal disease is reduced by tea polyphenols in the diet or in drinking water (Katoh, 1995).


43

Caries formation in rats is inhibited by the addition of green tea polyphenols at concentrations of 0.1 to 0.5% to the diet or drinking water. In humans, a double-blind study showed that rinsing the mouth after meals with 0.05 to 0.5% green tea polyphenols for 3 days inhibits dental plaque formation by 30 to 43% (Sakanaka, 1997). In two primary schools, children drinking only one cup a day of green tea after school lunch have reduced dental caries. The effectiveness of green tea catechins against dental caries also has been observed in other countries (Rosen et al., 1984; Elvin-Lewis and Steelman, 1986). Tea polyphenols added to sugar-containing foods, such as chocolate, candy, and biscuits, reduce the incidence of dental caries in rats previously infected with Streptococcus mutans. Therefore, tea polyphenols have been added to various confectioneries. In fact, tea polyphenols added to chewing gum are effective in decreasing dental plaque formation in humans (Sakanaka, 1997). The effect of tea catechins on methyl mercaptan (MSH), a main source of halitosis, has been studied (Ui et al., 1991). Deodorant activity decreased in the following order: EGCG > EGC > ECG > EC. Chewing gum containing tea catechins significantly depressed MSH production from saliva containing L-methionine and apparently was useful in reducing bad breath. The deodorizing effect of EGCG involves a chemical reaction between EGCG and MSH (Yasuda et al., 1995). The reaction involves introduction ofa methylthio and/or a methylsulfinyl group into the B ring of EGCG. During this reaction, a methylthio group is added to the orthoquinone form of the catechin generated by oxidation with atmospheric oxygen. H.

NERVOUS SYSTEM AND MEMORY

Temporary ischemia of the brain or heart has been reported to increase the amount of reactive oxygen species in these tissues, which causes injury (Oliver et al., 1990). Glucose oxidase converts D-glucose to glucuronic acid with the production of hydrogen peroxide. Tea catechins, such as CA and EC, at dosages of 1 to 100 ~M protect cultured newborn mouse cerebral nerve cells from death induced by glucose oxidase. The potency of EGCG is weaker than that of EC and CA. To study possible in vivo effects of green tea catechins, the learning ability of mice was assessed by analyzing memory impairment of mice subjected to oxidative stressors. Intracisternal injection of EC at a dosage of 0.4 ~mol/mouse lessens memory impairment caused by intracisternal injection of glucose oxidase, whereas intravenous injection of CA or EC at a dosage of 100 mg/kg body wt lessens memory impairment

44


caused by cerebral ischemia induced by occlusion of the common carotid arteries. EGCG is not active. These findings indicate that tea catechins can ameliorate injuries or impairments induced by reactive oxygen species through scavenging of intracellular reactive oxygen species and may be useful for protecting against senile disorders, such as dementia (Matsuoka et al., 1995). Chronic administration of decaffinated green tea in the drinking water of mice exposed to psychosocial stress significantly reduces physiological stress markers and behavioral responses (Henry and StephensLarson, 1984). Some flavonoids may have a low affinity for the benzodiazepine receptor and play a role in neurochemical responses (Medina et al., 1997). These studies raise the possibility that tea may have psychopharmacological effects that act independently or in conjunction with caffeine. A study with 19 healthy volunteers used psychometric tests to evaluate whether a subject's ability to distinguish discrete sensory data, sensorimotor reaction to a critical stimulus, shortterm memory, and subjective sedation changed following administration of black tea beverage. It was concluded that while caffeine is generally regarded as a mild central nervous system stimulant, other factors in tea are likely play a significant role in mediating responses with regard to cognition and psychomotor performance (Hindmarch et al., 1998).

I. OSTEOPOROSIS

High caffeine intake is reportedly a risk factor for reduced bone mineral density (BMD). However, tea is reported to protect against hip fractures (Kanis et al., 1999). A recent study involving 1256 women, 65 to 76 years of age, in the United Kingdom (Hegarty et al., 2000) showed that tea (probably black tea) drinkers, whether consuming one to six cups or more per day, have significantly greater mean BMD measurements. This finding is independent of smoking habits, use of hormone replacement therapy, coffee drinking, and whether milk is added to tea. These results suggest that flavonoids may influence BMD and tea drinking may protect against osteoporosis in older women. J. ANTIBACTERIAL ACTMTY

1. Antibacterial Activity

Methicillin-resistant Staphylococus aureus (MRSA) is a serious problem in hospitals because it is a major cause ofnoscomial infections. Most


45

common antibacterial agents, such as methicillin and other ~-lactams, are not effective for treatment of patients infected with MRSA. Although the reasons for the multidrug resistance of MRSA are not very clear, both genomic and nongenomic factors appear to be involved (Wu et al., 1996; Ito et al., 1999). Although individual catechins (EC, EGC, ECG, and EGCG) have antibacterial activity, the minimum inhibitory concentrations (MIC) are fairly high (128 ~g/ml or above). However, when each catechin is tested in combination with ~-lactams, low concentrations of catechins reduce the MICs of ~-lactams. MIC of oxacillin is reduced 250- to 500-fold by 80 ~M ECG and 8- to 120-fold by 110 ~M EGCG. EC and EGC are only slightly active, suggesting that the gallate group is important. It appears that gallated catechins restore the effectiveness of ~-lactams against MRSA. The effect of gallated catechins are specific; they are effective for other ~-lactams, but not for other types of antibacterial agents, such as erythromycin and tetracycline (Shiota et al., 1999). Clostridia are responsible for much human morbidity and mortality due to toxicity, mutagenicity, and carcinogenicity. They can cause biotransformation of ingested or endogenously formed compounds to harmful products like N-nitroso compounds or toxic aromatic metabolites. Microflora in eldery or cancer patients are mainly clostridia and eubacteria. ECG and EGCG, but not EC or GC, inhibit the growth of clostridia (Hara and Watanabe, 1989; Ahn et al., 1991). The growth of bacteria that infect vegetables is inhibited by EGC and EGCG, but not EC or ECG. Therefore, the ring B gallyl group seems to be more important than the gallate group for activity (Fukai et al., 1991). However, when 16 strains of foodborne pathogenic bacteria were studied, gallated catechins, such as ECG and EGCG, showed very potent antibacterial activities, while EC and EGC were not as active (Hara and Watanabe, 1989). Helicobacter pylori infection is associated with upper gastointestinal diseases, such as chronic gastritis, peptic ulceration, and gastric cancer. This bacterium is sensitive to various antibiotics in vitro, but most clinical studies find that it is difficult to eradicate H. pylori. Among six catechins tested for their bactericidal effect, EGCG is the most active; the MIC for some strains tested is 18 ~tM. EGCG is active at pH 7, but not at pH < 5. In infected Mongolian gerbils, H. pylori is eradicated in 10 to 36% of the catechin-treated animals. Mucosal hemorrhage and erosion are significantly decreased by catechin treatment (Mabe et al., 1999). Enterohemorrhagic Escherichia coli (EHEC) is ingested as a contaminant in food and colonizes the large intestine, producing and releasing

46


Vero toxins (Gyles, 1992), which cause hemorrhagic colitis in humans (O'Brien et al., 1984). EGCG and GCG, at 110 ~M or higher, significantly inhibit production and extracellular release of Vero toxins from cultured EHEC in vitro (Sugita-Konishi et al., 1999). CA, EC, ECG, or EGC did not affect the release of toxin, suggesting that the effect is dependent on both the gallate ester and the trihydroxy group on ring B. Catechin inhibition of toxin release is not selective; release of other proteins from bacterial cells is also inhibited. EGCG also has antifungal activity against Cryptococcus neoformans and the IC5o for this activity is 3.5 ~M (Li et al., 1999b).

2. Membrane Fluidity

Catechin inhibition of bacterial growth may be due to catechin interaction with cellular membranes. This may affect membrane fluidity and disrupts barrier function and cell morphology (Ikigai et al., 1993; Tsuchiya, 1999). The immune-enhancing activity of catechins is thought to involve catechin interaction with membranes of target cells (Brattig et al., 1984). Catechin interactions with membranes have been studied by using phosphatidylcholine liposomes (Ikigai et al., 1993). Strongly bactericidal EGCG causes leakage of 5,6-carboxyfluorescein from liposomes, but very weakly bactericidal catechins, such as EC cause little damage to liposomes. When the amounts of EGCG bound to bacteria cells per unit weight of bacteria are compared, gram-positive S. aureus binds 2.5 times more EGCG than does gram-negative E. coli. Gram-negative bacteria have a strong negative charge on their cell surface with a tight penetration barrier in the outer membrane against hydrophobic and large hydrophilic compounds. This barrier may contribute to the stronger resistance of gram-negative bacteria to EGCG (Ikigai et al., 1993). The effects of catechins on membrane fluidity have also been studied by using a fluorescence polarization method and liposomes (Tsuchiya, 1999). Catechins with gallate esters, EGCG, GCG, ECG, and CG, increase polarization more significantly than the corresponding nongallated catechins, EGC, GC, EC, and CA. Larger changes in polarization of liposomes are caused by cis-catechins (EGCG, ECG, EGC, and EC) than by trans-catechins (GCG, CG, GC, and CA). The effective dose for EGCG is 2.5 ~M, whereas it is 250 IxM for CA. Since an increase in polarization means a reduction of membrane fluidity, these observations are consistent with the suggestion that catechins act by reducing membrane fluidity.


47

K. ANTMRALACTIVITY Tea polyphenols inhibit the activity of influenza virus (Nakayama et al., 1990), vaccinia virus, herpes simplex virus, Coxsackie virus B6, and poliovirus 1 (John and Mukundan, 1979). EGCG and theaflavin digallate inhibit infection of cultured rhesus monkey kidney cells with rotaviruses and enteroviruses. Depending on the strain of virus, 50% inhibition of the infectivity of viruses is observed with 18 to 273 ~M EGCG. Since the effect is more pronounced when EGCG is added directly to virus than when EGCG is added prior or after cells are treated, the catechin effect is apparently due to its interference with virus adsorption to the cell (Mukoyama et al., 1991). Similar conclusions have been obtained in a study of EGCG inhibition of the infection of canine kidney cells by influenza virus. Based on studies using electron microscopy, EGCG causes agglutination of viruses and prevents the virus from adsorbing to kidney cells, blocking its infectivity (Nakayama et al., 1990, 1993). In a study of TPA-induced Epstein-Barr virus activation in human lymphoblastoid Raji cells in vitro, EGC has the highest inhibitory activity with 63% inhibition at 65 ~M, whereas GC, ECG, and EGCG have moderate activity. CA and EC activity is insignificant. Ethyl gallate inhibits about 30% at 50 ~M (Hagiwara et al., 1991). Catechins inhibit Moloney murine leukemia virus and human immunodeficiencyvirus (HIV) reverse transcriptase activity in an in vitro cell-free system (Nakane and Ono, 1990; Chang et al., 1994). The IC50 values for catechins are EGCG, 0.7 ~M; ECG, 0.3 ~M; and EGC, 8 ~M. The gallate group (ring C) appears to enhance inhibitory activity (Chang et al., 1994). Inhibition is not observed when serum albumin is added to assays, suggesting that the inhibition of HIV reverse transcriptase is a nonspecific phenomenon (Moore and Pizza, 1992). The effects of catechins and other polyphenols on HIV-1 replication in lymphocytes have also been studied in vitro (Hashimoto et al., 1996). While several polyphenols are active, polyphenols from green tea that have anti-HIV activity are active only at toxic concentrations. EGCG does inhibit HIV replication in lymphocyte cells. These finding are consistent with the observation that in vitro reverse transcriptase inhibition does not correlate with inhibition of HIV replication. As shown in Table II, catechins are inhibitors of various nucleic acid polymerases in cell-free systems (see Section VI,B,10). In general, EGCG and ECG are active at 0.2 to 1 ~M, and EC and EGC are active at about 10 ~M or not active. The biological significance of these observations remains to be determined.

48

S. LIAO,Y-H. KAO,AND R. A. HIIPAKKA TABLE II EGCG INHIBITIONOF ENZYMATICACTMTYIN CELL-FREESYSTEMSa Enzymes b

IC50

References

Dental caries-related enzymes Glucosyltransferase I

~6 ~M

Sakanaka et al. (1990)

Diabetes-related enzymes Aldose reductase c

38 ~M

Murata et al. (1994)

DNA or RNA polymerases and topoisomerase DNA polymerase I 0.2 ~M DNA polymerase-a, -~, -~/ 0.3, 0.3, 1.3 ~M RNA polymerase 0.3 ~M AMBV reverse transcriptase c 1 ~M HIV-1 reverse transcriptase 0.045 ~M MMLV reverse transcriptase 1.0 ~M RMLV reverse transcriptase 0.3 ~M Telomerase 1 ~M Topoisomerased 5-50 ~M

Nakane and Ono (1990) Nakane and Ono (1990) Nakane and Ono (1990) Moore and Pizza (1992) Nakane and Ono (1990) Moore and Pizza (1992) Nakane and Ono (1990) Naasani et al. (1998) Austin et al. (1992)

Fibrinolytic proteinases Plasminogen activator Plasma kallikrein Plasmin c Thrombin

Kinoshita Kinoshita Kinoshita Kinoshita

1.5 ~M 0.8 ~M 97 ~M 1.1 tLM

and and and and

Horie (1994) Horie (1994) Horie (1994) Horie (1993)

Gastrointestinal digestive enzymes a-Amylasee 20 ~M Esterase Km = 90 ~M H+,K+-ATPase 0.1 ~M

Hara and Honda (1990) Yang et al. (1999) Murakami et al. (1992)

Gelatinase/collagenase Collagenase Matrix metalloproteinase-2 Matrix metalloproteinase-9 Matrix metalloproteinase-12

Sazuka et al. (1997) Maeda-Yamamoto et al. (1999) Garbisa et al. (1999) Demeule et al. (2000)

1 ~M 6 ~M 0.3 ~M 1 ~M

Hormone metabolism-related enzymes Prolyl endopeptidase 1.5 ~M Type 1 5~-reductase 15 ~M Type 2 5a-reductase 74 ~M Tyrosinase 34 ~M Kinases Cyclin-dependent kinase 2 Cyclin-dependent kinase 4 Casein kinase-II kinase EGF receptor FGF receptor Protein kinase C MAP kinase

18 ~M 20 ~M 0.3 txM 1.1 ~M 2.2 ~M >44 ~M 60 ~M

Fan et al. (1999)

Liao and Hiipakka (1995) Liao and Hiipakka (1995) No et al. (1999) Liang et al. (1999) Liang et al. (1999) Maekawa et al. (1999) Liang et al. (1997) Liang et al. (1997) Liang et al. (1997) Yasokawa et al. (1999) (continues)

GREEN TEA: BIOCHEMICALAND BIOLOGICALBASISFOR HEALTHBENEFITS

49

TABLE II (continued) Enzymes b PDGF receptor Protein kinase A pp60v-src Lipid-related enzymes GB lipoxygenase Pancreatic lipase Lipoxygenase Squalene epoxidase Oxidases and peroxidases Horseradish peroxidase Xanthine oxidase Phases I and II enzymes CYP 2C6 CYP 1A2 CYP 2 C l l Glucuronosyltransferase NADPH-CYP450 reductase Respiratory chain-related enzyme NADH dehydrogenase

ICb0

References

2.3 }xM >44 ~M >22 trM

Liang et al. (1997) Liang et al. (1997) Liang et al. (1997)

10 ~M 11 tLM 10 #xM 0.7 ~M

Furuya et al. (1997) Shimura et al. (1994) Ho et al. (1992) Abe et al. (2000)

4 IxM >40 #~M

Mendez and Mato (1997) Hatano et al. (1990)

O.25 tLM 0.25 ~M 0.25 ~M 150 mM), requiring substantially higher concentrations of this substrate than those typically found in vivo (Kurahashi et al., 1997; Katayama et al., 1999). Finally, the upregulation of free arachidonic acid levels does not lead to corresponding increases in the levels of anandamide in cultured neurons, suggesting that arachidonic acid is not the direct precursor of anandamide (Di Marzo et al., 1994). In summary, the majority of evidence accumulated to date argues that the production of NAEs in vivo does not occur through the energy-independent condensation of free fatty acid and ethanolamine. A second proposed route for NAE biosynthesis involves a two-step enzymatic mechanism. First, a fatty acyl chain is transferred in a Ca 2+dependent manner from the sn-1 position of a phospholipid to the primary amine of phosphatidylethanolamine, forming an N-acylphosphatidylethanolamine (NAPE) (Fig. 2). This NAPE intermediate is then hydrolyzed by a phospholipase D-like enzyme to yield the corresponding NAE (Natarajan et al., 1981; Schmid et al., 1983; Di Marzo et al., 1994). This mechanism for NAE production was first characterized by Natarajan and coworkers in 1981, who reported the formation of saturated and monounsaturated NAEs through a route mediated by a Ca 2+dependent transacylase (CDTA) and a phospholipase D (Natarajan et al., 1981). The CDTA enzyme has since been characterized biochemically in both brain and testis microsomal preparations (Di Marzo et al., 1994; Sugiura et al., 1996b,c; Cadas et al., 1997). Although no CDTA activity has yet been purified or cloned, the enzyme displays several intriguing properties discernible in both cell and tissue extracts. The CDTA exclusively transfers acyl chains from the sn-1 position of phospholipids and exhibits a broad specificity for the transferred acyl chain (Sugiura et al., 1996b,c; Cadas et al., 1997). Serine hydrolase inhibitors like PMSF and DIFP either do not inhibit (Sugiura et al., 1996b) the CDTA or are fairly weak inhibitors (Cadas et al., 1997). Perhaps the most striking property of the characterized

100

MATTHEWP. PATRICELLIAND BENJAMIN F. CRAVATT 0

R Phosphatidylethanolamine

R Phosphatidylcholine CDTA

0

* j.o o=={ R

0

iYo

N

R

o-r-o

,o, PhospholipaseD

N-Acylphosphatidylethanolamine( N A P E )

x

R 0==( R

Phosphatidic acid

II

0

N-Acylethanolamine(NAE)

FIG. 2. The two-step pathway proposed for the biosynthesis of NAEs is shown. In the first step, a Ca2+-dependent transacylase (CDTA) enzyme transfers a fatty acid from the sn-1 position of phosphatidylcholine to the primary amine substituent of phosphatidylethanolamine to generate an N-acyl phosphatidylethanolamine (NAPE). The resulting NAPE is then hydrolyzed by a phospholipase D-like enzyme to yield the N-acyl ethanolamine (NAE) and phosphatidic acid.

CDTA is the strict requirement its activity shows for Ca 2+ (Sugiura et al., 1996b,c; Cadas et al., 1997). No CDTA activity is observed in the absence of Ca 2+, and EDTA, Mg2+, Cd 2+, Zn 2+, and Ag 1+ inhibit the enzyme (Cadas et al., 1997). Importantly, both crude tissue and cell culture systems have been found to accumulate NAPEs and NAEs in a coupled calcium-dependent manner, suggesting that the rate limiting step for the formation of NAEs is likely the CDTA-dependent generation of NAPEs (Di Marzo et al., 1994; Cadas et al., 1997). In general support of this notion, the relative levels of NAPEs correlate well with the levels of the corresponding NAEs in tissue extracts (Sugiura et al., 1996b,c; Cadas et al., 1997). The second step in NAE biosynthesis catalyzed by a PLD-like enzyme has not yet been extensively investigated. Several exogenous PLDs have been found to produce NAEs from NAPE- containing membranes (Cadas et al., 1996), and therefore, identifying the precise PLD

METABOLISM OF FATTY ACID AMIDES

101

responsible for NAE biosynthesis in vivo may be complicated by the presence of multiple enzymes capable of catalyzing the reaction. Notably, however, Petersen and Hansen have recently described a unique PLD activity in rat heart and brain microsomes that may represent a dedicated NAPE-hydrolyzing PLD involved in NAE production in vivo (Petersen and Hansen, 1999). It is worth noting that the relevance of the CDTA-PLD pathway to the production of endogenous anandamide was initially unclear, as phospholipids containing arachidonic acid at the sn-1 position had not been previously characterized in viva However, recent measurements of cellular phospholipid composition of brain and testis by Sugiura and colleagues (Sugiura et al., 1996b,c) and Cadas and colleagues (Cadas et al., 1997) have confirmed the presence of sn-1 arachidonoyl-containing lipids in these tissues. Further, N-arachidonoylphosphatidylethanolamine and several other NAPEs were also detected in these tissues (Di Marzo et al., 1994; Sugiura et al., 1996b,c; Cadas et al., 1997). Interestingly, however, the levels of N-arachidonoylphosphatidylethanolamine and anandamide were much lower than other NAPEs and NAEs. Establishing a firm link between increases in intracellular C a 2+ levels and NAE production would place these bioactive lipids at the center of neuronal signaling networks. While the CDTA's dependence o n C a 2+ is consistent with such a connection, the C a 2+ concentrations required for CDTA activity in vitro (0.5-3 mM) (Natarajan et al., 1981) are significantly higher than those typically found in vivo, even following the release of intracellular C a 2+ stores. This observation has led some to suggest that NAEs serve a role as neuroprotective agents in pathophysiological processes, since extremely high concentrations ofintracellular C a 2+ a r e typically restricted to damaged or dying cells (Hansen et al., 1998). In support of this notion, NAEs have been characterized as having some neuroprotective properties (Skaper et al., 1996; Sinor et al., 2000). Additionally, the levels of NAPEs and NAEs are significantly higher in ischemic and/or damaged tissues and some investigators have been unable to observe detectable levels of NAEs in fresh tissue (Epps et al., 1980; Schmid et al., 1995). Despite some doubts regarding the physiological production of NAEs, many laboratories have isolated detectable quantities of NAEs, including anandamide, even when suitable precautions were taken to eliminate postmortem NAE production (Sugiura et al., 1996c; Cadas et al., 1997; Yang et al., 1999). Additionally, several lines of evidence support the involvement of NAEs in physiological, as opposed to exclusively pathophysiological, processes. First, C a 2+ release in neuronal cell culture systems caused by glutamate or ionomycin is sufficient to cause

102

MATTHEW P. PATRICELLI AND BENJAMIN F. CRAVATT

increases in the levels of NAPEs and NAEs prior to detectable signs of neurotoxicity (Hansen et al., 1997). Additionally, two studies have documented the release of anandamide in specific brain regions in response to physiological stimuli (Giuffrida et al., 1999; Walker et al., 1999). On this subject, it is important to emphasize that the high levels of C a 2+ required for CDTA activity in vitro do not preclude this enzyme's involvement in the physiological production of NAPEs and NAEs. For example, the CDTA's high in vitro C a 2+ requirement may reflect a suboptimal activity for the enzyme that results from artificial phospholipid environments present in lipid reconstitution assays. Additionally, the precise subcellular distribution of CDTA is currently unknown. If this enzyme resides in close proximity to C a 2+ channels, it may experience extremely high localized concentrations of C a 2+ generated during normal signaling events. On this note, other proteins such as synaptotagmin, which have been shown to associate physically with Ca 2+ channels (Charvin et al., 1997), are believed to be involved in Ca 2+mediated signaling events in vivo despite displaying high micromolar binding affinities for C a 2+ (Sudhof and Rizo, 1996). Although the biological properties of NAEs other than anandamide and N-palmitoylethanolamine remain mostly unexamined, the abundance of these NAEs in vivo suggests that they may also have important biological functions. Notably, saturated and monounsaturated NAEs are produced by the CDTA-PLD pathway in much greater quantities than anandamide (Sugiura et al., 1996b,c; Cadas et al., 1997), possibly raising some concern about the relevance of this route to anandamide's biosynthesis. Whether anandamide is just one of many bioactive NAEs that are coordinately released by the CDTA-PLD pathway, or a separate and more specific biosynthetic route exists for anandamide production, is currently unknown. Regardless, the possibility remains that the physiological production of NAEs may proceed through an alternative pathway independent of the CDTA activity depicted to date through in vitro efforts. The resolution of this issue will likely require the molecular identification and detailed biochemical characterization of the enzymes participating in the proposed biosynthetic pathway(s) for NAEs. B. FAPAs At least two models have been proposed for the biosynthesis of FAPAs. First, Merkler and colleagues have hypothesized that FAPAs are endogenously derived from their glycine adducts (Merkler et al., 1996) (Fig. 3). This biosynthetic route would require first the production of

103

METABOLISM OF FATTYACID AMIDES O

0

HOJ~

NH2 +

?, ACGNAT

O

,.

H

.o

CoA

O PAM O

O

O

Glyoxalate

FAPA

FIo. 3. Merkler and colleagues have proposed a two-step biosynthetic route for fatty acid primary amides (FAPAs) through the intermediate generation of an N-acyl glycine. The N-acyl glycine would be formed from glycine and the fatty acyl-CoA through an as-of-yetunknown enzyme or in the liver by acyl-CoA glycine N-acyl transferase (ACGNAT). This acyl glycine would then be oxidatively cleaved by peptidyl glycine ~-amidating monooxygenase (PAM) to generate the FAPA and glyoxylate.

fatty acyl glycines by an as-of-yet-unidentified enzyme, followed by the oxidative cleavage of this acyl glycine by peptidyl glycine ~-amidating monooxygenase (PAM) to yield the corresponding FAPA and glyoxalate. PAM is a well-characterized enzyme involved in the production of C-terminally amidated neuropeptides (Eipper et al., 1992). Recent in vitro studies have demonstrated that PAM efficiently generates oleamide and other FAPAs from their corresponding glycine adducts, displaying a higher catalytic efficiency for these lipids than for its best peptide substrates (Wilcox et al., 1999). The presence of PAM in NlsTG2 cells (Ritenour-Rodgers et al., 2000), a cell line in which the in vivo production of oleamide has been demonstrated (Bisogno et al., 1997b), is consistent with a potential role for this enzyme in FAPA biosynthesis. However, before PAM can gain general acceptance as an enzyme involved in FAPA biosynthesis, clarification is needed regarding the postulated presence of fatty acyl glycines in vivo. To date, fatty acyl glycines have not been detected in mammalian tissues. Notably, liver enzymes have been characterized that transfer glycine to the acid groups of xenobiotics to facilitate their subsequent excretion (Gregersen et al., 1986; Kelley and Vessey, 1993; Mawal and Qureshi, 1994). It is conceivable that these or related enzymes could produce fatty acyl glycines in vivo, but the ability of such enzymes to accept fatty acyl CoAs as substrates has not been demonstrated, and their presence in tissues where

104

MA~'rHEWP. PATRICELLIAND BENJAMINF. CRAVATT

FAPAs are likely to be produced is currently unknown. Nonetheless, a PAM-dependent mechanism for FAPA biosynthesis is a biologically attractive model in that it mimics the biosynthesis of neuropeptides and merits further characterization to test its validity. A second possible route to FAPAs could occur through a glutaminedependent amination of an activated form of oleic acid. Glutamine serves as an ammonia source for many types of amination reactions in vivo (Walsh, 1979), and Sugiura and colleagues have observed a modest glutamine-dependent biosynthesis of oleamide from oleic acid in whole brain extracts (Sugiura et al., 1996a).

III. THE ENZYMATICINACTIVATIONOF FATTYACID AMIDES

A. FATTYACIDAMIDEHYDROLASE(FAAH) MEDIATEDINACTIVATION OF FAAs

The neurophysiological effects of FAAs, in conjunction with their isolation from cerebrospinal fluid and brain tissue, suggest that these compounds may serve important signaling roles in the central nervous system (CNS). However, if bioactive molecules like FAAs are to be generally accepted as in vivo participants in brain function, they must first fulfill several criteria. One expectation is that the molecules under consideration be closely linked to a mechanism for their expeditious inactivation. FAAs seem to satisfy this requirement, as a membranebound enzymatic activity that hydrolyzes these compounds to their corresponding fatty acids has been described (Schmid et al., 1985; Deutsch and Chin, 1993; Desarfiaud et al., 1995; Maurelli et al., 1995; Ueda et al., 1995; Cravatt et al., 1996). This hydrolytic activity was originally described by Schmid and colleagues during their initial characterization of saturated and monounsaturated NAEs as endogenous constituents of mammalian tissues (Schmid et al., 1985). A similar enzymatic activity that converted anandamide to arachidonic acid was later identified by Deutsch and colleagues and termed "anandamide amidohydrolase" (Deutsch and Chin, 1993). "Anandamide amidohydrolase" behaved as a membrane-bound enzyme, exhibited sensitivity to inhibitors of both serine and cysteine hydrolases, and hydrolyzed a broad range of NAEs with similar catalytic efficiencies (Deutsch and Chin, 1993; Desarnaud et al., 1995; Maurelli et al., 1995; Ueda et al., 1995). Shortly after the identification of this "anandamide amidohydrolase" activity, the FAPA oleamide was characterized as a rat CSF molecule that increased upon sleep deprivation and induced sleep when


105

administered to rats (Cravatt et al., 1995). Oleamide, like anandamide, was degraded by a membrane-bound hydrolytic activity (Cravatt et al., 1995; Patterson et al., 1996). This "oleamide hydrolase" activity exhibited properties that were essentially identical to "anandamide amidohydrolase," and it was suggested that these two activities may be attributed to the same enzyme (Maurelli et al., 1995). In 1996, "oleamide hydrolase" was successfully purified and molecularly characterized from rat liver membranes (Cravatt et al., 1996). The purification of "oleamide hydrolase" was accomplished by an affinity chromatography method that exploited the low-nanomolar Kis exhibited by trifluoromethyl ketone inhibitors of the enzyme (Patterson et al., 1996). Following several conventional protein chromatography steps, the "oleamide hydrolase" enzyme was affinity purified using a trifluoromethyl ketone column. Peptide sequence information on the purified enzyme was obtained by standard protein chemistry procedures and this information was used to isolate the enzyme's cDNA. When this cDNA was transfected into COS-7 cells, robust oleamide hydrolase activity was observed in cellular membrane fractiofis. Importantly, the transfected cells also displayed high levels of an anandamide hydrolase activity that exhibited biochemical and kinetic properties matching those previously described for "anandamide amidohydrolase." Thus, a single enzyme was indeed capable of degrading both NAEs and FAPAs. Based on the plurality of FAA substrates accepted by the cloned hydrolase, it was renamed fatty acid amide hydrolase, or FAAH (Cravatt et al., 1996). A more detailed analysis of FAAH's properties supported that this enzyme was responsible for the FAA hydrolytic activities described repeatedly in the literature. For example, FAAH's transcript as judged by Northern blotting was most abundant in the rat liver and brain, at intermediate levels in the testis and kidney, and undetectable in the skeletal muscle and heart (Cravatt et al., 1996). A nearly identical distribution of anandamide amidohydrolase activity had been previously described (Desarnaud et al., 1995; Hillard et al., 1995). Likewise, FAAH behaved biochemically like an integral membrane enzyme (Cravatt et al., 1996), a property also shared by anandamide and oleamide hydrolytic activities characterized in mammalian tissues. Finally, the expression pattern of FAAH in the rat brain as determined by Western blotting and in situ hybridization correlated well with the regional distribution of oleamide and anandamide hydrolytic activities within the CNS (Thomas et al., 1997). FAAH was found at high levels in several neuronal populations throughout the rat brain, especially in principle neurons of the hippocampus, cerebellum, cortex, and olfactory bulb

106

MATTHEW P. PATRICELLI AND BENJAMIN F. CRAVA2~r

(Egertova et al., 1998; Tsou et al., 1998). FAAH's transcript was notably absent in glia (Thomas et al., 1997), consistent with the low anandamide and oleamide hydrolytic activity exhibited by these cells in culture (Beltramo et al., 1997a). Immunocytochemical studies have revealed that in the cortex, hippocampus, cerebellum, and olfactory bulb, FAAH is primarily localized to the somatodendritic compartment of principle neurons (pyramidal, purkinje, and mitral cells, respectively) (Tsou et al., 1998; Egertova et al., 1998). Interestingly, CB1 receptors display a complimentary distribution in these brain regions, residing on axonal tracts presynaptic to neurons expressing FAAH (Fig. 4) (Egertova et al., 1998). These data lend descriptive support to the model that FAAs like anandamide may serve as retrograde messengers, possibly being produced and degraded postsynaptic to their presynaptic site of action. Pharmacological studies have also provided suggestive evidence that FAAH is the primary means by which FAAs are catabolized in viva For example, a FAAH-resistant analog of anandamide, methanandamide, displays enhanced bioactivities in both cell culture and whole-animal systems (Abadji et al., 1994; Romero et al., 1996; Calignano et al., 1998). Additionally, Willoughby and colleagues conducted an in vivo catabolism study ofanandamide in mice, revealing that the major degradation product observed for this FAA in the brain is arachidonic acid (Willoughby et al., 1997). Importantly, within 1 min following peripheral injection of [3H]-anandamide, significant radioactive was found in brain tissue, of which approximately 8, 6, and 86% were identified as anandamide, polar lipids, and arachidonic acid, respectively. Collectively, these biochemical and pharmacological investigations offer strong support that FAAH-mediated hydrolysis is the primary route by which FAAs are catabolized in the CNS, providing a rapid means for terminating the endogenous signaling functions of these compounds at their presumed sites of action in viva B. FAAH ASA SITE OF ACTIONFORNON-CB1-BINDINGFAAs? Considering that anandamide binds and activates CB1 receptors in vitro and in cell culture systems, it was a natural extension to propose that this FAA's in vivo pharmacology was mediated by the central cannabinoid system. Consistent with this notion, anandamide's general pharmacological effects in rodents resembled those of THC (and other exocannabinoids), with both compounds inducing hypothermia, cataplexy, analgesia, and hypomotility in rats (Smith et al., 1994). This "tetrad" of behavioral effects was originally defined by Smith and

METABOLISM OF FATTYACID AMIDES

107

FIG. 4, Cerebellum: (a) F A A immunoreactivity is present in the cell bodies (large arrows) and dendrites (small arrows) of purkinje cells. FAAH immunoreactivity is not evident in granule cells and basket cells. (b) Dense CB1 immunoreactivity is present in the molecular layer where stained parallel fibers surround the unstained dendrites (white arrows) of purkinje cells. The stained axon terminals of basket cells (small black arrows) can be seen around the unstained cell bodies (large black arrows) ofpurkinje cells. Hippocampus: (c) FAAH immunoreactivity is present in the cell bodies (large arrows) and dendrites (small arrows) of pyramidal cells in the CA3 region of the hippocampus. (d) CB1 immunoreactivity is present in beaded nerve fibers surrounding the unstained cell bodies of pyramidal cells (arrows) in the CA3 region of the hippocampus. Neocortex: (e) FAAH immunoreactivity is present in the cell bodies (large arrows) and dendrites (small arrows) of pyramidal cells in the frontal lobe of the neocortex. (f) CB1 immunoreactivity is present in beaded nerve fibers surrounding the unstained cell bodies of pyramidal cells (arrows) in the frontal lobe of the neocortex.

108


colleagues as a test for compounds that acted through the cannabinoid system in vivo (Smith et al., 1994). Perplexingly, however, subsequent studies revealed that other FAAs like oleamide, which do not bind or activate CB1 receptors in vitro, also displayed "cannabinoid" activity in the tetrad test (Mechoulam et al., 1997). Although these results could be interpreted in several ways, one explanatory hypothesis, the "entourage effect," emerged as an early favorite among endocannabinoid researchers (Mechoulam et al., 1997; Ben-Shabat et al., 1998). The entourage effect rationalized the cannabinoid pharmacology of non-CB1binding FAAs as follows. Although saturated and monounsaturated FAAs do not interact with the CB1 receptor in vitro, they are excellent FAAH substrates. Thus, these FAAs could compete for FAAH's active site with endogenously produced anandamide, thereby potentially increasing the concentrations of anandamide in viva This postulated increase in endogenous anandamide levels would in turn be responsible for the observed cannabinoid behavioral effects of non-CBl-binding FAAs. A corollary to this proposal suggested that the endogenous route for anandamide biosynthesis also took advantage of the entourage effect by generating, in addition to anandamide, numerous other NAEs to serve as competitive substrates for FAAH. Unfortunately, few lines of experimental evidence support the entourage effect as a source for the cannabinoid properties of non-CB1binding FAAs. For example, studies to date have failed to demonstrate an increase in endogenous anandamide levels following the administration of bioactive doses of other FAAs. Additionally, Adams and colleagues showed that anandamide's effects in the tetrad tests were not blocked by the CB1 receptor anatagonist SR141716A, despite this agent's efficacy at reducing the activity of exocannabinoids such as THC (Adams et al., 1998). Most importantly, recent data indicate that anandamide retains it pharmacological effects as measured in the tetrad test in CB1 receptor knockout mice (B. Martin, personal communication). Collectively, these data clearly demonstrate that several of anandamide's cannabinomimetic effects are not mediated by the CB1 receptor, challenging the fundamental assumptions upon which the entourage effect was originally based. Perhaps a simpler explanation for several of the shared behavioral effects ofanandamide and non-CBl-binding FAAs is a common site of action for these compounds distinct from the CB1 receptor (see below). Nonetheless, some curious pharmacological data still implicate the central cannabinoid system as a participant in at least some of the bioactivities induced by non-CBl-binding FAAs. For example, oleamide's sleep-inducing effects were recently shown to be blocked by SR141716A (Mendelson and Basile, 1999). The mechanism(s) by


109

which this CB1 antagonist impacts oleamide's sleep effects remains unknown. Overall, the abundance of pharmacological data obtained to date indicate that signaling pathways in addition to the CB1 recePtor system are affected by anandamide and/or oleamide in vivo. On this note, the vanilloid VR1 receptor has recently been identified as the site of action for anandamide's vasodilator effects (Zygmunt et al., 1999) and could conceivably participate in the analgesic and hypothermic properties of anandamide as well. Although it is not yet known whether oleamide acts as a ligand for the VR1 receptor, an oleoyl derivative of capsaicin olvanil, is a potent VR1 agonist (Brand et al., 1987). In summary, while competition among FAA substrates for FAAH could lead to some of the observed effects of these compounds when administered at high doses, substantial evidence supports the alternative possibility that many of the shared pharmacological properties of FAAs are mediated by nonCB1 systems in vivo. C. BIOPHYSICALPROPERTIESOF FAAH At the close of 1996, with the first FAAH cDNA in hand (Cravatt et al., 1996), experimental strategies for the recombinant expression of this enzyme were initiated by several laboratories. Initially, recombinant FAAH protein was produced in transiently transfected eukaryotic cell culture systems (Cravatt et al., 1996). However, it rapidly became clear to researchers in the field that such methods provided insufficient quantities of the enzyme to permit detailed biochemical, biophysical, and structural studies. Therefore, alternative recombinant expression systems were developed. Full-length FAAH protein was successfully expressed in both Escherichia coli (Patricelli et al., 1998) and baculovirus (Katayama et al., 1999) systems, allowing for the purification of large quantities of the enzyme (milligram amounts per liter culture volumes). With such expression systems established, a thorough examination of FAAH enzymological and biophysical properties could be undertaken. A comparative analysis of the primary structures of rat, mouse, human, and pig FAAHs reveals that these enzymes all share greater than 80% sequence identity (Cravatt et al., 1996; Giang and Cravatt, 1997; Goparaju et al., 1999). Each FAAH possesses a single predicted transmembrane (TM) domain (amino acids 9-29), a conserved amidase signature sequence (amino acids 215-257), and a polyproline stretch (amino acids 310-315) that matches consensus sites for interaction with src kinase (Feng et al., 1994) and Homer proteins (Tu et al., 1998) (Fig. 5).

110

Transmembrane Domain IIIlllllll 1 7-~~,~


AmidaseSignature Sequence ~ 5

SH3Binding Domain 579

134-LYGVPVSLI~E--206-NPWKSSKSPGC~GGEGALIGSGGSPLGLGTDIGC~II~FPSAFCGICGLKPT-257 FIG. 5. Fatty acid amide hydrolase (FAAH) is a 579-amino-acid integral m e m b r a n e enzyme responsible for the hydrolysis of FAAs. Three putative functional elements can be identified in FAAH's primary structure: a n N-terminal t r a n s m e m b r a n e domain, a central amidase signature sequence, a n d a proline-rich sequence t h a t matches the consensus sequence for interacting with the SH3 domain of src kinase and homer proteins. The amidase signature sequence (detailed sequence) contains FAAH's catalytic residues (boxed residues) as well as several other residues t h a t are highly conserved among other members of the amidase signature family (in bold).

FAAH's single predicted TM domain resides close to the enzyme's N-terminus, suggesting that a truncated form of FAAH in which its first 30 amino acids are removed might act as a soluble form of the enzyme. However, deletion of FAAH's TM domain did not effect the enzyme's membrane binding properties, instead producing a FAAH variant, ATM-FAAH, that showed an unaltered subcellular distribution in COS-7 cells and wild-type levels of enzymatic activity (Patricelli et al., 1998; Arreaza and Deutsch, 1999). Interestingly, the removal of FAAH's TM domain profoundly affected the enzyme's oligomerization state in solution, as well as its propensity to form SDS-resistant oligomers as detected by SDS-PAGE analysis (Patricelli et al., 1998). ATM-FAAH existed as a single oligomeric species with a sedimentation coefficient of 11.2S, consistent with a trimeric or larger state for the enzyme. In contrast, wild-type FAAH existed as a heterogeneous distribution of species between 15S and 28S. These data suggested that FAAH's TM domain engages in homotypic interactions, a hypothesis that was confirmed by fusing this region of the protein to GST (Patricelli et al., 1998). Self-association through FAAH's TM domain drove the FAAHTM-GST fusion protein into very large oligomers that were fully separable by size exclusion chromatography from GST (which is naturally a dimer). Additionally, unlike GST, the FAAH-TM-GST fusion protein formed SDS-resistant oligomers. The functional significance, if any, of the self-association of FAAH's TM domain remains unknown. It is possible the FAAH's TM domain stabilizes an oligomeric state of the enzyme required for optimal in vivo activity and/or helps direct the enzyme to its proper subcellular


111

compartment. Notably, the ability of transmembrane domains to form specific, often SDS-resistant, homooligomeric structures has been demonstrated for a small but growing number of proteins (Bormann and Engelman, 1992). A common feature of most self-associating transmembrane domains, including FAAH's, is the presence of two conserved glycine residues predicted to reside on one face of the transmembrane helix (Lemmon et al., 1992; Herbert et al., 1996; Russ and Engelman, 2000; Senes et al., 2000). Replacement of either of these glycines with other residues is often disruptive to the self-association properties of the transmembrane domain (Lemmon et al., 1992; Brosig and Langosch, 1998). Recently, MacKenzie and colleagues determined the solution structure of a micellar glycorphorin A transmembrane domain dimer, confirming the presence of two glycine residues on one helical face of this protein segment (MacKenzie et al., 1997). These glycines appear to form pockets in which bulkier valine substituents insert to maximize van der Waals interactions between the two associating transmembrane domains. Finally, it is important to stress that as is the case with FAAH's TM domain, the roles that other self-associating transmembrane domains play in protein function in vivo remain mostly a mystery. The strong association with membranes displayed by ATM-FAAH in the absence of any predicted transmembrane domains is a property reminiscent of other enzymes that act on hydrophobic substrates, such as prostaglandin H 2 synthase (PS) (Picot et al., 1994) and squalene cyclase (SC) (Wendt et al., 1997), both of which appear to bind membranes through nonpolar patches that penetrate one leaflet of the bilayer. These membrane interactions are thought to be critical for catalysis in vivo, facilitating both substrate binding and product release. Thus, it is intriguing to speculate that FAAH may possess a membranebinding domain similar in structure to those found in PS and SC and that such extensive membrane interactions may allow the enzyme to access bilayer-embedded FAAs in vivo. D. ENZYMOLOGICALPROPERTIESOF FAAH FAAH represents the first identified mammalian member of a large group of amidohydrolase enzymes termed the "amidase signature" (AS) family, which is defined by a highly conserved continuous sequence rich in serine and glycine residues (the AS sequence; Fig. 5) (Mayaux et al., 1990; Chebrou et al., 1996). More than 80 AS enzymes have been identified to date, including in addition to FAAH, proteins from archaebacteria (Sako et al., 1996), eubacteria (Klee et al., 1984; Mayaux et al., 1990; Kobayashi et al., 1993; Chebrou et al., 1996; Curnow et al., 1997;

112


Boshoff & Mizrahi, 1998), fungi (Corrick et al., 1987; Genbauffe and Cooper, 1991), birds (Ettinger and DeLuca, 1995), and plants (Newman et al., 1994). Additionally, recent genome sequencing efforts have uncovered six members of the AS family in Caenorhabditis elegans and six members in Drosophila melanogaster. 1. Characterization of FAAH's Core Catalytic Residues Prior to the molecular characterization of FAAH, little was known about the enzymological properties of the AS family. Representative AS enzymes displayed sensitivity to both serine and cysteine-directed reagents (Kobayashi et al., 1993; Koutek et al., 1994; Patterson et al., 1996; Deutsch et al., 1997), leaving even the chemical identity of the AS family's catalytic nucleophile obscure. Over the past few years, an extensive series of mutagenesis, kinetic, and chemical labeling studies on both wild-type and mutant forms of FAAH have helped elucidate the remarkably distinctive catalytic mechanism employed by this enzyme and correlatively the AS family as a whole (Goparaju et al., 1999; Omeir et al., 1999; Patricelli and Cravatt, 1999; Patricelli et al., 1999; Patricelli and Cravatt, 2000). These multidisciplinary experimental efforts have revealed that FAAH is a serine hydrolase, using $241 as its catalytic nucleophile (Patricelli et al., 1999). Evidence supporting that $241 serves as FAAH's nucleophile can be summarized as follows: (1) mutation of $241 to alanine produces a FAAH variant with no detectable amidase or esterase activity (Goparaju et al., 1999; Omeir et al., 1999; Patricelli et al., 1999) and (2) $241 is the only FAAH residue labeled by the active site-directed irreversible inhibitor ethoxy oleoyl fluorophosphonate (Patricelli et al., 1999). The total conservation of $241 among AS enzymes indicates that this residue likely serves as the nucleophile for the entire AS family. Interestingly, mutagenesis studies demonstrated that FAAH does not utilize a histidine residue as its catalytic base (Patricelli et al., 1999), distinguishing the enzyme from most other serine hydrolases (including protease, esterases, and lipases), which possess a serinehistidine-aspartic acid catalytic triad (Dodson and Wlodawer, 1998). Instead of the traditional catalytic triad, FAAH appears to use a lysine residue, K142, as its catalytic base, making it the first identified mammalian hydrolytic enzyme to employ a serine-lysine dyad for catalysis (Patricelli and Cravatt, 1999; Patricelli and Cravatt, 2000). The most compelling evidence in support of K142 serving as FAAH's catalytic base can be summarized as follows: (1) mutation of K142 to alanine produced a catalytically deficient enzyme (35,000 reduction in amidase activity at pH 9.0) whose residual activity showed linear dependence on


113

11

oi J

/

1I K142A 4

5

6

7

8

9

10

pH

FIG. 6. The pH dependence ofkcat observed for FAAH with oleamide (diamonds) and the K142A m u t a n t with oleoyl methyl ester (triangles) and the pH dependence of k2 observed for the K142E m u t a n t with oleamide (open circles) are shown. Single-residue ionization models were used to fit the pH dependence of FAAH and the K142E m u t a n t , resulting in pKa values of 7.9 and 5.7, respectively. The pH dependence ofkcat for the K142A m u t a n t was fit to a line with a slope of 0.9. The altered pH rate profiles of the K142 m u t a n t s support a role for this residue as a catalytic base in FAAH's hydrolytic reaction.

solvent hydroxide concentration (Patricelli and Cravatt, 1999) (Fig. 6); (2) mutation of K142 to glutamate produced a catalytically deficient enzyme whose residual activity showed dependence on a residue with a pKa ~ 5-6 (Patricelli and Cravatt, 1999); (3) both the K142A and K142E m u t a n t s displayed significantly reduced fluorophosphonate (FP) reactivities, indicative of a decrease in the strength of the $241 nucleophile (Patricelli and Cravatt, 1999); and (4) K142 is the only conserved ionizable residue among AS enzymes that when m u t a t e d yields an enzyme with extreme reductions in both hydrolase activity and F P reactivity (Patricelli and Cravatt, 2000). The total conservation of K142 among AS enzymes indicates that this residue likely serves as the catalytic base for the entire AS family.

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Subsequent research efforts have focused on identifying other FAAH residues that play important roles in the enzyme's hydrolytic mechanism. Toward this end, all potentially catalytic residues in FAAH that are conserved among members of the AS family have been mutagenized and their individual roles in catalysis assessed by chemical labeling and kinetic methods (Patricelli and Cravatt, 2000). This comprehensize investigation has revealed that only five FAAH residues appear to be important for catalysis: K142, $217, $218, $241, and R243. The most central residues, $241 and K142, function as FAAH's nucleophile and general base/acid catalyst, respectively, composing the enzyme's postulated serine-lysine catalytic dyad (Patricelli and Cravatt, 1999). Although the roles of $217, $218, and R243 are currently unclear, distinct functions for these residues can be gleaned from the kinetic properties of mutant enzymes (Patricelli and Cravatt, 2000). For example, mutation of $217 to alanine caused a 2000-fold decrease in FAAH's kca t for oleamide and severely decreased the FP reactivity of FAAH, while mutation of $218 to alanine caused a 100-fold decrease in kcat without affecting FAAH's FP-reactivity. Mutagenesis of R243 to alanine produced an intriguing FAAH variant that maintained wild-type FP reactivity and esterase activity, but hydrolyzed amide substrates at more than 100-fold reduced rates. Based on the data currently available, some roles for $217, $218, and R243 can essentially be excluded (e.g., $218 and R243 are not involved in nucleophile activation). However, detailed structural information will be required to determine more precisely the functions of these catalytically important residues. 2. Substrate Specificity of F A A H Several early studies demonstrated that FAAH was capable of hydrolyzing a relatively broad range of FAPAs and NAEs with similar efficiencies (Deutsch and Chin, 1993; Desarnaud et al., 1995; Maurelli et al., 1995; Ueda et al., 1995, Cravatt et al., 1996). In general, the enzyme displayed a preference for mono- or polyunsaturated fatty acyl chains; however, saturated FAAs were still fairly good FAAH substrates. Subsequently, more detailed analyses of FAAH's substrate specificity have revealed that the enzyme displays some rather strict requirements with regard to the nature of both the substrate's acyl chain and leaving group. Boger and colleagues have synthesized a wide range of trifluoromethyl ketone (Boger et al., 1999) and ~-keto heterocycle (Boger et al., 2000) inhibitors of FAAH in order to probe the enzyme's acyl chain specificity. The results of both studies demonstrate that essentially all of the binding energy for these inhibitors is derived from the first 8 or 9 carbons of the acyl chain, with each methylene unit in

METABOLISM OF FA'FrY ACID AMIDES

115

this segment of the inhibitor contributing an approximately fivefold decrease in Ki value. In contrast, extending the inhibitor's acyl chain past position 9 did not greatly impact its Ki value. Interestingly, a site of unsaturation in the acyl chain in the vicinity of carbons 9-12 increased potency considerably. We have found a similar trend among a panel of p-nitro-anilide FAAH subtrates, whose Km values decreased 5- to 10fold for each methlyene unit added to the acyl chain up to 9 carbons and then remained constant with further extensions (our unpublished observations). Thus, FAAH's acyl chain specificity clearly indicates that descriptions of this enzyme as a "nonselective amidase" are misrepresentative. In contrast, FAAH appears to have evolved a substrate binding pocket optimally suited for the inactivation of the entire family of endogenous FAAs in viva In one rather comprehensive study, Lang and colleagues characterized the FAAH-mediated hydrolysis of arachidonoylamides bearing a wide variety of leaving groups ranging from ammonia to hydroxyanilines (Lang et al., 1999). Primary and unbranched secondary amides were hydrolyzed with high efficiency by FAAH, but the enzyme was inactive against tertiary amides. Branching of the substrate at either the carbon alpha to the amide nitrogen or alpha to the carbonyl greatly reduced FAAH's hydrolytic efficiency, except in the case of hydroxyanilides, which were fairly robust FAAH substrates. It is currently not clear whether these leaving group effects on FAAH's substrate specificity are the result of steric or electrostatic influences on the enzymatic reaction. The trends observed, however, support the notion that FAAH has evolved a substrate binding site well suited for the hydrolysis of both FAPAs and NAEs. One of the most interesting and potentially biologically relevant enzymatic properties exhibited by FAAH is its ability to degrade structurally similar amides and esters with equivalent efficiencies (PatriceUi and Cravatt, 1999). Analysis of the kinetic parameters of FAAH-mediated amide and ester hydrolysis has revealed that the enzyme exhibits a slightly higher kcat/Km value for oleamide than oleoyl methyl ester (Patricelli and Cravatt, 1999). These data imply that FAAH reacts with, or is acylated by, these substrates at similar rates, an extremely unusual property for a serine hydrolase. Most serine hydrolases react with ester substrates at rates more than 1000-fold faster than amides (Bender et al., 1962). FAAH's curious ability to normalize the acylation rates of its amide and ester substrates gains considerable importance when one recognizes that the enzyme likely hydrolyzes both FAAs and fatty acid esters (FAEs) concurrently in vivo. For example, several biochemical studies have shown that FAAH degrades a second family

116

MATTHEWP. PATRICELLIAND BENJAMINF. CRAVATT

of endogenous cannabinoids, the 2-acyl glycerols (2-AG) (Mechoulam et al., 1995; Sugiura et al., 1995; Goparaju et al., 1998). IfFAAH acted as a typical serine hydrolase and displayed a much higher reactivity with such FAE substrates relative to FAAs, the enzyme might encounter difficulty hydrolyzing the latter class of lipids in vivo (Patricelli and Cravatt, 1999). Therefore, the highly unusual ability of FAAH to react with amides and esters at similar rates may empower the enzyme to function as both an amidase and esterase in vivo, potentially allowing the enzyme to coordinate the activities of two distinct families of neuromodulatory compounds (the FAAs and 2-AGs).

IV. TRANSPORT OF FATTY ACID AMIDES

Predictions based on FAAH's primary sequence place the majority of the protein, including the active site region, in the cytosolic compartment of the cell. Additionally, immunocytochemistry studies have shown that FAAH appears to reside primarily on intracellular membrane compartments rather than on the plasma membrane (Cravatt et al., 1996; Egertova et al., 1998). Thus, it is possible that FAAs must first cross the plasma membrane before gaining access to FAAH for hydrolysis. By analogy to other neurotransmitters such as serotonin and epinephrine, it has been proposed that the catabolism of FAAs may require the presence of a plasma membrane transport process to facilitate the interaction of FAAs with FAAH. Several studies have characterized a putative "anandamide transporter" in cell culture systems and have presented evidence that a selective protein transporter exists to allow cells to uptake anandamide (Di Marzo et al., 1994; Beltramo et al., 1997b; Bisogno et al., 1997a; Hillard et al., 1997). The anandamide transport process displayed four properties that have been interpreted as evidence for a protein-facilitated event: (1) the temperaturedependence of transport, (2) the saturability of transport, (3) the structural specificity of transport, and (4) the sensitivity of transport to chemical inhibitors. Although the presence of putative transport mechanisms for FAAs clearly merits continued investigation, it is also important to stress that the current evidence in support of the existence of such transporters is inconclusive. In the transport studies mentioned above, anandamide was treated as if it were a classic water-soluble neurotransmitter rather than a highly hydrophobic and membrane-permeable neutral fatty acid derivative. The complex nature of studying the cellular uptake of water-insoluble compounds has been well documented in the


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field of fatty acid transport. After more than 50 years of study by numerous laboratories and the cloning of several postulated fatty acid transport proteins, there is still no general agreement as to the existence of dedicated fatty acid transport proteins (for reviews on this topic, see Hamilton, 1998; Abumrad et al., 1999; Hamilton and Kamp, 1999; McArthur et al., 1999). Measurement of FAA transport is subject to the same complexities and pitfalls that have plagued the fatty acid transport field and, as such, should be approached with extreme experimental caution. Several properties of FAAs must be considered and accounted for when studying transport, including the rate of passive diffusion of FAAs across membranes, the equilibrium levels of FAAs in cellular membranes at a given FAA concentration (partitioning), the solubility limits of the FAAs in both membranes and solution, and the rate of cellular catabolism of FAAs (Fig. 7). When these properties are considered collectively, the current evidence in support of a proteinmediated FAA transporter can also be viewed as consistent with a passive diffusion mechanism for FAA uptake. One of the more pervasive observations used to support a proteinmediated anandamide transport system is the temperature-dependent

/~ 1

Fatty Acid Amide

Cell Membrane

Inner membrane Compartment(s)

FIG. 7. The accumulation of FAAs in cells involves several physical processes t h a t can occur through either passive diffusion or protein-facilitated mechanisms. FAAs, like fatty acids, display low solubility in aqueous media and will rapidly be absorbed into cell memb r a n e s (1). Before entering the cell interior, the polar head group of the FAA m u s t traverse the lipid bilayer (2), a process t h a t h a s not been studied for FAAs but is fairly rapid for free fatty acids (tl/2 ~ mseconds). In the event t h a t FAAH resides on a n intracellular m e m b r a n e c o m p a r t m e n t t h a t is noncontiguous with the plasma membrane, the FAA m u s t leave the cell m e m b r a n e a n d e n t e r the m e m b r a n e of the intracellular compartment (3) prior to hydrolysis by FAAH (4). The m e a s u r e m e n t of FAA accumulation into cells t h r o u g h the use of radioactive or fluorescent labels will yield a rate dependent on the rates and equilibrium constants for one or more of these steps.

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nature of this process (Di Marzo et al., 1994; Beltramo et al., 1997b; Bisogno et al., 1997a; Hillard et al., 1997). Although a protein-facilitated transport process should be reduced in activity at lower temperatures, the passive diffusion of FAAs into and across membranes would be predicted to show a similar temperature dependence. For example, both the lipid/aqueous partitioning of fatty acids in membranes and their rates of membrane crossing have been shown to be highly temperaturedependent in model membrane systems (Kleinfeld et al., 1998). Interestingly, when the temperature is lowered from 37 to 4°C, the rate of passive diffusion of fatty acids across erythrocyte ghost membranes (Kleinfeld et al., 1998) is decreased to a similar level to that observed for anandamide transport over this temperature range (Di Marzo et al., 1994; Beltramo et al., 1997b; Bisogno et al., 1997a; Hillard et al., 1997). It is also worth noting that the relatively modest two- to threefold decrease in anandamide transport over this temperature range indicates that passive diffusion is highly competitive with any "protein-mediated" process postulated to maintain activity exclusively at higher temperatures. Finally, the rapid enzymatic metabolism of FAAs and their incorporation into acyl-CoAs and phospholipids will greatly impact the passive diffusion of FAAs and would also constitute temperaturedependent events (DeGrella and Light, 1980a,b). On this subject, one postulated fatty acid transport protein was subsequently found to be involved in the metabolism of fatty acids rather than their direct transport (Choi and Martin, 1999). Importantly, the overexpression of this protein measurably increased fatty acid "transport" rates in cell systems. Numerous studies on anandamide transport have reported that this process is saturable (Di Marzo et al., 1994; Beltramo et al., 1997b; Bisogno et al., 1997a; Hillard et al., 1997), a quality taken to support a protein-mediated event. Although true saturation kinetics is clearly a promising indicator of a protein-dependent process, the general nature of FAA diffusion and the specific way in which these saturation experiments have been conducted challenge the importance of the results obtained to date on the "saturability" of anandamide transport. First, the observation that anandamide transport exhibits saturation kinetics does not necessitate that a transport protein exists. As stated above, the metabolism of anandamide subsequent to its passive diffusion into the cell could greatly affect the cellular accumulation of this compound. Thus, the saturation of anandamide transport could indirectly reflect the saturation of intracellular enzymes (e.g., FAAH) involved in the catabolism of this compound. In most anandamide uptake studies, no evidence is presented that the measured accumulation


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of cellular radioactivity is still present as anandamide over the time period of the experiment (Di Marzo et al., 1994; Beltramo et al., 1997b; Hillard et al., 1997). Second, the very nature of the observed "saturation" of anandamide transport remains obscure, as the studies conducted to date fail to clearly define whether the rate of anandamide transport is actually saturated as opposed to the potential saturation of cell membranes with anandamide. In order to address this important issue, studies on anandamide transport should not, as they most commonly do, rely on kinetic measurements using single time-point assays. If these time points are not acquired during the initial rate (linear or zero order) phase of the process, and anandamide accumulation has already proceeded either partially or completely to equilibrium, the saturation measured may reflect the solubility limit of anandamide in the cell membranes. Finally, two studies of anandamide transport report Km values of 30-50 ~M (Bisogno et al., 1997a; Hillard et al., 1997), which are fairly close to the aqueous solubility limit of anandamide. In these cases, the saturation observed could be due to the insolubility of anandamide at the higher concentrations assayed for transport. The third criterion presented as evidence for carrier-mediated anandamide transport is the "structural specificity" of the transporter. It is important to stress that few studies have directly measured the transport of FAAs other than anandamide. Nonetheless, at least two reports have described a transporter process that displays a high degree of specificity for anandamide (Di Marzo et al., 1994; Piomelli et al., 1999). However, both of these investigations appear to have compared unequal concentrations of FAAs in their transport studies. Most strikingly, a recent study in which several FAAs were tested for uptake normalized the addition of these compounds based on their radioactivity (10-50 x 106 dpm/ml) rather than their molar concentrations (Piomelli et al., 1999). Since the specific activities of the FAAs tested varied by more than five orders of magnitude, and the degree of transport was measured by recording the fractional depletion of the FAAs from cell culture media, it is not surprising that only those FAAs present at lower concentrations (due to their higher specific activity) were observed to be "transported" to a significant extent. Finally, structural specificity is not necessarily an exclusive property of carrier-mediated uptake, as a passive diffusion mechanism should also show differences in rates and concentrations of saturation for various FAAs, dependent on their individual physical and chemical properties (e.g., partition coefficients, solubility limits, and rates of metabolism) (Kleinfeld et al., 1998; Hamilton and Kamp, 1999). In consideration of these factors, the current data that

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the transport of anandamide displays structural specificity remains inconclusive. Several studies of anandamide transport have reported that the process can be chemically inhibited and therefore must be proteinmediated. To date, however, inhibitors ofanandamide transport are all either general cell metabolic inhibitors (Beltramo et al., 1997b), FAAH inhibitors (Bisogno et al., 1997a), or are FAAs themselves (Beltramo et al., 1997b; Beltramo and Piomelli, 1999). In the first two cases, the results are not necessarily indicative of a protein transporter since inhibition of the downstream metabolism of FAAs would affect passive or protein-facilitated diffusion mechanisms. The FAA-based inhibitors of anandamide transport could in a similar manner either saturate the downstream metabolic processes or simply saturate the membranes preventing anandamide accumulation. To date, no inhibitors with IC5o values that are significantly lower than the the Km value measured for anandamide transport (1-50 txM) have been reported. Both passive diffusion and protein-facilitated transport mechanisms would be subject to weakly potent inhibition in this manner by structural analogs of anandamide. Perhaps the most potent inhibitor of anandamide transport reported to date is albumin (Di Marzo et al., 1994). This is an important observation in that fatty acid transport studies invariably measure transport in the presence of BSA to enhance the solubility of the fatty acids and minimize their nonspecific partitioning into cell membranes. The fact that anandamide transport is essentially abolished in the presence of BSA is highly suggestive that the transport measured may reflect the passive accumulation of the compound in cell membranes due to its extreme hydrophobicity rather than a proteinmediated transport process. It is curious to note that there have been no reports of cell lines lacking anandamide transport activity. Indeed, a number of different cell systems have been reported to exhibit carrier-mediated anandamide transport, including neurons (Di Marzo et al., 1994; Beltramo et al., 1997b), astrocytes (Beltramo et al., 1997b), astrocytoma lines, basophil leukemia cells (Bisogno et al., 1997a), and macrophages (Bisogno et al., 1997a). It is not clear why such a broad range of cell lines, including transformed cells, would maintain the production of a protein to specifically transport anandamide. Unfortunately, in the absence of a cell line that does not "transport" anandamide, it is difficult to rule out a passive diffusion mechanism for the cellular uptake of this FAA. Considering the chemical properties of FAAs, it is worth questioning whether these compounds require a dedicated transport system in order

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to access FAAH at intracellular sites. It is well known that long-chain fatty acids exist mainly in membranes or bound to albumin rather than free in solution. Additionally, due to a shift in the pKa of their acid group, fatty acids exist mainly as uncharged compounds when in membranes and can flip-flop through bilayers at millisecond time scales (Kleinfeld et al., 1998). FAAs would most likely behave in a similar manner to fatty acids, although the neutral amide group would abrogate the need for any protonation event prior to membrane crossing. Extrapolating from their chemical similarity to fatty acids, FAAs should exist mainly within membranes and should freely diffuse across them at some rate. However, if FAAs act in vivo as fast-acting neurotransmitters in the classic sense, it is possible that their inactivation may necessitate a more rapid form of cellular uptake than can be provided by passive diffusion. In this regard, it is interesting to consider that the slowest step in the diffusion of fatty acids across membranes is their release from the bilayer (Kleinfeld et al., 1998). The consequence of this property is that fatty acids, and presumably FAAs as well, spend the majority of their time in membranes and may not move quickly between noncontiguous membrane compartments. Fatty acid binding proteins facilitate the movement of fatty acids between membrane compartments in viva If FAAH exclusively resides on intracellular membrane structures, a similar mechanism may be required to facilitate the movement of FAAs from within the plasma membrane to intracellular membranes bearing FAAH. Further characterization of the properties of FAA transport in both cellular and model membrane systems, as well as the continued evaluation of the biological roles and physiological occurrence of FAAs, should eventually resolve whether a protein-dependent facilitated FAA transport process exists in viva

Vo CONCLUSIONSAND FUTURECHALLENGES

An extensive body of scientific literature now exists describing the remarkable pharmacological properties of FAAs. These endogenous lipids can affect a broad range of behavioral processes, including nociception (Calignano et al., 1998; Jaggar et al., 1998; Richardson et al., 1998a,b; Walker et al., 1999), sleep-wake (Cravatt et al., 1995; MurilloRodriguez et al., 1998; Basile et al., 1999), thermoregulatory (Crawley et al., 1993; Fride and Mechoulam, 1993; Smith et al., 1994), and learning/memory systems (Terranova et al., 1995; Murillo-Rodriguez et al., 1998). Nonetheless, translating the impressive pharmacology displayed

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by FAAs into a meaningful assessment of their endogenous functions will require a far greater understanding of the proteins involved in regulating the levels and activities of these signaling lipids in viva This chapter has attempted to critically evaluate the current state of knowledge on FAA biosynthetic and catabolic pathways, highlighting both the major advances made and the considerable challenges still remaining. The biosynthetic pathways leading to the production of FAAs are largely unknown. Due to the low abundance of FAAs and their rapid degradation in biological systems, it has been difficult to robustly and quantitatively monitor the production of FAAs. Significant progress has been made despite these obstacles, with multiple candidate enzymes and/or activities identified that are believed to participate in FAA production. Presently, the isolation and molecular characterization of the CDTA enzyme postulated to produce NAPEs is perhaps the most pressing issue with regard to the biogenesis of NAEs. The ability to genetically disrupt or selectively inhibit this CDTA would provide a means for directly testing the participation of this enzyme in the production of NAEs in viva Uncovering the route by which FAPAs are biosynthesized faces perhaps even greater challenges. It is at present unknown where FAPAs are synthesized or in what tissues they are most abundant. Additionally, unlike NAEs, which appear to be synthesized and released in response to C a 2+ signaling, it is not clear what types of biological events (other than sleep deprivation) lead to the release of FAPAs. The identification of fatty acyl glycines in vivo, and the characterization of an enzyme capable of their production, would add significant support to the proposed PAM-dependent pathway for FAPA biosynthesis. The biological relevance of this pathway could then be tested through the chemical and/or genetic manipulation of one or both of the participating enzymes and a subsequent evaluation of the effects on FAPA production in vivo. The most well-characterized aspect of FAA metabolism is clearly the degradation of these compounds by FAAH. The biochemical and cell biological properties of this enzyme have been the subject of several recent studies, yielding valuable insights into not only the enzyme's structure and function, but also the potential roles played by its FAA substrates in viva Nonetheless, several important aspects of FAA degradation remain fertile ground for future research, including the possible presence of other enzymes or pathways for FAA inactivation, the participation of FAAH in the hydrolysis of 2-AGs, and the presence of putative proteinfacilitated mechanisms for FAA uptake. Resolution of these first two issues should be facilitated by the generation of genetically engineered


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mice t h a t lack FAAH. T h e s e a n i m a l s will also provide p e r h a p s t h e first direct a s s e s s m e n t of t h e g e n e r a l i m p o r t a n c e of FAA s i g n a l i n g in m a m mals. FAA t r a n s p o r t studies w o u l d benefit g r e a t l y from t h e c h a r a c t e r i z a t i o n of t h e physical p a r a m e t e r s of FAA diffusion across m e m b r a n e s , as well as the d e v e l o p m e n t of i m p r o v e d m e t h o d s for m e a s u r i n g FAA u p t a k e a n d trafficking a m o n g cellular c o m p a r t m e n t s . I n s u m m a r y , a l t h o u g h the p h a r m a c o l o g i c a l p r o p e r t i e s of FAAs h a v e b e e n well c h a r a c t e r i z e d , t h e a c t u a l biological roles p l a y e d b y t h e s e end o g e n o u s c o m p o u n d s r e m a i n m o s t l y a m a t t e r of speculation. Uncovering t h e m e c h a n i s m s responsible for FAA p r o d u c t i o n a n d d e g r a d a t i o n in vivo should g r e a t l y e n r i c h o u r u n d e r s t a n d i n g of t h e physiological functions of this i n t r i g u i n g f a m i l y of bioactive lipids. REFERENCES Abadji, V., Lin, S., Taha, G., Griffin, G., Stevenson, L. A., Pertwee, R. G., and Makriyannis, A. (1994). (R)-methanandamide: A chiral novel anandamidepossessing higher potency and metabolic stability. J. Med. Chem. 37, 1889-1893. Abumrad, N., Coburn, C., and Ibrahimi, A. (1999). Membrane proteins implicated in longchain fatty acid uptake by mammalian cells: CD36, FATP and FABPm. Biochim. Biophys. Acta 1441, 4-13. Adams, I. B., Compton, D. R., and Martin, B. R. (1998). Assessment of anandamide interaction with the cannabinoid brain receptor: SR 141716Aantagonism studies in mice and autoradiographic analysis of receptor binding in rat brain. J. Pharmacol. Exp. Ther. 284, 1209-1217. Arafat, E. S., Trimble, J. W., Andersen, R. N., Dass, C., and Desiderio, D. M. (1989). Identification of fatty acid amides in human plasma. Life Sci. 45, 1679-1687. Arreaza, G., Devane, W.A., Omeir, R. L., Sajnani, G., Kunz, J., Cravatt, B. F., and Deutsch, D. G. (1997). The cloned rat hydrolytic enzyme responsible for the breakdown of anandamide also catalyzes its formation via the condensation of arachidonic acid and ethanolamine. Neurosci Lett. 234, 59-62. Arreaza, G., and Deutsch, D. G. (1999). Deletion of a proline-rich region and a transmembrane domain in fatty acid amide hydrolase. FEBS Lett. 454, 57-60. Basile, A. S., Hanus, L., and Mendelson, W. B. (1999). Characterization of the hypnotic properties of oleamide. NeuroReport 10, 947-951. Beltramo, M., and Piomelli, D. (1999). Anandamide transport inhibition by the vanilloid agonist olvanil. Eur. J. Pharmacol. 364, 75-78. Beltramo, M., di Tomaso, E., and Piomelli, D. (1997a). Inhibition of anandamide hydrolysis in rat brain tissue by (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2Hpyran-2-one. FEBS Lett. 403, 263-267. Beltramo, M., Stella, N., Calignano, A., Lin, S. Y., Makriyannis, A., and Piomelli, D. (1997b). Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 277, 1094-1097. Bender, M. L., Schonbaum, G. R., and Zerner, B. (1962). Spectrophotometric investigations of the mechanism of a-chymotrypsin catalyzed hydrolyses: Detection of the acyl enzyme intermediate. J. Am. Chem. Soc. 84, 2540-2550. Ben-Shabat, S., Fride, E., Sheskin, T., Tamiri, T., Rhee, M. H., Vogel, Z., Bisogno, T., De Petrocellis, L. et al. (1998). An entourage effect: Inactive endogenous fatty acid

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Willoughby, K. A., Moore, S. F., Martin, B. R., and Ellis, E. F. (1997). The biodisposition and metabolism of anandamide in mice. J. Pharmacol. Exp. Ther. 282, 243247. Yang, H. Y., Karoum, F., Felder, C., Badger, H., Wang, T. C., and Markey, S. P. (1999). GC/MS analysis of anandamide and quantification of N-arachidonoylphosphatidylethanolamides in various brain regions, spinal cord, testis, and spleen of the rat. J. Neurochem. 72, 1959-1968. Yost, C. S., Hampson, A. J., Leonoudakis, D., Koblin, D. D., Bornheim, L. M., and Gray, A. T. (1998). Oleamide potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor activity but does not alter minimum alveolar anesthetic concentration. Anesth. Analg. 86, 1294--1300. Zygmunt, P. M., Petersson, J., Andersson, D. A., Chuang, H., Sorgard, M., Di Marzo, V., Julius, D., and Hogestatt, E. D. (1999). Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400, 452-457.

VITAMINSANDHORMONES,VOL. 62

Three-Dimensional Organization of the Aquaporin Water Channel: What Can Structure Tell Us about Function? ALOK K. MITRA Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 I. Movement of Water across the Membrane--Discovery of Aquaporin-1 II. M a m m a l i a n Aquaporins and Their Transport Function III. Investigations of Amino Acid Residues Involved in Solute Transport in Aquaporins IV. Structural Studies of Aquaporins A. Amino Acid Sequence Analysis and Spectroscopic Studies of Aquaporins B. Oligomeric Structure and the Functional Unit for Solute Transport in Aquaporins C. Analysis of Aquaporin 3D Structure at High Resolution V. Unresolved Questions and Future Directions References

I. MOVEMENT OF WATER ACROSS THE MEMBRANE----DISCOVERY OF AQUAPORIN-1

The entry and exit of water across the lipid bilayer membrane is a fundamental physiological process necessary for maintaining cell homeostasis, which is crucial to the survival of an organism. Historically, several observations over many decades (see Agre et al., 1993, 1995; Verkman et al., 1996; King and Agre, 1996, for review) suggested the presence of water-specific channels or pore in some tissues. These were (a) unusually high osmotic water permeability of, for instance, red blood cell membranes and renal proximal tubular epithelium, which is characterized by low Arrhenius activation energy (Ea) and is too rapid to be explained by passive diffusion of water across the bilayer (Finkelstein, 1987); (b) reversible inhibition of the high water permeability across red blood cells by mercurial reagents (Goldstein and Solomon, 1960, Macey and Farmer, 1970); and (c) radiation inactivation studies of renal brush-border membrane vesicles and erythrocytes (van Hoek et al., 1991, 1992) which indicated that a protein of ~30kDa is responsible for the high water permeability. Additional evidence in favor of a protein responsible for water transport was provided when, upon injection of heterologous mRNAs from kidney reticulocytes and amphibian bladder 133

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into Xenopus oocytes, which is known to have low water permeability, increased water permeability (Zhang et al., 1990, 1991) was elicited. However, the chemical nature of this water transporter remained unknown until an abundantly present 28°kDa membrane protein in erythrocyte membranes (~2 × 105/cell) called CHIP28 (CHannel forming Integral membrane Protein of 28 kDa), which was discovered accidentally during characterization of the Rh blood group antigen (Denker et al., 1988) was further investigated. First, cloned RNA of CHIP28 (Preston and Agre, 1991) upon injection into X e n o p u s oocytes led to high mercurialsensitive water permeability (Preston et al., 1992). Subsequently, direct proof that CHIP28 completely accounts for the characteristics of osmotic water permeability oferythrocyteswas provided from measurements on proteoliposomes reconstituted with CHIP28 purified from red blood cells (Zeidel et al., 1992; Van Hoek and Verkman, 1992). CHIP28 was later renamed AQP1 (Agre, 1997) to signify it as an archetypal member of a family (aquaporin) of homologous proteins belonging to the MIP superfamily of integral membrane proteins (Pao et al., 1991; Reizer et al., 1993), named after the Major Intrinsic Protein MIP26 of the lens (Gorin et al., 1984). The members of this family include proteins from both eucaryotes and procaryotes and are characterized by sequence related N- and C-terminal halves, each containing an absolutely conserved tripeptide sequence Asn-Pro-Ala (the "NPA" box). The internal sequence homology probably arose by a tandem, intragenic duplication event (Wistow et al., 1991; Reizer et al., 1993).

II. MAMMALIANAQUAPORINSANDTHEIRTRANSPORTFUNCTION

After the identification of AQP1, several aquaporins were identified from mammalian tissues by cDNA screening (reviewed in Brown et al., 1995; Echevarria and Ilundain, 1998). The deduced amino acid sequences of mammalian aquaporins show 20-40% sequence identity when their amino termini are aligned (Reizer et al., 1993) with the homology in the N-terminal half being somewhat higher than that in the C-terminal half. Apart from the "NPA" boxes several other conserved motifs of various lengths (Fig, 1) can be identified in the aligned sequences of mammalian aquaporins. The observed tissue-specific expression of different aquaporins with rare overlap has led to speculation about their function in normal physiology and disease (Verkman et al., 1996; King and Agre, 1996). The physiological importance of AQP1 was questioned, however, because of the grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) (Preston et al., 1994; Smith et al., 1994). However, it was recently shown that

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transgenic mice lacking AQP1 displayed severely impaired ability to concentrate urine (Ma et al., 1997) indicating that AQP1 is required for normal functioning of the kidney. Based on measurements of water permeability (Zeidel et al., 1992, 1994; Yang and Verkman, 1997) using AQPl-reconstituted proteoliposomes and Xenopus oocytes expressing AQP1, the magnitude of the AQPl-mediated water flux, ~10 -13 cm3/s/AQP1 is slightly higher than that for gramicidin. This measured flux corresponds to the movement of ~2 × 106 water molecules/ms/AQP1. No significant increases in the permeabilities to urea, protons, hydroxyl ions, ammonium ions, and salts were observed over control experiments showing the exquisite specificity of AQP1 for only water. Although, cAMP-dependent water permeability and cation conductance in Xenopus oocytes injected with AQP1 cRNA was reported by Yool et al. (1996), several researchers failed to duplicate these results (Agre et al., 1997). Recent experiments reported by Yasui et al. (1999) on Xenopus oocyte-expressed AQP6 indicate an onset of anion conductance either by exposure to mercurial reagents or upon acidification. In the case of mammalian glycerol transporter AQP3, Zeuthen and Klaerke (1999) find thatXenopus oocyte-expressed protein acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel upon acidification (pH 6.1), implying gating by H +. Cahalan and Hall (2000) have observed that both Ca 2+ and pH in the physiological range (7.2 to 6.5) regulate the water permeability of MIP26 (AQP0) via a histidine residue (His40) in the sequence. CO2 permeability has been reported for AQP1 (Nakhoul et al., 1998; Prasad et al., 1998) but has been challenged by results obtained from measurements in transgenic AQPl-deficient mice and reconstituted proteoliposomes (Yang et al., 1999). At the present time, within the mammalian aquaporin family apart from AQP1, the vasopressin-sensitive renalcollecting duct aquaporin AQP2, the mercurial-insensitive AQP4, and AQP5 are thought to exclusively transport water, with AQP4 being the most prolific water transporter (Yang et al., 1997). On the other hand, several other members are more promiscuous, transporting in addition glycerol (AQP3 and AQP7), urea (AQP8), and even larger solutes, e.g., polyols, purines, and so on, in the case of AQP9 (Tsukaguchi et al., 1998). Currently, this has led to broad classification into the so-called "aquaporin" and "aquaglyceroporin" families. The inhibition of transport properties for AQP1 and other aquaporins by submillimolar amounts of mercurial reagents such as HgC12 and pCMBS suggested the involvement of cysteine residues in the solute pathway. Systematic substitution of the four cysteine residues in AQP1 showed that extracellular Cys189 in the second NPA box region is the mercury-sensitive residue (Preston et al., 1993). Also, in AQP2, Cysl81

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analogous to Cys189 in AQP1 is responsible for mercurial inhibition (Bai et al., 1996). In the case of AQP3, which is primarily a glycerol transporter, mercurial inhibition is less pronounced; however, enhanced inhibition could be induced by substitution of analogous Y212 to Cys (Kuwahara et al., 1997). Inhibition of glycerol transport in the case of MIP26 (AQP0) by mercurial reagents was observed but was not characterized (Kushmerick et al., 1998). On the other hand, interestingly, Yasui et al. (1999) report for AQP6 up to twofold enhancement of otherwise low basal level water permeability in response to HgC12. This excitatory rather than inhibitory effect of mercurial reagents for AQP6 is a novel feature in the aquaporin family.

III. INVESTIGATIONSOF AMINOACID RESIDUES INVOLVEDIN SOLUTE TRANSPORT IN AQUAPORINS

Site-specific mutagenesis to probe elements in sequence and structure that are important for function has been applied to probe structural/functional relationships for many membrane proteins. A synthesis of the molecular biological approach of generating AQP1 variants directed by 3D structural information is necessary to define residues involved in the selectivity filter and those that form the water pore. Although atomic-resolution structural information is still lacking, a number of studies utilizing the effects of site-directed mutagenesis have been carried out in order to understand the involvement of specific residues in aquaporin function. Notwithstanding the lack of knowledge of whether the functional alterations due to substitution are due to local or global conformational effects, some insight into elements involved in the solute pathway has emerged from such studies. Using the heterologous X e n o p u s oocyte expression system, it was demonstrated that conservative single-site substitutions for AQP1 in the NPA-box regions had deleterious effects on water transport (Jung et al., 1994; Abrami et al., 1996), suggesting that these two regions are critical for water transport. Substitution of Cys189 by larger amino acids such as Trp significantly inactivated AQP1 but not smaller amino acids such as Gly or Ala. This suggested that Cys189 is located near the aqueous pore and that the mercurial binding or replacement by larger amino acids directly or indirectly causes an occlusion of the pathway. In addition, recombinant Cys189Ser, Ala73Ser AQP1, where Ala73 (in the 2-3 loop) is the cytoplasmic N-terminal analog of Cys189, displayed mercurial sensitivity (Jung et al., 1994). Theoretical studies using sequence analysis also indicate similar importance of the two NPA regions in the aquaporin family (Froger et al., 1998). The importance of residues near the NPA

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box was also demonstrated in the case of AQP4 by Shi and Verkman (1996), who showed that substitutions of some of the residues near the NPA box by cysteine can induce mercurial sensitivity to this otherwise mercurial insensitive water channel. In the case of AQP2, it has been suggested (Bai et al., 1996) that in addition to the NPA loops, the extracellular 3-4 loop is also important for water transport. It is noted that this loop is significantly longer in the case of glycerol facilitators and may participate in solute selectivity (Froger et al., 1998). To date extensive studies directed at the possible functional roles of residues in the transmembrane segments of aquaporins have not been reported.

IV. STRUCTURALSTUDIES OF AQUAPORINS

In order to understand at the atomic level how a biological macromolecule functions, a detailed knowledge of its three-dimensional structure is essential. This has proven to be true for soluble proteins such as enzymes, electron transport proteins, or protein-nucleic acid assemblies such as viruses where the 3D structures have helped define their function. The structure/function relationships for integral membrane proteins are relatively more difficult to understand since the transbilayer region embedded in the lipid usually regulates function. The discovery of AQP1 has catalyzed a focused interest in investigating the molecular structure of aquaporins and components of the polypeptide sequence implicated in water and solute transport. A. AMINO ACID SEQUENCE ANALYSISAND SPECTROSCOPICSTUDIES OF AQUAPORINS

Several techniques such as predictive theoretical methods, spectroscopic, ultrastructural, immunohistochemical, and molecular biological approaches have been used to provide low- to medium-resolution views of aquaporin structures. The hydropathy analyses of the MIP members predict the presence of six major hydrophobic, putative membranespanning segments (Reizer et al., 1993) and two minor hydrophobic segments. Based on Kyte-Doolittle hydropathy plots (Preston and Agre, 1991; Verkman and Mitra, 2000) for AQP1 (Fig. 2) a model for the disposition of the segments of the polypeptide sequence in the bilayer and extrabilayer space can be generated, as shown in Fig. 3. Such a topology was supported by investigations carried out in several laboratories. First, immunohistochemical experiments using antibodies to the C- and N-termini indicated that the AQP1 polypeptide chain is arranged in the bilayer with the N- and C-terminus cytoplasmic (Smith and Agre, 1991;


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Nielsen et al., 1993; Zeidel et al., 1994). Next, segments of the polypeptide chain that face the cytoplasm or the extracellular space were identified by utilizing epitope tagging and aquaporin-reporter chimeras (Preston et al., 1994; Shi et al., 1995). The results were consistent with the polypeptide chain threading the bilayer six times, corresponding to the six major hydrophobic segments. Three of the loops, namely those connecting segments 1 and 2 (loop 1-2), segments 3 and 4 (loop 3-4), segments 4 and 5 (loop 4-5), and the charged carboxy- and aminoterminals correspond to the hydrophilic signals in the hydropathy plot (Fig. 2). On the other hand, the loops containing the first NPA box which connect segments 2 and 3 (loop 2-3) and that containing the second NPA box which connect segments 5 and 6 (loop 5-6) correspond to the minor hydrophobic signals and are therefore likely to be largely oriented within the bilayer. Thus, based on the observed important effects on water transport imparted by the residues in these segments of the polypeptide chain, it was envisioned that the two loops dip into the bilayer from the two sides and line the water pathway, forming an "hourglass" (Jung et al., 1994; Engel et al., 1994). Such a topology model is expected to be shared by other aquaporins and MIP members and has been shown to be the case for two other aquaporins, AQP2 (Bai et al., 1996) and AQP4 (Shi et al., 1995). Based on sequence analysis of MIP members Reizer et al. (1993) first indicated that the major hydrophobic segments in the polypeptide sequence possibly are s-helical as, for instance, in the case of bacteriorhodopsin. Spectroscopic studies using circular dichroism (CD) and Fourier transform infrared spectropscopy (FTIR) (van Hoek et al., 1993; Haris et al., 1995) suggested ~40% s-helical content for AQP1. Later, Cabiaux et al. (1997), from a carefully attenuated total reflection- (ATR) FTIR study, arrived at a helical content of ~45% with no elements of E-structure present. The estimates of the secondary structure elements from analyses of the spectra are dependent on the basis sets that are derived from only a handful of available high-resolution membrane protein structures. Therefore the estimates have considerable uncertainty. This notwithstanding, the results from spectroscopic studies from various laboratories are consistent with each of the six major hydrophobic segments adopting a s-helical conformation. This has been subsequently proven to be correct from high-resolution electron crystallographic studies (see Section IV,C). These same observations strongly contradict theoretical predictions suggesting a porinlike E-barrel structure (Fischbarg et al., 1995) with minimal s-helical content; however, elements of E-structure in the interhelix loops cannot be ruled out.


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B. OLIGOMERICSTRUCTUREAND THE FUNCTIONALUNIT FOR SOLUTETRANSPORTIN AQUAPORINS

Measurement of Stokes radius and sedimentation coefficients in Triton X-100 micelles (Smith and Agre, 1991) first indicated that AQP1 exists as a multisubunit complex. Determination of protein size after correction of detergent binding yielded an apparent mass of ~ 135 kDa, which suggested that AQP1 molecules are tetrameric in Triton X-100. Single-particle images of purified AQP1 in the detergent octyl ~-D glucoside (OG) also indicated tetrameric assembly (Walz et al., 1994a). Different results with respect to the oligomeric assembly were obtained by van Hoek et al. (1993), who showed by size-exclusion chromatography that in the detergent OG native AQP1 migrated as dimers and monomers at a 1 : 1 ratio, whereas upon deglycosylation by treatment with PNAGaseF AQP1 molecules migrated exclusively as monomers. Both deglycosylated and native, partially glycosylated AQP1 transported water with equal efficiency (van Hoek et al., 1995), indicating that glycosylation has no role in function, as has been shown also to be the case for AQP2 (Bai et al., 1996). Freeze-fracture electron microscopy (FFEM) of reconstituted proteoliposomes containing purified AQP1, CHO cells expressing AQP1, and kidney proximal tubule and thin descending limb of Henle provided a low-resolution, direct visualization of AQP1 quaternary structure in the membrane as a tetrameric assembly of individual units (Verbavatz et al., 1993). The tetrameric organization for AQP1 in the lipid bilayer membranes was later established for both native and deglycosylated AQP1 at a higher resolution by electron crystallographic studies and was also demonstrated in the case of AQP0 and AQPZ based on similar studies (see below). On the other hand, the bacterial glycerol facilitator glpF solubilized in OG showed sedimentation properties consistent with monomeric forms (Lagree et al., 1998) and also in situ (Bron et al., 1999) based on FFM analysis. A recent study by Mathai and Agre (1999) has indicated that the Ala73Met substitution in the first NPA loop (2-3) of recombinant, tandem AQP1 dimers causes the disruption of the normal tetrameric structure. This result suggests that even though Ala73 in the hydrophobic loop is expected to be buried, it is important for normal oligomeric assembly of AQP1. In the case of an insect aquaporin, AQPcic, Lagree et al. (1999) observe that substitution of two residues (Y222P and W223L) at the extracellular face of helix 6 disrupts the tetrameric organization in OG. Also, unlike the wild-type AQP2, oocyte-expressed AQP2-R187C, a mutant in recessive nephrogenic diabetes insipidus, is not only monomeric but

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also fails to heterotetramerize with wild-type AQP2 in the endoplasmic reticulum (Kamsteeg et al., 1999). As noted above, the radiation inactivation studies (van Hoek et al., 1991, 1992) of water-permeable cells, which indicated a molecular weight of ~30 kDa for the principal species responsible for facilitated water transport, suggested that monomers are the functional units in the case of AQP1. In addition, several lines of evidence emanating from studies utilizing molecular biological approaches, e.g., water-transport properties of oocytes expressing wild-type and mutant AQP1 mixtures (Preston et al., 1993; Zhang et al., 1993; Jung et al., 1994) and heterodimers comprised of wild-type and mutant subunits (Shi et al., 1994), were consistent with the notion that the water-selective pore in AQP1 is enclosed by a monomer. This is similar to the case of porins but is in contrast to the oligomeric ion channels (Green and Millar, 1995) such as the K + channel (Yang et al., 1995; Doyle et al., 1998) or the ligand-gated nicotinic acetylcholine receptor (Miyazawa et al., 1999), where the solute pathway is surrounded by the component subunits. It is to be noted, however, that since isolated, functional AQP1 monomers have not yet been identified, one cannot discount the possibility that the tetrameric quaternary structure may be indirectly involved in the architecture of the active, functional AQP1 monomeric channel. It has been noted by Lagree et al. (1999) for the insect AQPcic that switch from water to glycerol transport induced by site-specific substitution is accompanied by a loss oftetramerization. In view of these observations, further investigations are warranted to firmly establish the exact role of the quaternary organization within the aquaporin members. C. ANALYSISOF AQUAPORIN3D STRUCTUREATHIGH RESOLUTION Although, there has been burgeoning research on aquaporins in recent years, much of our high-resolution knowledge about the structure/function relationship of aquaporins is currently limited to the archetype AQP1. This is directly related to the fact that milligram amounts of highly purified AQP1 can be obtained from red blood cells (Zeidel et al., 1992; van Hoek et al., 1993, 1995) that are amenable for 2D crystallization through reconstitution into lipid bilayer membranes. Hence what follows is a detailed description of our current level of structural information on AQP1 determined by electron crystallography. 1. Electron Crystallography of 2D Membrane Protein Crystals Unlike soluble proteins, integral membrane proteins are usually recalcitrant to the growth of large, well-ordered 3D crystals, which are

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necessary for high-resolution X-ray crystallographic analyses. Recent examples of high-resolution (< 4 A) membrane protein structures determined by X-ray crystallography can be seen, for example, in Buchanan et al. (1999), Chang et al. (1998), Ferguson et al., (1998) and Doyle et al. (1998). The difficulty in this case is partly due to the fact that such crystals can be generated only in the presence of detergents which are required to keep the protein in solution. An alternative approach is to grow thin, one-molecule-thick 2D crystals in lipid bilayers (reviews, e.g., Jap et al., 1992; Ktihlbrandt, 1992; Hasler et al., 1998) and to solve the structures using electron crystallography. Examples of membrane protein structures solved to high resolution by electron cyrstallography applied to 2D crystals in the lipid bilayer include the LHC II from spinach chloroplasts (Ktihlbrandt et al., 1994) and bacteriorhodopsin from Halobacterium salinarium (Grigorieffet al., 1996; Kimura et al., 1997). The membrane protein in such a 2D crystal is surrounded by lipids rather than detergent micelles, which allows for a direct assay of function such as solute transport (Walz et al., 1994b) and its modulation, opening of the channel in the bilayer transduced by ligand binding (Berriman and Unwin, 1994; Unwin, 1995). Another notable advantage of electron crystallography is that phases can be directly obtained from the images, unlike in the case of X-ray, where phases must be determined indirectly by methods such as isomorphous replacement. The availability of the phase information partially compensates for the lack of data at the highest resolution (typically ~3.5/~ and beyond) because of low contrast in the images. 2. Method of Analysis of 2D Crystals by Electron Microscopy

Usually, screening for optimum conditions for successfully growing 2D crystals is carried out by negative-stain electron microscopy. For this purpose, the specimen, after being allowed to settle on a carbon film overlaying an electron microscope grid, is embedded in a heavy metal salt such as uranyl acetate or sodium phosphotungstate and then examined in an electron microscope. The presence of crystalline areas, the degree of order, and the extent of the coherent areas are judged by an examination of a recorded image in an optical diffractometer. Structural analysis from 2D crystals is briefly outlined below since excellent reviews (e.g., Amos et al., 1982) and papers detailing this method already exist in literature. Since embedding in stain produces a surface relief, only low-resolution information is preserved. For higher resolution analysis, specimens dried in sugars or preserved unstained and forzen-hydrated in vitrified buffer are used. Typically, the analysis is first carried out in projection using data derived from

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electron micrographs of crystals viewed in the direction of the electron beam (nominally untilted). For this purpose, areas of recorded images that diffract to the highest resolution in an optical diffractometer are digitized and then Fourier transformed in a computer. The amplitudes and phases of the Fourier components are extracted after application of algorithms [e.g., suite of programs form the Medical Research Council at Cambridge (Henderson et al., 1990)] that correct for lattice distortion and the effects of the levels of lens defocus used in recording the images (contrast transfer function). Inverse Fourier transformation of the amplitudes and phases then yields a map of the protein density viewed in a direction perpendicular to the plane of the bilayer. In order to determine the structure in three dimensions, the specimen is tilted with respect to the electron beam and many two-dimensional projected views are recorded. The data from the tilted views are extracted as described above and combined to generate a three-dimensional density map. The analysis of negatively stained crystals in projection, albeit at low resolution, is very useful for a rapid determination of the planegroup symmetry and the dimensions of the lattice, the overall shape of the molecule, indications of possible intermolecular interaction, and the oligomeric state. However, no information about the structure in the bilayer can be derived. This is because the polar heavy-metal stain cannot significantly permeate the hydrophobic lipid bilayer. Alternate approaches of embedding in sugar such as glucose (e.g., Henderson et al., 1990), trehalose (e.g., Kimura et al., 1997, Nogales et al., 1998), or tanin (e.g., Kfihlbrandt et al., 1994) or preservation in a frozen-hydrated state (Dubochet et al., 1988) in vitrified buffer (e.g., Unwin, 1995; Cheng et al., 1997; Auer et al., 1998; Zhang et al., 1998) are used to achieve higher resolution and to visualize the complete three-dimensional fold of the molecule. 3. History of Electron Crystallography of 2D Crystals of AQP1 Two-dimensional crystals of AQP1 were generated by three groups using different conditions for crystallization. These crystals were subjected to electron cryocrystallographic analyses to determine the structure in projection (Jap and Li, 1995; Walz et al., 1995; Mitra et al., 1995) using different methods for specimen preservation. Walz et al. (1995) used samples of glucose-embedded, native, partially glycosylated AQP1 from h u m a n erythrocytes crystallized in lipids extracted from E. coli. Glucose embedding was also employed by Jap and Li (1995), who used samples of native, bovine AQP1 (previously called CHIP29) crystallized in dimyristoyl phosphatidylcholine (DMPC). We (Mitra et al., 1995) crystallized deglycosylated human erythrocyte AQP1 (van Hoek


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FIG. 4. Projection density map of AQP1 obtained by different groups. (a) Map at 3.5/~ resolution reproduced from Jap and Li (1995), (b) map at 3.5/~ resolution reproduced from Hasler et al. (1998), and (c) map at 3.7/~ resolution reported in Ren et al. (2000) (reproduced by permission from the Journal o f Structural Biology).

et al., 1995) in dioleoyl phosphatidylcholine (DOPC) bilayers and examined the frozen-hydrated crystals while preserved unstained in vitrified buffer. These differences notwithstanding in the conditions of crystallization and specimen preparation, the three projection maps revealed overall similarities (Fig. 4) in the density features. The aquaporin monomers in the 2D crystals assemble as tetramers, as observed in vivo (Verbavatz et al., 1993); however, adjacent tetramers, because of the lattice symmetry (p4g plane group), have opposite orientation to that in vivo (Verbavatz et al., 1993). Inspite of the up-and-down orientation of AQP1 tetramers, it was possible to show that protein formed functional channels (Walz et al., 1994b) by virtue of the fact that AQP1 and other aquaporins are bidirectional (Meinild et al., 1998) (see also below). The multiple density peaks in the projection maps suggested the presence of six or more helical segments (Jap and Li, 1995; Walz et al., 1995; Mitra et al., 1995); however, the rather elongated and not clearly separated peaks indicated that the putative helices are likely to be significantly tilted. This was borne out from the three-dimensional density maps published by us and the other two groups at ~6-7 A resolution. We examined frozen-hydrated, ice-embedded specimens as before and accumulated 3D data from diffraction patterns and images recorded from crystals tilted up to 45 ° to determine the unperturbed 3D structure (Cheng et al., 1997). The other two groups used specimens preserved in trehalose and the tilted projections composed of images and electron diffraction pattern (Walz et al., 1997) and only images (Li et al., 1997). Two-dimensional crystallization and structural studies in projection on four other aquaporins, namely mammalian MIP (AQP0), bacterial

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AQPZ, insect aquaporin AQPcic, and aTIP from plants, have been reported. Two-dimensional crystals of MIP (AQP0) were generated (Hasler et al., 1998) using protein extracted from sheep lens and purified in decylmaltoside. Crystallization was achieved by reconstitution into E. coli lipid bilayers by dialysis at 37°C. The 2D crystals of AQP0 exhibited a lower p4 symmetry than the p4212 symmetry ofAQP1 (Walz et al., 1995; Mitra et al., 1995). When compared to AQP1, the projection map of freeze-dried AQP0 crystals at 9 A resolution showed differences in packing consistent with lower symmetry. However, overall, the p e a k s in the monomer densities have similar dispositions except at the periphery and in the interior of the monomer that may be related to the functional difference between AQP0 and AQP1. Recombinant AQPZ expressed in E. coli and purified in OG was reconstituted by dialysis at room temperature into a 1:1 mixture of synthetic lipids POPC and DMPC to yield 2D crystals (Ringler et al., 1999). The crystals exhibited the same symmetry p4212 as AQP1 2-D crystals and a projection map at 8/~ displayed very similar characteristics of the monomer density when compared to those for AQP1. Two-dimensional crystals of AQPcic naturally formed in native membranes of a filter chamber of CicadeUa virdis and the degree of order was enhanced by alkali treatment followed by incubation at 4°C for 1 week (Bron et al., 1999). A 15-/~-resolution projection map was obtained which showed that the AQPcic is a tetramer in which each monomer is composed of two unequal density domains. No information about the plane-group symmetry was provided, a-TIP from plant vacuole membrane was purified in diheptanoylphosphocholine and was reconstituted into lipid bilayers to generate helical crystals (Daniels et al., 1999). Flattened tubes displayed p2 symmetry and c222 pseudosymmetry, and it was inferred that the unit cell houses two tetramers. A projection density map at 7.7/~ resolution revealed that a heart-shaped ring forms each subunit and is composed of density peaks that were interpreted to represent a-helices. 4. Description of the Near-Atomic Resolution 3-D Map of AQP1 a. AQP1 Is Formed by a Barrel of Six Transmembrane a-Helices. We discuss below in more detail our current knowledge of the 3D density of AQP1 and what the structure can tell us about how AQP1 functions. Inclusion of high-resolution 3D data generated from 2D crystals embedded in unstained, vitrified buffer and titled up to 60 ° allowed the computation of a 4-A-resolution 3D density map of AQP1. Four AQP1 at monomers are arranged symmetrically around a four-fold axis oriented perpendicular to the bilayer. The overall density for each monomer is

AQUAPORIN WATER CHANNEL

147

FIG. 5. Three-dimensional density map of ice-embedded, frozen-hydrated AQP1 at a n in-plane resolution of 4/~ a n d at a resolution of ~ 7 / ~ normal to the bilayer. One monomer with the densities for the tilted t r a n s m e m b r a n e a-helices (A-F) and portions of adjacent monomers are shown. The density enclosed by the helix barrel and located near the middle of the bilayer (asterisk) is attributed to the NPA box region of the polypeptide chain.

approximately cylindrical (~30/~ in diameter and ~60/~ high). The prominent feature in a monomer (Fig. 5) is a barrel formed by approximately six cylindrical, tilted (18-30 °) rods (A to F ~36-44 A long) representing the six transmembrane a-helices. This helix barrel characterizing the core structure of the monomer encloses a central density (Fig. 5) that has been attributed to the NPA box regions (Walz et al., 1997; Li et al., 1997; Cheng et al., 1997). Within a monomer, the helices pack tightly near the middle of the bilayer in three pairs: A-B, C-D, and E-F. The small-sized amino acids Gly and Ala, which are quite abundant especially in segments 2, 3, 5, and 6 (Fig. 3), may encourage

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such observed close packing of the helices within the hydrophobic core. Consistent with the improved resolution, densities for the a-helical segments, some more than the others, display discernible protrusions along their length. All of the helices show some degree of curvature, especially helix D and helix F. These bends in the helices could be due to the presence of a number of glycine and/or proline residues located within the putative membrane-spanning regions of the polypeptide chain (Fig. 3). The putative sites of interhelix contacts mediated by side chains are also revealed. On the proximal side (Fig. 3), intramonomer, interhelix contacts possibly mediated by side chains are seen between helices A and B, C and D, and E and F and near the middle of the monomer between helices B and C and between helices E and F. The region near the fourfold axis at the interface of the four monomers appears to lack significant protein density (Fig. 5); however, its diameter is smaller than seen in the lower resolution map (Cheng et al., 1997) because of the appearance of protrusions on density for helices C and D. b. The 3D Structure Reveals S y m m e t r y within an A Q P 1 Monomer. The polypeptide sequences of aquaporins and those of the MIP family members, in general, display internal sequence homology between the N- and C-termini, which is due to an intragenic duplication event (Wistow et al., 1991). In the 3D structure also we see evidence of internal symmetry. Examination of our 7/~ 3D density map revealed that in the hydrophobic core of an AQP1 monomer, there is a symmetric disposition of protein density. Thus planes of 3D density, parallel to the bila~er and equidistant above and below a particular plane (located ~3 A away from the center of the mass), can be superposed by a rotation of 180 ° around an axis inclined by ~10 ° to a lattice. This axis of symmetry approximately bisects the helix pairs C/D and A/F (Fig. 5) and passes through the central density. This (twofold) symmetry is local (pseudosymmetry) and is strongest within a span of ~14/~ near the center of an AQP1 molecule. We carried out a more careful analysis of the current, higher resolution density map to scrutinize the presence of an in-plane pseudo-twofold axis of symmetry. This analysis showed that the presence of an in-plane pseudo-twofold axis of symmetry is strongly revealed only when data up to a resolution of 6 A were used. However, the signal was not significant when 3D data to the highest resolution were included (data not shown). This result indicates that the local symmetry is strong only when features such as the spatial locations and presumably the backbones of the a-helical segments of the two halves of the polypeptide chain (Mitsuoka et al., 1999) are compared. Alternatively, the location of the helices belonging to the tandemly repeating motifs are indeed constrained by the pseudosymmetry but the


149

symmetry is obscured at higher resolution by the significant differences in the density due to the dissimilarity of the side-chain composition. In membrane proteins, intramolecular pseudosymmetry has been seen also in LHC II (Kiihlbrandt et al., 1994) between two homologous helices where the twofold axis is oriented perpendicular to the membrane plane. It is known that AQP1 transports water in either direction. The observation of two-fold symmetry in the membrane plane and tandem repeats in the sequence may be coupled to provide a simple explanation for how such a bidirectional transport across the bilayer may be achieved. c. Density Attributed to the NPA Box Region within the Helix Barrel and Extrabilayer Densities Representing Interhelix Linkers. Strong bands of density, some of which are continuous and comparable in strength to that for the transmembrane helices, can be identified at the extremities of some of the helices. These suggest the locations of some of the ordered interhelical loops and those within the helix barrel (central density) define the putative NPA box region (Fig. 6). Consistent with this observation, we note that, both in the case of solubilized AQP1 (in our hands) and lipid-reconstituted 2D crystals ofAQP1 (Walz et al., 1996), interhelical loops are protected from limited proteolysis and therefore are likely to be structurally rigid and/or are apposed close to the bilayer surface. On the proximal side of Fig. 3, a clear linkage between helices B and C is observed, whereas on the distal side helices D and E appear to be linked. The density within the helix barrel, which is attributed to the NPA box regions of the polypeptide chain, is located in front of the lipid-facing helices A and F. This density appears as two V-shaped segments arranged tip-to-tip, similarly to that seen by Mitsuoka et al. (1999), and is better resolved than in our earlier density map. On the proximal side of Fig. 3, the central density extending from the edge of helix F is revealed as an almost continuous segment that broadens to a dimension comparable to that of a transmembrane a-helix and may harbor a short segment in a-helical conformation. Mitsuoka et al. (1999) have suggested, based on a density map calculated at a similar (4.5 A) resolution, that this relatively wider portion of the central density represents a short a-helix. Likewise, on the distal side, the central density extending from the edge of helix A widens near the middle of the bilayer and again the relatively wider portion of this density may harbor a short a-helix, as has also been suggested by Mitsuoka et al. (1999). d. Models for the AQP1 Topology. Because of the limited vertical resolution of the current 3D map, unambiguous alignment of the primary sequence into density proved to be difficult. In order to arrive at rational

FIo. 6. Central density within the helix barrel attributed to the NPA box regions and viewed parallel to the bilayer. The chicken wire density is at 1 s t a n d a r d deviation while the solid density is at 2 s t a n d a r d deviations of the m e a n density of the map.


151

model(s) for the threading of the polypeptide chain (topology) we exploited the presence of observed linkages in the 3D density map discussed above and employed the so-called "hourglass" arrangement, in which the NPA containing interhelix loops fold into the bilayer (Jung et al., 1994), and assumed that the relative dispositions of the transmembrane helices are similar between homologous aquaporins. Based on observed linking densities that are more clearly visualized in our higher resolution map, clues from protease sensitivity, and amino acid sequence and muatagenesis experiments (e.g., Zeidel et al., 1994; Pao et al., 1991; Jung et al., 1994; Froger et al., 1998; Deen et al., 1994; Bai et al., 1996), two classes of models were deduced which are shown schematically in Fig. 7. The two classes correspond to two possible

FIG. 7. Proposed models for the a s s i g n m e n t of the six major hydrophobic segments to the six helices, A to F, seen in the density m a p of a monomer. (a) Class 1 models corresponding to the proximal side extracellular. (b) Class 2 models corresponding to the proximal side cytoplasmic.

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FIG. 7. (Continued) orientations of the AQP1 monomer in the 3D density map, i.e., proximal side extracellular (class 1) or cytoplasmic (class 2). All the four proposed models require the presence of a "crossover" 3-4 linkage. Density for such a linker in the location implied in class i models (proximal edges of helices A and E) is not apparent in the 3D map, but is ascribed to the distal connection between helix F to helix C or helix B via the central density in the class 2 models. Such an arrangement in class 2 models is consistent with the suggested functional role for the 3-4 linker in water transport in AQP2 (Bai et al., 1996) and in influencing solute selectivity in glycerol transporters, where this linker is significantly longer (Froger et al., 1998). The irregular arrangement of helices in the proposed models do not necessarily pose a problem in membrane topogenesis because the insertion process in vivo proceeds with the sequential threading of helix pairs (see review by Bibi, 1998 and papers cited therein). It is also noted, for example, that in


153

the X-ray crystal structure of subunit III of cytochrome c oxidase from Paracoccus dentrificans an irregular arrangement of seven transmemb r a h e s-helices have been observed (Iwata et al., 1996). Although the current models are derived based only on the observed connections in the higher 4-A-resolution 3D density map, it is interesting that one of the models (second model) in both classes implies the existence of twofold symmetry although no assumptions of symmetry were applied in the derivation. We had argued earlier (Cheng et al., 1997) that strict imposition of the in-plane pseudo-twofold symmetry is consistent with only a handful of topology models and one of the preferred models reported in Verkman and Mitra (2000) is identical to model 2 of class 1. Recently, based on analyses of the periodicity of sequence conservation and hydrophobicity in the polypeptide sequences of aquaporins, Heymann and Engel (2000) (see also Engel et al., 2000) have provided a topology model that is valid for both orientations and implies a pseudo-two-fold symmetry but is different from a model proposed earlier (Heymann et al., 1998) which ascribed a sidedness to the AQP1 monomer in their 3D density map (Walz et al., 1997). We note that our model 2 in class 1 agrees with the model proposed by Heymann and Engel (2000). The agreement notwithstanding, the correctness of the topology can only be established when either ancillary information regarding the relative locations of marker residues, obtained, e.g., from labeling or mutagenesis approaches, are available or when the atomic resolution structure is solved. e. Location and Architecture of the Channel. All published lines of evidence (Preston et al., 1993; Shi et al., 1994; van Hoek et al., 1991; Zhang et al., 1993) indicate that the AQP1 monomer contains the functional channel. The volume of low density (negative density) representing the solvent-accessible region enclosed by the helix barrel shown in the form of surface rendering in Fig. 8 has a vestibular shape (Mitra et al., 1995; Cheng et al., 1997). This volume is relatively wide on both the cytoplasmic and the extracellular sides but narrows down to a diameter of ~6/~ near the center of the molecule. This narrow region located in the hydrophobic core of the molecule may represent the physical constriction which defines the selectivity filter. The vestibular region is bounded by the tightly packed helices C and D near the fourfold axis and by the wall of density within the barrel attributed to the NPA boxes (Fig. 6), thereby ensuring entry and exit to the narrow constriction only from the top and bottom of the monomer. Because the demarcation of protein and solvent regions is somewhat arbitrary, the fact that the side chains have not been positioned due to limited vertical resolution of the map may mean that the actual diameter of the constriction is smaller. The side

154

ALOK K. MITRA

FIG. 8. The solvent-accessible region is represented as a light-gray-colored volume enclosed by the six-helix barrel, which has a vestibular shape. This is represented in the form of a smoothed, surface rendered volume of low (negative) density. The narrowing of the vestibule to ~ 6 / ~ near the center of the bilayer where the channel location is proposed is shown. The helices C and D, nearest to the fourfold axis, have been peeled offto illustrate more clearly the narrowing of the channel.


155

chains that define the channel wall may include residues contributed by helices C and D, which display bumps pointing to the putative pore region (Fig. 5), and based on the proposed threading models helices (Fig. 7) 1, 2, 4, and 5, which are proximal to the suggested pore location, may house functionally important residues. The appropriate architecture and/or selectivity of the AQP1 channel in part may be made up of some of these channel-lining residues in addition to amino acid residues in the vicinity of the two conserved NPA motifs that have been previously shown (Jung et al., 1994) to be important in water transport. f. Residues in the Transmembrane Helices That May Be Important for Water Transport Function in AQP1. We may speculate on the nature and identities of the channel-lining residues (Heymann et al., 1998) since we do not yet have an atomic-resolution structure. We note that the low activation energy (4-5 kcal/mol) for osmotic water transport suggests that the pathway followed by water is relatively polar, similar to that of bulk water (Macey, 1984). In other words, the biophysical measurements suggest that a water molecule approaching the channel, and in its passage through the aqueous pore, thermodynamically behaves as if it is diffusing in bulk water. Welling et al. (1996) have proposed a macroscopic model for the AQP1 channel which is characterized by a lining made up of hydrating molecules (hydrated side chains) that is sufficiently small for only water molecules to enter and exit by diffusion. In such a model, because the pore is lined with water molecules, interaction with the pore walls is not significantly different from that in free solution. The predictions of this model agree well with experimental data for AQP1 and other water-filled channels and in the absence of an atomic resolution model, such a macroscopic model is a possible alternative. A hydrated pore within the hydrophobic core of the molecule which is lined with polar or charged residues and sufficiently narrow for only water molecules to enter and exit by diffusion can adequately represent such an environment. Therefore, we may hypothesize that polar and charged amino acids participate to generate such an environment within the putative transmembrane domain and may be integral to the architecture of the aqueous pathway. Polar and charged residues have been shown in many cases to be key components in the structure and function of membrane proteins. Charged residues have been implicated, e.g., in light harvesting complex (Kfihlbrandt, 1994), nicotinic acetylcholine receptor (Imoto, 1988), muscarinic acetylcholine receptor (Page, 1995), Ca2+/Na+-ATPase (Andersen and Vilsen, 1995), photosynthetic reaction center (Okamura and Feher, 1992), and bacteriorhodopsin (Braiman et al., 1988; Balashov et al., 1993) while polar residues have been implicated, e.g., in nicotinic acetylcholine receptor

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(Charnet et al., 1990; Unwin, 1995; Imoto et al., 1988), Ca2+/Na +ATPase (Andersen and Vilsen, 1995), and cytochrome oxidase (Hosler et al., 1996). Residues that are expected to be located near the middle of the bilayer where the narrow water-selective pore is proposed to be are shown in Fig. 9a and a model for the azimuthal orientations of the helices was generated (Fig. 9b) based on consensus secondary structure assignment, alignment of the sequences of aquaporin homologs, the criterion that conserved residues are likely to be involved in proteinprotein contact, and that polar and charged residues are either buried or tend to position near the edge of the bilayer. Such a model places polar and charged residues such as Glul7, Thr21, Thr22, and Ser28 in helix 1; Set59 and Thr62 in helix 2; Glnl01 and Thrl09 in helix 3; Glu142, Thr146, and Gln148 in helix 4; and Ser175 and Hisl80 in helix 5 in the middle and the inside surface of the helix barrel. Some of these may line the pore and be structurally and/or chemically involved in channel function. Other conserved nonpolar residues, especially glycines and prolines (e.g., in helices 2, 3, 4, 5, and 6; Fig. 3), may be critical to the channel architecture and thereby influence selectivity indirectly. These residues are in addition to those belonging to the NPA box regions that have already been proposed (Froger et al., 1998; Jung et al., 1994) to be important for water transport. The complete, channel environment will certainly include other residues involved in the appropriate architecture of the channel and important for imparting variability in water permeability within the homologs (Yang and Verkman, 1997). Due to significant sequence homology, it is likely that elements of the 3D structure, especially the fold of the polypeptide chain visualized for AQP1, are conserved among the aquaporins. However, chemical/structural changes localized at the entrance or exit of the narrowest part of the channel and/or distributed globally effecting the vestibular architecture (Fig. 8) may be exploited to elicit the observed variability in the nature of the solute permeability. Undoubtedly, a concerted approach employing site-specific mutagenesis, dictated, for instance, by the proposed topology model and the candidate residues mentioned above (improved resolution of the density map to identify atomic details and biophysical measurements), should help provide further insight into how this ancient family of proteins play a fundamental role in cell homeostasis. V. UNRESOLVEDQUESTIONSAND FUTURE DIRECTIONS

Apart from the need to have an atomic resolution structure, there are many issues that require further research. Although, the consensus is


159

that the channel is enclosed by the monomer, the role in solute transport, if any, of the relatively protein-depleted region surrounded by the four monomers near the fourfold axis is not known. The exact structure/function relationship of the quaternary organization in AQP1 is not established yet. As noted above, sucrose-density gradient measurements and FFEM indicate that the glycerol transporter GlpF does not tetramerize (Lagree et al., 1998; Bron et al., 1999) and that specific residues in the second NPA loop can act as switches, dictating the quaternary organization (Lagree et al., 1999). The effect of binding of mercurial reagents to aquaporins is not known in detail. Mapping of the conformational modulation, if any, upon binding of mercurial reagents is essential to understanding mechanistically how the inhibition process takes place. Factors in the sequence and structure that impart mercurial insensitivity (as in AQP4) and how the recently observed enhancement rather than inhibition in the case of AQP6 are achieved at the molecular level warrant study. Another issue is whether there are conformational changes in the protein during solute transport. Such an investigation requires elaborate molecular dynamics simulation when a high-resolution 3D structure becomes available. These studies are important toward rational design of compounds for targeted and possibly tissue-specific control of aquaporin-mediated water/solute flow. The pharamacological import of such an endeavor cannot be overstated. Biochemical regulation, e.g., by phosphorylation is an area that needs to be investigated given that consensus sequences for protein kinases A and C exist in several aquaporins. The issue of whether effect on function is directly related to phosphorylation, as in the cases of plant aquaporins PM28A (Johansson et al., 1998) and s-TIP (Maurel et al., 1995), or indirectly related due to trafficking, as in the case of AQP2 (Lande et al., 1996), needs to be addressed before any elaborate structural studies are attempted. The recent exciting results relating to regulation of transport for AQP3 (Zeuthen and Klaerke, 1999), AQP6 (Yasui et al., 1999), and AQP0 (Cahalan and Hall, 2000) should instigate experiments to explore whether similar or other gating phenomena exist for other aquaporins. The promiscuity of the newly discovered AQP9 channel with regard to solute transport (Tsukaguchi et al., 1998) has raised important questions about the solute pathway, i.e., what elements of polypeptide sequence are exploited to effect acute selectivity in some, but broad selectivity in others, within a family of very homologous members. Is there a generic pore with a similar overall structure in all aquaporins and variation of residues within and at the mouth of the pore that are responsible for presence or absence solute selectivity? Undoubtedly, a concerted approach employing site-specific mutagenesis, high-resolution structural studies, and biophysical measurements

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should help provide further insight into how this ancient family of proteins play a fundamental role in cell homeostasis. Recent successes in the expression of several recombinant aquaporins are expected to significantly help this endeavor (Laize et al., 1995, 1997; Coury et al., 1998; Lagree et al., 1998; Borgnia et al., 1999). ACKNOWLEDGMENTS I thank G. Ren for carrying out much of the work related to the determination of the high-resolution 3D density map of AQP1 and V. Lagree for useful comments on the chapter. The research was supported by a grant from the National Institutes of Health, in part by grants from the National Science Foundation and the American Heart Association, and by a special fellowship from the Cystic Fibrosis Foundation. A.I~M. is an established investigator with the American Heart Association. REFERENCES Abrami, L., Berthonaud, V. et al. (1996). Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: Inhibition studies. Pflugers Arch. 431(3), 408-414. Agre, P. (1997). Aquaporin nomenclature workshop: Mammalian aquaporins. Biol. Cell. 89, 255-257. Agre, P., Brown, D., and Nielsen, S. (1995). Aquaporin water channels: Unanswered questions and unresolved controversies. Curr. Opin. Cell Biol. 7, 472-483. Agre, P., Lee, M. D., Devidas, S., and Guggino, W. B. (1997). Aquaporins and ion conductance. Science 275, 1490. [letter; comment] Agre, P., Preston, G. M. et al. (1993). Aquaporin CHIP: The archetypal molecular water channel. Am. J. Physiol. 265(4/2), F463-F476. Amos, L. A., Henderson, R., and Unwin, P. N. T. (1982). Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog. Biophys. Mol. Biol. 39, 183-231. Andersen, J. P., and Vilsen, B. (1995). Structure-fimctionrelationships of cation translocation by Ca(2+)- and Na+,K(+)-ATPases studied by site-directed mutagenesis. F E B S Lett. 359(2/3), 101-106. Auer, M., Scarborough, G. A., and Kiihlbrandt, W. (1998). Three-dimensional map of the plasma membrane H+-ATPase in the open conformation. Nature 392, 840-843. Bai, L., Fushimi, K., Sasaki, S., and Marumo, F. (1996). Structure of aquaporin-2 vasopressin water channel. J. Biol. Chem. 271, 5171-5176. Balashov, S. P., Govindjee, R. et al. (1993). Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle. Biochemistry 32(39), 10,331-10,343. Berriman, J., and Unwin, N. (1994). Analysis of transient structures by cryomicroscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56, 241-252. Bibi, E. (1998). The role of the ribosome-translocon complex in translation and assembly of polytopic membrane proteins. Trend. Biol. Sci. 23, 51-55. Borgnia, M. J., Kozono, D., Maloney, P., and Agre, P. (1999). Purification and functional reconstitution of bacterial aquaporins. Biophys. Soc. Abstr. 76, W-Pos204. Braiman, M. S,, Mogi, T. et al. (1988). Vibrational spectroscopy of bacteriorhodopsin mutants. I. Tyrosine-185 protonates and deprotenates during the photocycle. Proteins 3(4), 219-229. Bron, P., Lagree, V., Froger, A., Rolland, J.-P., Hubert, J.-F., Delamarche, C., Deschamps, S., Pellerin, I., Thomas, D., and Haase, W. (1999). Oligomerization state of MIP


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VITAMINS AND HORMONES, VOL. 62

Cytostatic p21 G Protein-Activated Protein Kinase ~/-PAK JOAN ROIG AND JOLINDA A. TRAUGH Department of Biochemistry, University of California, Riverside, Riverside, California 92504 I. Background II. Comparison of PAK Proteins III. Autophosphorylation and Activation of ~/-PAK IV. ~/-PAKHas Cytostatic Activity A. Injection of ~-PAK into Early Frog Embryos B. Xenopus X-PAK and the Cell Cycle C. Expression of Recombinant ~/-PAKin Mammalian Cells V. Activation of ~/-PAKin Response to Stress A. DNA Damage and Mitotic Arrest B. Hyperosmolarity C. Sphingosine D. Serum Starvation and Contact Inhibition E. Activation of ~/-PAKby Cleavage with Caspase 3 during Programmed Cell Death VI. The Individual Roles of ~/-PAKand SAPK/p38 in the Stress Response VII. Substrates for ~/-PAK A. Identification of a Recognition/Phosphorylation Sequence for ~/-PAK B. Protein Substrates for ~/-PAK C. Effects of Phosphorylation on Substrate Activity VIII. Working Model A Role for ~/-PAKas a Master Switch IX. Conclusions and Remaining Questions References

The p21-activated protein kinase ~-PAK, also known as PAK2, has very different properties from the other two highly conserved isoforms of the PAK family, a-PAK (PAK1) and B-PAK (PAK3). ~/-PAK has cytostatic activity, as shown by inhibition of cleavage of early frog embryos following microinjection of ~/-PAK and by inhibition of growth when expressed in mammalian cells. ~/-PAK is activated in response to a variety of stresses including radiation- and chemicallyinduced DNA damage, hyperosmolarity, addition of sphingosine, serum starvation, and contact inhibition. Activation occurs through at least two signaling pathways, depending on the type of stress, one of which requires phosphoinositide 3-kinase and/or tyrosine kinase activity. During apoptosis ~/-PAK is cleaved by caspase 3 and activated and appears to have a role in the apoptotic response. ~/-PAK is present in the cytosol, associated with the membrane and 167

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JOAN ROIG AND JOLINDA A. TRAUGH

in secretory granules. A wide variety ofsubstrates have been identified for ~-PAK. We propose y-PAK may be involved in coordinating the stress response, possibly in conjunction with other stress response proteins. ©2o01AcademicPress.

I. BACKGROUND

The first member of a new family of protein kinases was identified in 1994 by Lim and coworkers (Manser et al., 1994); this enzyme bound to and was activated by the small G proteins Cdc42 and Racl, but not by Rho; thus the nomenclature p21-activated protein _kinase (PAK). During this same period, Jakobi et al. (1996) were in the process of obtaining the cDNA sequence for the protein kinase PAK. PAK had been purified from rabbit reticulocytes and smooth and skeletal muscle as an inactive enzyme (Tahara and Traugh, 1981; Tuazon et al., 1982; Tuazon and Traugh, 1984). It could be proteolytically activated by limited digestion with trypsin, chymotrypsin, or a calcium-activated protease; hence the nomenclature _protease-activated _kinase I (Tahara and Traugh, 1981, 1982). Limited tryptic digestion produced cleavage of the regulatory domain of PAK I, producing an active catalytic fragment, p37 (Jakobi et al., 1996). During the period of 1994-1996, three isoforms of PAK were identified by sequence analysis, as indicated in Table I. a-PAK (also called PAK1) has a calculated molecular weight of around 60,660 and migrates at 68 kDa on SDS-PAGE. ~-PAK (PAK3) is around 60,690 Da and migrates at 65 kDa. ~-PAK (PAK2, PAK I) has more variability between species, with an average calculated molecular weight around 58,800, and migrates at 58-60 kDa on SDS-PAGE. To avoid confusion, the nomenclature containing Greek letters is utilized herein, a-PAK is enriched in brain but is also present in other tissues; ~-PAK is present primarily in brain. ~/-PAK appears to be ubiquitous and is present in all cells and tissues of higher eukaryotes examined. ~-PAK has been cloned and sequenced from human, rat, and rabbit. The three forms of PAK are highly homologous, with an N-terminal regulatory domain containing a small G protein-binding site and a C-terminal catalytic domain. Although the homology is high, there is no indication that the three gene products have similar functions. a-PAK is activated in response to growth factors, such as EGF and PDGF, and by insulin and has a role in cell growth, membrane ruffling, cytoskeletal reorganization, and motility (Manser et al., 1997; Sells

-PAK

169

TABLE I ISOFORMSOFMAMMALIANPAK Molecularweight Isoforma

Species

a-PAK PAK1 hPAK65 ~-PAK PAK3

Human Rat Mouse Human

-PAK PAK2 PAKI

Calculated SDS-PAGE Accession (Da) (kDa) numbers 68

Rat Mouse

60,661 60,578 60,737 60,689 60,693 60,711 60,684

Human Rat

60,578 57,960

60

Rabbit

58,027

65

Reference

Q13153 P35465 088643 075914 AAF67008 Q62829 Q61036

Brownet al. (1996) Manseret al. (1994) Burbeloet al. (1999) Allen et al. (1998) Jiang et al. (1999)b Manseret al. (1995) Bagrodiaet al. (1995a) Burbelo et al. (1999) Q13177 Martinet al. (1995a,b) Q64303 Teo et al. (1995) AAA65442 Sellset al. (1995)b Q29502 Jakobiet al. (1996)

aThe nomenclaturefor the individualisoformsis not alignedwith the rest of the table. b Accessionnumber only.

et al., 1997; Zhao et al., 1998; Galisteo et al., 1996; D h a r m a w a r d h a n e et al., 1997; Tsakiridis et al., 1996; Daniels et al., 1998). Following stim-

ulation with PDGF, ~-PAK becomes translocated into the membrane, specifically to the lamellipodia and filopodia, and this localization requires phosphoinositide 3-kinase (PI 3-kinase) activity (Dharmawardhane et al., 1997). ~-PAKinteracts with the adapter protein Nck, which links ~-PAK with receptor tyrosine kinases (Galisteo et al., 1996). Targeting of ~-PAK to the membrane induces neurite outgrowth in PC12 cells (Daniels et al., 1998; Nikolic et al., 1998). ~-PAK has also been shown to interact with a number of proteins including phosphatase PP2A (Westphal et al., 1999). At this time, little is known about the activation and function of ~-PAK. In contrast to ~-PAK, ~-PAK is activated in response to a variety of stresses, including ionizing radiation, UV radiation, and DNA-damaging drugs (Roig and Traugh, 1999); hyperosmolarity (Roig et al., 2000a); serum starvation and contact inhibition (Rooney et al., manuscript in preparation, Roig and Traugh, unpublished data) and in response to sphingosine (Tuazon et al., 1999). -PAK is also activated during FAS induction of apoptosis by cleavage with caspase 3 (Rudel and Bokoch, 1997; Lee et al., 1997; Rudel et al., 1998) and during heat shock (Chan et al., 1998).

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JOAN ROIG AND JOLINDA A. TRAUGH I I . COMPARISON OF P A K PROTEINS

~/-PAK has a 72% identity with ~-PAK and 71% with ~-PAK (see Fig. 1 for alignment of human PAKs). The catalytic domain of T-PAK is highly conserved (>99%) between human and rabbit, and the proteins are 91% identical (Fig. 1). The catalytic domain of ~/-PAK is 68% identical to the catalytic domain of the Ste20 protein kinase from S a c c h a r o m y c e s cerevesiae (accession code NP011856; Johnston et al., 1994). In contrast, the regulatory domains of a-, ~-, and ~/-PAK contain highly conserved regions as well as regions with important differences in sequence. One of the highly conserved regions in the regulatory domain is the p21-binding domain (which corresponds to residues 73 to 108 in ~/-PAK); the sequence is identical between all isoforms, except for residues 76 and 98 (Fig. 1). The GTP- bound forms of Cdc42 and Rac-1 associate with this domain in ~- and ~-PAK, resulting in stimulation of autophosphorylation, with a concomitant activation of the protein kinase (Manser et al., 1994, 1995). In contrast to ~- and ~-PAK, ~/-PAK is activated primarily by Cdc42 (Jakobi et al., 1996; Teo et al., 1995), while others report Racl also activates ~/-PAK (Martin et al., 1995a). Burbelo et al. (1995) have identified a short sequence within the G protein binding domain which is conserved in all proteins which bind Cdc42 or Rac. This core Cdc42/Rac interaction and binding sequence, ISXPXXXXHXHVGXD (residues 74-87 in ~/-PAK), is denoted CRIB. Overlapping the small G protein binding domain is an autoinhibitory domain (identified as residues 83-149; Frost et al., 1998) or 73-132 (Zhao et al., 1998) in ~-PAK. Taken together, a minimal autoinhibitory domain would correspond to residues 82-132 of 7-PAK. This domain inhibits autophosphorylation and activation through interaction with the catalytic site (Tu and Wigler, 1999). This suggests a mode of regulation of PAK activity, in which binding of Cdc42 or Racl would disrupt the inhibitory interaction between the regulatory and the catalytic domain, resulting in autophosphorylation and activation. Removal of the majority of the regulatory domain of ~/-PAK by cleavage with caspase 3 (Walter et al., 1998) or by limited proteolysis with trypsin (Tahara and Traugh, 1981) also results in autophosphorylation and activation of -PAK. Another common feature of PAKs is the acidic stretch of residues following Ser174 in ~-PAK, Ser171 in ~-PAK, or Thr169 in ~/-PAK, which constitute a recognition/phosphorylation site for the protein kinase casein kinase II (CKII) (Tuazon and Traugh, 1991; Pinna, 1990). The number of acidic residues is variable; ~/-PAK has 8 residues, while c~- and ~-PAK have 9 and 13, respectively. There is also a putative

~-PAK

171

Pm4

h~-PAK h~-PAK hT-PAK r~- P A K

Pro2

: 62 : 57 : 61 : 61

: : : :

.........

p2~l -binding

domain

........

CRI~

h(X-PAK

:

:

h~-PAK

:

h~-PAK rT-PAK

:

: :

:

:

hp-P~

:

hT-PAK ry-pAK

:

Pro3

AcidlG

: 184 : 185

: 177 : 177

:

Pro4 h~-PAK hT-PAK rT- P A K

•

•

PYoS : 244 : 225 : 225

?

hu-PAK h~-PAK hT-PAK r~-PAK

: 310 : 308 : 289 : 289

hC~-pAK

:

h~-P.'~

:

hT-pAK rT-PAK

: :

374 372 : 353 : 353 : :

I h~-PAK h~.= P~K

126 121 125 125

| z

:

502

:

: : :

500 481 481

GL~-Bindlng---

ha-PAK h~-PKK h~-PAK rT-PAK

: : : :

: : : .-

545 544 524 524

FIG. 1. Sequences for a-, ~-, and 7-PAK from human and 7-PAK from rabbit are compared. Black indicates identity between all forms; dark gray indicates identity between two forms; light gray indicates identity between human and rabbit 7-PAK. The autophosphorylation sites for 7-PAK from rabbit are indicated by arrowheads. The caspase cleavage site is identified by an arrow. A minimal sequence for the autoinhibitory domain is identified.

172

JOAN ROIG AND JOLINDA A. TRAUGH TABLE II SH3 BINDINGDOMAINSIN "~-PAK PXXP Regions PAK

1

2

a

12pPAPP 11pPAPP llpPAPP

4°pLPPNPEE 34pMAPEE 41pSVPEE

3

4

168pAVP 2°9pVTP 165pLAP 213pAAP __ __

5 22°PISP __ __

binding site for the 6-subunit of the heterotrimeric G-protein in Ste20 and in the C-terminus of all three isoforms (residues 505-518 of ~-PAK) (Leeuw et al., 1998; Wang et al., 1999). There are consistencies and differences in the PXXP motifs in prolinerich domains of ~-, 6-, and ~/-PAK, which are potential binding sites for proteins containing SH3 domains (Table II). The first proline-rich region contains two overlapping PXXP sites (PPAPP), which are conserved in all three isoforms. The adaptor protein Nck has been shown to bind ~-PAK at this site and to target it to the membrane (Bokoch et al., 1996; Galisteo et al., 1996). ~-PAK also binds Nck (Roig and Traugh, unpublished data), presumably at the same site, since this is the only fully conserved PXXP region in both proteins. All three forms also have a second proline-rich region, PXXPEE, in which the amino acids represented by X are different for each isoform (Table II). In ~-PAK this site is composed of two overlapping PXXP motifs (PXPPXPEE). ~-PAK has three additional PXXP motifs; the third region is shared by 6-PAK, with different internal amino acids. The fourth region is offset slightly in ~- and 6-PAK, while the fifth is present only in ~-PAK (Table II). These differences in PXXP motifs between the PAK isoforms suggest that the protein kinases can be targeted to different proteins and sites within the cell and thus could respond differently to the same stimuli. In addition to the described features, ~/-PAK has a caspase cleavage site after Asp 212; this cleavage site is not present in other isoforms of PAK (Fig. 1) (see Section V,E for details). The fact that mammals possess at least three forms of PAK may allow the different members of the PAK family to transduce a variety of signals and to participate in different physiological processes. The existing differences in sequence, along with the differential expression and localization of a-, 6-, and ~PAK, will probably account for most of the differences in activation and function that are emerging between the different members of the PAK family.

~-PAK

173

III. AUTOPHOSPHORYLATIONAND ACTIVATIONOF ~-PAK

Cdc42 and Racl are members of the Rho family of small GTPases, which are activated by different cellular stimuli (Lim et al., 1996; Van Aelst et al., 1997; Mackay et al., 1998). To examine the mechanism of activation of ~-PAK by Cdc42, the native form of inactive ~-PAK was purified from rabbit reticulocytes (Tahara and Traugh, 1981) and the recombinant enzyme from rabbit was expressed and purified from baculovirus-infected insect cells (Jakobi et al., 2000; Walter et al., 1998). -PAK exists in different phosphorylated forms which migrate between 58 and 60 kDa on SDS-PAGE. The rate of migration in SDS gels is dependent on the state of autophosphorylation and is due to phosphorylation of the regulatory domain, as shown by caspase cleavage of autophosphorylated ~-PAK (Walter et al., 1998). Multiple isoelectric forms of ~-PAK are detected by isoelectric focusing followed by SDS-PAGE and indicate multiple phosphorylated forms. Following autophosphorylation in response to binding of Cdc42(GTP), the two-dimensional profile of ~-PAK is characterized by an acidic shift (Gatti et al., 1999). -PAK is autophosphorylated in the presence and absence of Cdc42. Autophosphorylation in the absence of Cdc42 is significant, but results in negligible protein kinase activity (Tuazon et al., 1998). Autophosphorylation in the presence of Cdc42(GTP~] S) stimulates autophosphorylation (up to 7 mol of phosphate are incorporated per mol ~-PAK), and the protein kinase activity is stimulated up to 20-fold. Sequencing of the tryptic phosphopeptides obtained from ~-PAK phosphorylated in the presence of Cdc42(GTP) resulted in the identification of eight sites (see Fig. 1). Seven of the sites are seryl residues located in the regulatory domain; a single threonine (Thr-402) is phosphorylated in the catalytic domain (Gatti et al., 1999). In the absence of Cdc42(GTP), ~-PAK is autophosphorylated at five sites (serines 19, 20, 55, 192, and 197), all of which are also phosphorylated in the active enzyme. Three additional autophosphorylation sites correlate with activation of ~-PAK, Ser141, Ser165, and Thr402 (Gatti et al., 1999). Thr402 is present in the activation loop of the catalytic domain and is a highly conserved phosphorylation site among protein kinases. Six of the eight autophosphorylation sites identified in ~-PAK are common with the sites reported for ~-PAK (Manser et al., 1997), while Ser19 and Ser165 appear to be uniquely phosphorylated in ~]-PAK. Ser165 is located in the region corresponding to PXXP region 3 in ~- and ~-PAK (Fig. 1). This residue is present in rabbit and rat ~ -PAK, but is replaced by a proline in h u m a n ~-PAK. An additional autophosphorylated serine in ~-PAK is present five residues after the ~-PAK equivalent of Serl41; serine is also present at the same position in ~-PAK.

174


Cdc42(GTP~ S) stimulates autophosphorylation and activation of recombinant wild-type ~-PAK expressed in insect cells. The m u t a n t K278R, where the invariant lysine in the active site is replaced by arginine, shows neither autophosphorylation nor activity (Tuazon et al., 1997; Walters et al., 1998; Jakobi et al., 2000). Replacement of Thr402, the conserved threonine residue located in the activation loop, with alanine (T402A) greatly reduces autophosphorylation and phosphorylation of peptide and protein substrates, indicating that Thr402 is involved in autophosphorylation and activation by Cdc42. Some protein substrates, such as histone 2B, histone 4, and myelin basic protein, stimulate autophosphorylation, activating ~-PAK independently of Cdc42. These basic substrates overcome the requirement for activation of ~-PAK by autophosphorylation at Thr402, as shown with T402A (Jakobi et al., 2000). The degree of activation of ~-PAK is dependent on the concentration of ~/-PAK and substrate. King et al. (2000) have shown that 3-phosphoinositide-dependent kinase-1 (PDK1) can phosphorylate the activation loop of ~-PAK in vitro, suggesting that this can lead to activation of PAK. The in vivo significance of these observations remains to be determined, as the proposed site in PAK1 (Thr-423) is the autophosphorylation site equivalent to Thr-402 in ~-PAK. Recently, the X-ray crystal structure of a complex between the C-terminal kinase domain and a fragment of the N-terminal regulatory domain containing the auto-regulatory fragment of ~-PAK was solved (Lei et al., 2000). Although the structure represents the inactive protein kinase, the authors modeled the activation of~-PAK by comparison with another Cdc42 binding protein, WASP, whose solution structure in complex with Cdc42(GTP) was recently solved. In the inactive state, the auto-regulatory region interacts with the catalytic site, stabilizing the protein kinase as an inactive dimer. Binding of Cdc42(GTP) is proposed to induce conformational changes, disrupting the protein : protein interaction resulting in a rearrangement of the catalytic site, thereby leading to activation of the protein kinase. IV. ~-PAK HAS CYTOSTATICACTIVITY A. INJECTIONOF ~]-PAK INTOEARLYFROGEMBRYOS To examine whether ~-PAK was involved in induction of a cytostatic state, active ~-PAK was microinjected into one blastomere of two-cell frog embryos (Rooney et al., 1996). Injection of femtomole amounts of purified active ~-PAK (p58), or the active catalytic domain of ~-PAK (p37), arrested cleavage in the injected blastomere at mitotic metaphase,

~-PAK

175

whereas the noninjected blastomere progressed through mid-cleavage. The arrest was observed within 30 min following microinjection. Microinjection of heat-inactivated catalytic domain (p37), or purified inactive ~-PAK (p60), did not arrest cleavage. Injection of other protein kinases did not alter cell cycle progression in early embryos. Oocytes, zygotes, and developing embryos from the frog Lepidobatrachus laevis contain the small G-proteins Cdc42 and Rac2, but not Racl, as shown by Western blotting. A protein related to ~]-PAK (p58/p60) is specifically recognized by antibody against the regulatory domain of active ~-PAK from rabbit (Rooney et al., 1996). The ~-PAK homolog is present and active in oocytes and early zygotes; around 60 min postfertilization the protein is reduced, and is almost undetectable at 80 min postfertilization and in the two-cell stage embryo. In the fourcell embryo, PAK levels are beginning to increase; however, PAK is primarily inactive. In the mature oocyte, 66% of the PAK is in an active form, as determined by activity assays following chromatography on DEAE-cellulose; the late zygote contains only minor amounts of active and inactive enzyme, while in the four-cell embryos, only 41% of the PAK is active (Rooney et al., 1996). Thus, the levels of protein and activity of the -PAK homolog are high in oocytes and early zygotes, but are greatly reduced prior to cleavage. Rooney et al. (1996) propose that the ~/-PAK homolog is regulated by activation with factors, like small G-proteins, through autophosphorylation and inactivated by dephosphorylation and by degradation. These results suggest that ~-PAK and its homologs have cytostatic properties and are involved in maintenance of cells in a nondividing state. B. Xenopus X-PAK ANDTHE CELLCYCLE Additional insight into the mechanism of cell cycle arrest by ~-PAK comes from studies in Xenopus by Faure et al. (1997, 1999). In a search for Xenopus homologs of the p21-activated kinases, the authors identified proteins which have high homology with the mammalian PAK proteins. One of them, X-PAK1, is described as a protein of 65 kDa containing a kinase domain and a regulatory domain capable of binding to Cdc42 and Racl. X-PAK is 69% identical with ~-PAK and 65% identical with ~-PAK and ~-PAK. The catalytic domain of X-PAK1 is highly conserved (92% homology) when compared to the catalytic domain of mammalian PAKs. X-PAK1 is proteolytically cleaved into two fragments, a catalytic and regulatory domain, and activated in Xenopus extracts undergoing

176


apoptosis (Faure et al., 1997). The 7-PAK cleavage site in h u m a n (HVD212GAA) and in rabbit (HVD212SGA) is conserved in X-PAK1 (HVD213SGA), suggesting X-PAK1 may be a Xenopus homolog of mammalian 7-PAK. Recently, another 7-PAK homolog, X-PAK2, has been identified in Xenopus, with an overall identity of 88% with 7-PAK (Cau et al., 2000). It also has a caspase cleavage site and is approximately the same molecular weight as 7-PAK, although it is not known whether X-PAK2 is cleaved during apoptosis. Faure et al. (1997) show that microinjection of a catalytically inactive m u t a n t of the X-PAK1 catalytic domain (X-PAK1-CterK/R) facilitates progesterone- and insulin-induced release of oocytes from the G2/ prophase arrest. Release from arrest at meiosis I involves two different but related mechanisms; in response to progesterone, MAPK is activated leading to c-Mos accumulation, that in turn activates the MAPK cascade in a positive feedback loop (Faure et al., 1999). In parallel, MPF (p34Cdc2/cyclin B) is activated through dephosphorylation of p34 cdc2 by Cdc25. Microinjection of X-PAK1-Cter K/R prevents c-Mos accumulation and activation of MAPK and MPF in progesterone-treated oocytes, which remain in G2. X-PAK appears to prevent the initial step in activation of the c-Mos/MAPK feedback loop and interferes with the activation of MPF at different levels. X-PAK activity may prevent MPF activation by interfering with an early step necessary to t u r n on the positive feedback loop. This is similar to the effect described for protein kinase A (Faure et al., 1999) and could be understood if X-PAK is a regulator of the MAPK cascade (since MAPK activation can lead to MPF activation). The authors suggest X-PAK may act as a regulator of Raf-1. This is a real possibility since 7-PAK has been identified as the protein kinase that phosphorylates mammalian Raf-1 at Ser338 (King et al., 1998). Unfortunately, the authors erroneously call the identified protein kinase PAK3, instead ofPAK 2 (7-PAK), as a result of mistakes in the GenBank nomenclature which have now been corrected. For a discussion of the possible role of PAK enzymes in the control of MAPK cascades see Section VI. Faure et al. (1999) also show that the cytostatic properties of X-PAK1 are not limited to oocytes, as active X-PAK arrests cell cycle progression in cycling Xenopus egg extracts. X-PAK blocks G2fM progression by inhibiting the inactivation of p34¢dc2/cyclin A complexes and maintaining MPF in an inactive state, as it does in meiosis. Cau et al. (2000) have shown X-PAK2 is activated by Cdc42 and inactivated by MPF through what seems to be a positive feedback loop. It is of interest to know whether other X-PAKs (Faure et al., 1997) are homologs of specific mammalian PAKs and/or have similar properties to X-PAK1.

~-PAK

177

C. EXPRESSIONOF RECOMBINANT~-PAK IN MAMMALIANCELLS The intracellular localization and physiological function of ~-PAK have been examined by expression of wild-type ~-PAK and the kinaseinactive catalytic site m u t a n t K278R in HEK 293 and COS-7 cells. Expressed wild-type ~-PAK is present in both the soluble and particulate fractions, while K278R exists only in the soluble fraction and is expressed at an eight-fold higher level than the wild-type enzyme (Huang and Traugh, unpublished data). The cells containing wild-type ~-PAK do not divide over a 4-day posttransfection period, as determined by immunofluorescent detection of HA-tagged recombinant ~]-PAK. Cells containing K278R continue dividing. When examined by immunofluorescent microscopy, wild-type ~-PAK is localized in a region surrounding the nucleus, while K278R is distributed throughout the cell. Analysis of ~-PAK activity and protein following sucrose density gradient centrifugation shows that endogenous and wild-type ~-PAK are distributed in the high-density fractions associated with the endoplasmic reticulum (ER), the intermediate-density membrane fractions, and the low-density fractions (soluble), while K278R is present only in the soluble fractions. ER-associated ~-PAK has a higher specific activity than the soluble form of ~-PAK, suggesting that localization is related to -PAK activation (Huang and Traugh, 1999).

V. ACTIVATIONOF ~]-PAK IN RESPONSETO STRESS

The data presented in Section IV indicate that ~-PAK has cytostatic activity and that microinjection of ~-PAK into early frog embryos leads to G2/M arrest. Inactivation of ~-PAK should then allow the cell to go through mitosis or meiosis, while failure to inactivate ~-PAK would maintain cells in an arrested state. Recently, different types of stress have been identified which result in activation of ~-PAK (Fig. 2). These are discussed in detail below. A. DNA DAMAGEANDMITOTICARREST

DNA damage induced by agents such as ionizing radiation (IR), ultraviolet light (UV), or DNA-damaging drugs results in a cellular response that includes activation of different signaling pathways, induction of gene transcription, arrest of cell growth, and DNA repair (Woloschak, 1997; Wang, 1998). Different types of radiation activate different signaling pathways and induce the transcription of different gene products,

178

JOANROIGANDJOLINDAA.TRAUGH

IonizingRadiation Serum-starvation DNAdamaging " ~ / Agents f Fas-ligand /

y-PAK ~.~

HeatShock Sphingosine Hyperosmolarity FIG.2. Summaryof the activatOrsof ~-PAK. suggesting that intracellular signaling is complex. The cytostatic response to DNA damage allows DNA repair prior to cell division; excessive DNA damage results in cell death through apoptosis (Woloschak, 1997; Wang 1998). Roig and Traugh (1999) have shown that different types of DNA damage can rapidly activate ~/-PAK. IR, a DNA-damaging agent that induces double-strand breaks, activates ~/-PAK at moderate levels (1-5 Gy) within 15 min in 3T3-L1 fibroblasts. Higher levels of IR are required to activate ~-PAK in U937 leukemia cells, which leads to apoptosis. UV radiation and the DNA-damaging drugs 1-~-D-arabinofuranosyltyrosine (AraC) and cisplatinum activate ~/-PAK in 3T3-L1 cells at doses that slow progression through the cell cycle (Roig and Traugh, 1999). UV induces pyrimidine dimers, while AraC is a pyrimidine nucleoside which incorporates into DNA and inhibits replication by site-specific termination of DNA strands (Kufe et al., 1984). Cisplatinum is an alkylating agent, also used for cancer therapy, which forms DNA interstrand links (Sherman et al., 1987). Using the inhibitors genistein and wortmannin, ~/-PAK activation in response to IR or AraC has been shown to be dependent on tyrosine kinase and/or PI 3-kinase activity (Roig and Traugh, 1999). In contrast, ~/-PAK activation in response to UV light and cisplatin does not appear to depend on tyrosine kinase and PI 3-kinase activity. The data suggest that there are at least two signaling pathways leading to ~/-PAK activation in response to different types of DNA damage. ~-PAK is not activated by IR or AraC in 3T3-L1 cells and is only slightly affected by UV radiation, suggesting differential regulation of c~- and ~/-PAK in response to different stimuli (Roig and Traugh, 1999).

-PAK

179

These data are consistent with the present understanding of the cytostatic response to DNA damage in mammalian cells leading to DNA repair. One pathway, activated by IR and AraC, depends on the PI 3-kinase-related enzymes DNA-dependent protein kinase and ATM and the tyrosine kinase c-Abl (which is not activated by UV) (Wang, 1998). Other signaling pathways activated by UV do not require PI 3-kinase related enzymes. 3T3-L1 cells irradiated with moderate doses of IR (1 and 5 Gy), resulting in an early transient activation of ~/-PAK, also show a transient arrest of the cell cycle (Roig and Traugh, unpublished data). 3T3-L1 cells irradiated with doses that lead to irreversible arrest of the cell cycle at G2/M (20 and 100 Gy) induce sustained activation of~/-PAK for more than 24 h, suggesting a relationship between ~/-PAK activation and cell cycle arrest. This is supported by data showing activation of~/-PAK during mitotic arrest of 3T3-L1 cells by the microtubule-associating drug nocodazole (Roig and Traugh, unpublished data). B. HYPEROSMOLARITY

Other types of cell stress that do not lead to DNA damage can also induce activation of~-PAK. Hyperosmotic stress results in cell shrinkage and a greater interaction between proteins, while swelling of cells has a dilution effect. Cells utilize ion transport systems and osmolytes (such as sorbitol and amino acids) to adjust cell osmolarity to maintain the cell volume against outside influences (Waldegger et al., 1997; Haussinger et al., 1994). In studies with hepatocytes, insulin and several growth factors have been shown to increase cell volume. Swelling stimulates protein synthesis and amino acid uptake, inhibits protein degradation, and stimulates the expression of new gene products. Cell shrinkage has the exact opposite effect, wherein the cell cycle is arrested, protein synthesis is inhibited, and protein degradation is stimulated. Utilizing 3T3-L1 cells as a model system, hyperosmotic stress has been shown to activate ~/-PAK (Roig et al., 2000a); the activation mechanism is different from the activation of~-PAK in response to DNA damage. In 3T3-L1 fibroblasts, there are two pools of ~/-PAK: The majority of the protein kinase is soluble (95% of the total ~/-PAK protein) and has low specific activity, while ~/-PAK associated with the particulate fraction has a significantly higher specific activity (up to 32-fold). Sorbitolinduced hyperosmolarity promotes translocation of a small fraction of ~/-PAK (~5%) from the soluble to the particulate fraction; this translocation parallels activation of the protein kinase. Thus, only a localized fraction of the protein kinase is activated, in contrast to the more

180


general activation observed in response to DNA damage (Roig and Traugh, 1999). The mechanism of translocation of ~-PAK to the particulate fraction in response to hyperosmolarity involves the small G-protein Cdc42. Cdc42 is activated by binding of GTP and is translocated to the particulate fraction of the cell in parallel with ~-PAK (Roig et al., 2000a). Translocation of ~-PAK is induced in vitro by GTP~ S, an activator of Cdc42, and transient transfection of constitutively active Cdc42 with ~-PAK induces ~-PAK activation and translocation in viva Inactive Cdc42 acts as a dominant negative mutant, inhibiting both translocation and activation of ~-PAK in response to hyperosmolarity. Activation, but not translocation of ~-PAK, is dependent on PI 3-kinase activity as shown with the PI 3-kinase inhibitor wortmannin. Roig et al. (2000a) have proposed a model for ~-PAK activation in response to osmotic stress, wherein ~]-PAK associated with the plasma membrane is activated through a mechanism that involves PI 3-kinase and Cdc42. Active Cdc42 would associate with ~-PAK, transporting -PAK to the membrane, where it would be activated by a process that involves PI 3-kinase activity. Osmotic imbalance and subtle changes in cell volume influence the expression of numerous genes, including those encoding transporters, enzymes, and signaling molecules, as well as alter the rate of proteolysis, protein synthesis, and cell growth (Kultz et al., 1998; Waldegger et al., 1997). Activation of ~-PAK in response to this cellular stress suggests that the protein kinase has a role in some of the cellular responses induced by hyperosmolarity, primarily at the membrane level. C. SPHINGOSINE

Sphingosine is produced in cell membranes as a result of sphingolipid catabolism in response to different signals, such as serum, insulin, PDGF, and phorbol esters (Spiegel and Merrill, 1996; Kolesnick and Kronke, 1998). Sphingosine formation has been associated with both growth induction and growth arrest. Sphingosine can stimulate proliferation of different cell types, but also has cytostatic and cytotoxic properties (Olivera and Spiegel, 1993; Coroneos et al., 1995; Merrill et al., 1996). High levels of sphingosine can induce apoptosis in different cell types (Ohta et al., 1995; Coroneos et al., 1995). Sphingosine stimulates ~]-PAK and ~-PAK activity both in vivo and in vitro (Tuazon et al., 1999; Bokoch et al., 1998). Sphingosine acts directly on ~-PAK in vitro, inducing autophosphorylation and consequent

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activation of the protein kinase activity. The response is observed at concentrations as low as 10 ~M and is maximal at 200-400 ~M (Roig et al., unpublished data; Tuazon et al., 1999). As shown by two-dimensional phosphopeptide mapping, ~/-PAK is autophosphorylated on multiple sites in response to sphingosine, and the autophosphorylated sites are similar, if not identical, to those observed in response to Cdc42(GTP) (Gatti et al., 1999). Sphingosine also activates ~/-PAK in 3T3-L1 cells. These cells respond to concentrations of sphingosine (15 ~M) which result in cell cycle arrest, with a small percentage of the cells entering apoptosis. Similar to the data obtained with osmotic stress, sphingosine induces translocation and activation of a small fraction of ~/-PAK. The active enzyme is associated with the membrane-containing particulate fraction, while the activity of the soluble enzyme is not enhanced by sphingosine (Roig et al., unpublished data; Tuazon et al., 1999). These results suggest that sphingosine has a role in activation of ~-PAK in response to cell stress. D. SERUMSTARVATIONAND CONTACTINHIBITION To examine regulation of ~/-PAK by serum starvation, the postribosomal supernatant from dividing and growth-arrested 3T3-L1 cells was chromatographed on DEAE-cellulose and analyzed for ~/-PAK activity and protein (Rooney et al., manuscript in preparation). Active ~/-PAK was assayed directly while total ~/-PAK was determined following activation by cleavage. ~/-PAK protein migrates at 58-60 kDa on SDSPAGE as detected by Western blotting. In serum-fed 3T3-L1 cells ~40% of the ~/-PAK is active. Serum starvation for 1½ h increases ~/-PAK activity by 2.5-fold and the majority of ~/-PAK (~70%) is active. A similar enhanced level of ~/-PAK activity is observed in contact-inhibited cells. Insulin treatment of serum-starved cells for 15-30 min results in a fourfold decrease in PAK activity with a concomitant reduction in ~/-PAK protein. As shown with inhibitors, this is due to degradation of ~-PAK through the proteosome pathway. The data indicate that the presence and absence of serum and insulin regulate ~/-PAK activity and that ~/PAK is regulated by cycling between active and inactive states and via degradation. Serum starvation of 3T3-L1 cells for 1.5 h also reduces the rate of protein synthesis. Treatment of serum-deprived cells with 10 nM insulin stimulates the rate of protein synthesis 2-fold (Chang and Traugh, 1997). Serum starvation also inhibits protein synthesis, both at the initiation and elongation steps (Morley and Traugh, 1993; Chang and

182

JOAN ROIGAND JOLINDAA. TRAUGH TABLE III ACTIVATIONOF ~]-PAK IN RESPONSETOSTRESS Activation of ~-PAK

Treatment

Reversible

Soluble

Particulate

Requirement for TYR K or PI 3-K

Activation of ~-PAK/JNK

IR/AraC UV/cisplatin Hyperosmotic stress Sphingosine

Yes No Yes

Yes Yes No

Yes Yes Yes

Yes/yes No/no ND/yes

No/no Yes/yes No/yes

Yes

No

Yes

NDa

ND

a

ND, not determined.

Traugh, 1998). These are the same conditions under which ~-PAK is activated 2.5-fold compared to exponentially growing cells. Addition of serum or insulin to the serum-deprived cells rapidly down-regulates -PAK activity and at the same time protein synthesis recovers to >90% of that in exponentially cells (Chang and Traugh, 1997). -PAK is also activated in 3T3-L1 cells that have exited the cell cycle in response to prolonged serum starvation (Roig and Traugh, unpublished data). These cells are arrested in Go. Thus, although ~-PAK appears to have a central role in arrest of the G2/M transition of the cell cycle, an additional role for ~-PAK in growth arrest may be through maintaining cells in Go. A summary of the activation and localization of~-PAK in response to the various stresses is shown in Table III. Under conditions where the treatment is reversible, ~-PAK can be activated in the particulate fraction (hyperosmolarity) or the soluble and particulate fraction (IR/Ara C), but is dependent upon upstream activator(s) such as PI 3-kinase or PI 3-related kinases and/or tyrosine kinases. Under these cytostatic conditions, no activation of ~-PAK is detected. With UV and cisplatinum, the DNA damage is more difficult to repair, and the signaling pathway for activation of ~-PAK kinase is decidedly different, with no requirement for PI 3-kinase or tyrosine kinase activity. Activation of ~-PAK is also observed under these conditions. E. ACTIVATIONOF ~]-PAK BY CLEAVAGEWITH CASPASE 3 DURING PROGRAMMED CELL DEATH

The caspases play integral roles in apoptosis or programmed cell death, which include a number of morphological changes such as

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183

nuclear condensation, membrane and cytoskeletal rearrangement, and the formation of apoptotic bodies (as reviewed in Nagata, 1997). In response to death stimuli, the caspase family of cysteine aspartatedirected proteases becomes activated. These morphological changes are paralleled by biochemical changes. Death factors (such as TNF and Fas), DNA damage, and strong stress can initiate the apoptotic responses. Caspase 3 is activated upon cleavage to p17 and p l 0 (FernandesAlnemri et al., 1994) and has been shown to participate in apoptosis through cleavage of a number of proteins including DNA-activated protein kinase and protein kinase C8, among others (Ghayur et al., 1996; Porter et al., 1997). In addition to the role of ~-PAK in cytostasis and cell cycle arrest, ~/-PAK has been implicated in apoptosis or programmed cell death. -PAK is cleaved and activated in Jurkat T cells undergoing apoptosis in response to crosslinking of the Fas receptor (Rudel and Bokoch, 1997; Lee et al., 1997). Other stimuli which induce ~/-PAK cleavage include C2 ceramide, TNF-a, and UV treatment (Lee et al., 1997; Rudel and Bokoch, 1997). Cleavage of~/-PAK by caspase 3 in vivo and in vitro produces two fragments (Rudel and Bokoch, 1997; Lee et al., 1997; Walter et al., 1998). The polypeptide consisting of residues 1-212 contains the major portion of the regulatory domain and migrates at 27 kDa (p27), whereas the polypeptide from 213 to 524 contains the entire catalytic domain plus 34 amino acids of the regulatory domain and migrates at 34 kDa (p34). Following cleavage, both peptides become autophosphorylated; the regulatory domain (p27) migrates with an apparent molecular weight of 32 kDa on SDS-PAGE following autophosphorylation on serine, while the catalytic domain (p34), autophosphorylated on threonine, does not change in migration (Walter et al., 1998). Mutation of Asp-212 to asparagine inhibits cleavage of~/-PAK (Lee et al., 1997; Rudel and Bokoch, 1997). A similar site is not present in either a- or [3-PAK. Thus, caspase cleavage and activation is a characteristic of ~/-PAK and takes place in a region of the protein kinase that is highly divergent between the different PAK enzymes (Fig. 1). Cleavage releases autoinhibition of the catalytic domain by the regulatory domain. Activation of ~/-PAK via caspase cleavage is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step and activation coincides with autophosphorylation of Thr402 in the catalytic domain (Walter et al., 1998). Expression of the endogenously active p34 cleavage product of~/-PAK induces some of the hallmarks of apoptosis (shrinkage, rounding up, and detachment) (Lee et al., 1997). Dominant-negative mutants of p34 or

184


p60 suppress the effects of p34. Rudel and Bokoch (1997) used dominant negative mutants ofa-PAK to study the role of~/-PAK in apoptosis. The authors assume that a dominant-negative mutant of a-PAK, although not cleaved and activated during apoptosis, will interfere with the normal function of ~/-PAK during the process. Expression of dominantnegative mutant a-PAK under the control of an inducible promoter inhibits the morphological changes that accompany Fas-induced apoptosis, while accelerating phosphatidylserine externalization in the membrane. Nuclear modifications such as DNA fragmentation are not affected (Rudel and Bokoch, 1997). However, one must take into account that ~- and ~/-PAK are different in function and it is not clear at this time whether inactive ~-PAK is truly affecting ~/-PAK functions. Active ~-PAK also activates the stress-activated protein kinase JNK, although this activation is weak (~twofold) (Rudel et al., 1998). Other studies (Chan et al., 1998) show that heat shock induces a stress response in mammalian cells that can also result in apoptosis. ~/-PAK is cleaved to form a constitutively active catalytic fragment; inhibition of activation of caspase 3 with inhibitors diminished the cleavage and activation of ~/PAK. Chang et al. (1999) also show that -/-PAK is cleaved and activated during apoptosis as a result of severe hyperosmotic shock induced by high concentrations of NaC1. Caspase inhibitors or antioxidants block activation of caspase 3, cleavage and activation of ~-PAK, and DNA fragmentation. The data suggest a role for oxidative stress in the death process. In summary, in the cases where the mechanism of activation of~/-PAK has been studied (i.e., Cdc42, sphingosine, and caspase activation of ~/-PAK), the protein kinase is activated only after autophosphorylation. Thus, ~/-PAK activation, although accomplished by different pathways and mechanisms, ultimately appears to involve autophosphorylation. ~/-PAK is activated in a number of physiological contexts related to cytostasis and apoptosis, but only in the last instance is it activated by caspase cleavage. To ensure that apoptosis is nonreversible, ~-PAK (and other enzymes) is activated or inactivated irreversibly. Thus, the holoenzyme is transiently activated to induce cytostasis, while it is cleaved and constitutively activated during cell death. Recently, ~-PAK has been identified as the Nef-associated protein kinase (NAK), which when complexed with Nef is active (Renkema et al., 1999). The Nef protein of the primate immunodeficiencyvirus is involved in the pathogenesis of acquired immunodeficiency syndrome (AIDS). This complex has been implicated in disease progression in cultured cells and enhanced viral infection in monkeys (Savai et al., 1997).

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185

VI. THE INDMDUAL ROLES OF ~ - P A K AND SAPK/p38 IN THE STRESS RESPONSE

The stress-activated protein kinases SAPK/JNK and p38 form two subgroups of MAP kinases that are activated in response to cellular stress (hyperosmolarity, UV exposure, DNA damage, heat, and oxidant stress), inflammatory cytokines (TNF-~ and IL-I~), and vasoactive peptides (thrombin) through protein kinase cascades architecturally similar to the Ras/Raf/MEK/ERK cascade (Kyriakis and Avruch, 1996; Schaeffer and Weber, 1999). At least two distinct SAPK cascades have been well defined. Activation of the SAPK and p38 pathways leads to phosphorylation of different transcription factors, as well as a number of other proteins, which eventually result in growth arrest, apoptosis, or activation of immune and reticuloendothelial cells. Like ~]-PAK, p38 has been shown to have cytostatic properties when injected into frog oocytes (Takenaka et al., 1998). The PAK activators Cdc42 and Racl have been shown to activate the SAPK and p38 pathways when transfected into mammalian cells (Bagrodia et al., 1995b; Coso et al., 1995; Minden et al., 1995; Zhang et al., 1995). A major question is whether any of the PAK enzymes act upstream of the SAPK and p38 pathways, similar to the yeast homolog Ste20, that transduces signals to a MAPK module (Herskowitz, 1995; Leberer et al., 1997). Overexpression of~-PAK (Brown et al., 1996; Frost et al., 1996; Zhang et al., 1995), ~-PAK (Bagrodia et al., 1995b), or ~-PAK (Frost et al., 1996) has been reported to lead to activation of SAPK or p38 in mammalian cells. Polverino et al. (1995) have shown that SAPK can be activated by addition of c~-PAKto extracts from X e n o p u s oocytes. Dominant negative forms of ~-PAK can interfere with SAPK/JNK or p38 activation induced by extracellular signals, e.g., interleukin I, or by cotransfection with Cdc42 or Racl (Zhang et al., 1995). Expression of ~-PAK induces an indirect activation of Mekkl, an upstream activator of SAPK (Siow et al., 1997). Although these data suggest that one or more PAKs can be upstream activators of SAPK and/or p38, it has been argued that the observed effects are small, cell type dependent, and may be due to overexpression of PAK protein (Minden and Karin, 1997; Avruch, 1998). Other studies fail to link ~-PAK and SAPK activation. Teramoto et al. (1996) report that coexpression of ~-PAK with constitutively active Cdc42 or Racl diminishes SAPK activity. Zhou et al. (1998) question the ability of PAK to activate SAPK and show that active ~-PAK does not activate the stress-activated protein kinase under the conditions used in those experiments.

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JOAN ROIGAND JOLINDAA. TRAUGH

A possible explanation for this contradictory data may be that only one form of PAK may be acting upstream of the SAPK cascade. ~/-PAK would be a prime candidate, as it is activated in response to different types of stress, in contrast to a-PAK, which is activated by growth factors and hormones. However, Roig and Traugh (1999) and Roig et al. (2000a) have shown that ~/-PAK can be activated at early time points (15-120 min), under stress conditions that do not induce SAPK/p38 activation. With UV treatment, ~-PAK and SAPK/JNK (but not a-PAK or p38) are concomitantly activated at early times in 3T3-L1 cells. Another SAPK/JNK activator, the protein synthesis inhibitor anisomycin does not activate ~/-PAK (Roig and Traugh, 1999). The DNA-damaging drug cisplatinum strongly activates ~-PAK, but induces only a slight stimulation of SAPK/JNK. Of the DNA-damaging treatments tested, only UV radiation results in coactivation of ~/-PAK and SAPK/JNK (Roig and Traugh, 1999). These results argue against ~-PAK involvement in SAPK/JNK activation, at least in 3T3-L1 cells, and suggest that the mechanism through which ~-PAK induces cytostasis is distinct from the activator of SAPK/JNK or p38.

V I I . SUBSTRATESFOR " ] - P A K

A. IDENTIFICATIONOFARECOGNITION/PHOSPHORYLATION SEQUENCE

FOR"y-PAK The determinants for recognition and phosphorylation of substrates by ~/-PAK were identified by examining the kinetics of phosphorylation of a series of synthetic heptapeptides (Tuazon et al., 1997). Using peptides patterned after the sequence KKRKSGL, the site phosphorylated by ~-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro (Leis et al., 1984; Fu et al., 1988ab), Tuazon et al. (1997) have shown that the sequence contains basic amino acids in the - 2 and - 3 positions. These are represented by K/RRXS, in which the - 2 position is an arginine, the - 3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the - 1 or - 4 position improves the rate of phosphorylation by increasing the Vm~xand decreasing the Kin. An acidic amino acid in the - 1 position increases the rate (2.5-fold), as does an acidic residue in the - 4 position, although to a lower extent (1.6-fold); an acidic residue at position - 1 diminishes the rate somewhat, but is still a suitable substrate. Proline in the - 1 or ÷ 1 position has a deleterious effect and inhibits phosphorylation by ~/-PAK. Protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue, such as protein kinase

~-PAK

187

A and protein kinase C, are negatively affected by an acidic residue in the - 1 position. Thus, a peptide containing the sequence KRES can be used as a specific substrate for ~-PAK (Tuazon et al., 1997). B. PROTEINSUBSTRATESFOR ~-PAK

A number of proteins have been identified as substrates for ~-PAK in vitro. These include the Rous sarcoma nuclear capsid protein NC (Leis et al., 1984), histone 2B and histone 4 (Tahara and Traugh, 1981), myelin basic protein (Teo et al., 1995; Tuazon et al., 1998), myosin light chain from smooth muscle (gizzard) (Tuazon and Traugh, 1984), prolactin (Oetting et al., 1986), Raf 1 (King et al., 1998) and the Abelson tyrosine kinase (c-Abl) (Roig et al., 2000b). Translation initiation factors eIF-3, eIF-4B, and eIF-4G (Tuazon and Traugh, 1989) are also phosphorylated by ~-PAK. Functional effects of phosphorylation on specific substrates of~-PAK have been described, as summarized below. Most of the phosphorylation sites which have been identified are consistent with the KRXS sequence, determined using heptapeptide substrates (Tuazon et al., 1997). In addition to Rous and avian sarcoma virus protein NC (Leis et al., 1984), a KRXS site has been identified in histones 2B and 4, myelin basic protein (Traugh et al., unpublished data) and in c-Abl (Roig et al., 2000b). In contrast, the site phosphorylated in myosin light chain from smooth muscle does not contain either KRXS or RRXS adjacent to the phosphorylatable threonine. Recent studies from our laboratory indicate that basic residues N-terminal to the phosphorylatable serine or threonine can be an alternative recognition site (Traugh et al., unpublished data). C. EFFECTS OF PHOSPHORYLATIONON SUBSTRATEACTIVITY

1. R o u s a n d A v i a n S a r c o m a Virus Nuclear Capsid Protein

Rous sarcoma virus nuclear capsid protein NC, a protein consisting of 89 amino acids, is phosphorylated by ~-PAK on Ser40, which is directly preceeded by a highly basic region KKRK. This basic region has been implicated in the binding of NC to single-strand regions of the viral RNA (Leis et al., 1984). Greater than 98% of the phosphate incorporated into NC in vivo is associated with this site, as shown by two-dimensional phosphopeptide mapping, microsequence analysis, and manual Edman degradation. Phosphorylation of Ser40 enhances the affinity of NC for viral RNA by >60-fold, changing the conformation and exposing the KKRKS region (Fu et al., 1985). This allows a tight association between the highly basic region and the viral RNA. Interestingly, all site-directed

188


mutations of Ser40 give a phenotype similar to that of phosphoserine and bind RNA (Fu et al., 1988a,b). The ability to regulate binding to the viral RNA by the host protein kinase ~-PAK suggests that this phosphorylation event has evolved to regulate a stage of development of the virus. In fact, site-directed mutations of the basic residues show that the KKRKS region is required for packaging of the virus (Fu et al., 1988b). When mutants of Ser40 are expressed in vivo, RSV is unable to replicate and the amount of phosphate associated with NC is twice that of the wild-type enzyme (Fu et al., 1988a). This is due to phosphorylation of Ser76 and Ser77 by ~-PAK. These sites are minimally phosphorylated in the wild-type protein in vivo (1-2% of the total phosphate incorporated). 2. Myosin Light Chain, ~I'PAK, and the Cytoskeleton A number of reports implicate ~-PAK in the control of cytoskeletal dynamics (Manser et al., 1997; Sells et al., 1997; Zhao et al., 1998), and it is clear now that the protein kinase has a central role in the control of the actin cytoskeleton. In contrast, there is little data relating ~/-PAK to the cytoskeleton, although this cannot be excluded, since the protein kinase is able to phosphorylate myosin light chain in smooth muscle (Tuazon and Traugh, 1984). The 20-kDa regulatory light chain of myosin from smooth muscle is phosphorylated by myosin light chain kinase (MLCK) in a Ca2+/ calmodulin-dependent manner. Myosin light chain is also phosphorylated by ~-PAK, independently of C a 2+ and calmodulin. Similar results are obtained using actomysin. Phosphorylation of the myosin light chain by ~-PAK stimulates the actin-activated Mg-ATPase activity of actomyosin in the absence of calcium, to an extent similar to that of MLCK in the presence of calcium. Both ~-PAK and MLCK phosphorylate myosin light chain at Ser 19 (Tuazon and Traugh, 1984). Thus, under conditions of low calcium, g-PAK could phosphorylate Ser 19 to maintain contractile activity in smooth muscle. Recently, Ser 19 has been confirmed as a phosphorylation site for ~]-PAK, and studies with endothelial cells suggest possible actin rearrangement upon expression of ~-PAK (Chew et al., 1998). 3. Prolactin Prolactin has also been shown to be phosphorylated by ~-PAK (Oetting et al., 1986). When the growth-promoting activities ofdephosphorylated and standard rat prolactin (partially phosphorylated) are measured, dephosphorylated prolactin has a significantly higher growth promoting activity than phosphorylated prolactin (Wang and

-PAK

189

Walker, 1993). This observation is supported by studies with recombinant wild-type h u m a n prolactin and mutants of the in vivo phosphorylation site Ser179 (which correlates with Ser177 in rat). Using S179A, Chen et al. (1998) show non-phosphorylated wild-type prolactin produced in E. coli is more active in growth promotion than the standard prolactin, whereas S179D inhibits growth. In recent studies, Lorenson et al. (2000) show that ~-PAK is present in secretory granules. In addition, ~-PAK phosphorylates prolactin at the critical serine residue, Ser179 in bovine. Thus, nonphosphorylated prolactin is a growth factor, whereas prolactin phosphorylated by ~-PAK inhibits growth by competiting with nonphosphorylated prolactin for the prolactin receptor. 4. Tyrosine Kinase c-Abl

Roig et al. (2000b) identified a functional interaction between the nonreceptor tyrosine kinase c-Abl and ~-PAK, opening the possibility of cross-talk between two pathways that to date were not known to be related. As shown by immunoprecipitation, c-Abl and ~-PAK interact in vivo, and both protein kinases can phosphorylate each other, c-Abl is phosphorylated by ~-PAK at a site implicated in regulation of protein : protein interactions and subcellular localization, suggesting that the activity of the tyrosine kinase may be affected by this modification. Indeed it is shown that cotransfection of Cdc42 and ~-PAK induces activation of a portion of the cellular pool of c-Abl tyrosine kinase. It remains to be determined whether this activation occurs only at certain subcellular localization sites where PAK and c-Abl could coexist with GTP-bound forms of Rac or Cdc42/Rac. An apparent negative regulatory feedback mechanism is also described, c-Abl phosphorylates ~-PAK in vitro on tyrosine and induces phosphorylation of ~]-PAK on tyrosine in vivo. Phosphorylation of ~-PAK by c-Abl down-regulates ~-PAK activity. As shown with inhibitors specific for the proteasome, ~-PAK is ubiquitinated and degraded through the proteasome pathway. Inactivation of ~/-PAK by c-Abl results in stabilization and accumulation of ~-PAK (Roig et al., 2000b).

VIII. WORKINGMODEL--A ROLE FOR~-PAK ASA MASTERSWITCH -PAK is activated under a wide variety of stress conditions. ~/-PAK has also been shown to have cytostatic properties which halt cells in

190


G2/M. Under these conditions, damage to the cell machinery can be repaired and toxic effects reversed. Under more extreme (irreversible) conditions, the cells move into apoptosis, where ~/-PAK is cleaved to a constitutively active form which can assist in driving the cell down the apoptotic pathway. ~/-PAK is located in the cytosol and is associated with the endoplasmic reticulum, the plasma membrane, and secretory granules. Although it has not been shown to be localized to the nucleus at this time, this cannot be ruled out, as ~-PAK phosphorylates nuclear proteins. However, this could also occur during mitosis when the nuclear membrane is broken down or prior to translocation to the nucleus. -PAK phosphorylates numerous proteins. These proteins are associated with the nucleus, the cytosol, and secretory granules. As indicated above, ~-PAK can also phosphorylate viral proteins. The diversity of these protein substrates suggests that ~-PAK could have regulatory properties which embrace and coordinate multiple aspects of cell physiology. For instance, phosphorylation of histones (as well as other nuclear proteins) could be involved in inhibition of cell division at G2/M. Inhibition of protein synthesis could occur through phosphorylation of translational initiation factors and ribosomal proteins. Phosphorylation of transcription factors could stimulate the production of stress-related or stress-induced proteins by enhanced transcription of the mRNAs encoding these proteins. Association of active ~-PAK with the ER, membranes, and secretory particles suggests that phosphorylation of ER and membrane-associated proteins by ~/-PAK could regulate functions involved in transport and secretion. We propose a model wherein ~/-PAK can be acting, either alone or in concert with other protein kinases, phosphatases, and tumor suppressors, as a master switch turning on cytostasis and potentially mediating a transition from cytostasis to apoptosis. Cytostasis allows time for repair of damage (e.g., DNA and membrane) and for removal of toxic or noxious substances or compounds. The period of time would last as long as the stress by maintaining ~/-PAK in an active state. Under conditions of excess stress, damage is not readily repairable. Under these conditions ~-PAK could be involved in the transition to apoptosis through cleavage by caspase 3. In this model, ~/-PAK is regulated independently of ~-PAK and is involved in activities different from those of ~-PAK. Obviously, there are a number of experiments that are required to validate this model. The fact that ~/-PAK is functionally different from ~-PAK is just becoming apparent, and further studies will differentiate between their similarities and differences.

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191

IX. CONCLUSIONS AND REMAINING QUESTIONS

A number of critical questions remain regarding the activation and function of ~-PAK. A critical issue is identification of the receptors involved in mediating the responses to various stresses and the GTPases that are activated in response to each signal. With regard to activation of the individual PAKs in vivo, which of the G proteins, Cdc42 and/or one of the Rac proteins, are responsible? What is the cellular localization of the PAKs and of these G proteins? Differences in the localization of ~-PAK and ~]-PAKhave already been shown. Is this due to the G protein, to differences in localization domains on the PAKs, and/or to targeting sites within the cell? What are the upstream activators of ~-PAK in response to the different signals? PI 3-kinase and tyrosine kinase activity have been implicated in some cytostatic responses. Is PI 3-kinase itself involved, or are the PI 3-related kinases DNA-activated protein kinase and ATM involved, and which tyrosine kinase? For other cytostatic responses no upstream signals have been identified; these signals need to be determined. In response to different signals, arrest or promotion of cell growth occurs through the integration of multiple pathways that affect cell division, gene expression, cytoskeleton organization, and cellular metabolism. How ~-PAK fits into these signaling pathways is one of the central issues in the study of the cytostatic role of the protein kinase. Although some of the physiological signals that induce ~-PAK activation and the signals involved in activation of the enzyme are beginning to be determined, little is known about the mechanism of action. What is the role of the protein kinase in induction of cell cycle arrest? What are the signaling pathways that ultimately cause inhibition of the cell cycle? These are questions that have only begun to be answered. The fact that most of the reported experiments involve overexpression of a PAK isoform (generally ~-PAK) could lead to some of the contradictions in the literature. Until the time that a systematic comparison between the localization, substrate specificity, and physiological effects of all of the PAK enzymes is carried out, some caution must be used in the generalization of experimental designs that rely on the overexpression of a single type of PAK in order to determine PAK function. A new family of PAK-binding proteins (PIX/cool) has been described recently by Bagrodia et al. (1999); these proteins'bind to the proline-rich region in ~-PAK beginning with Pro186 (Manser et al., 1998). It will be interesting to show whether ~-PAK has similar partners and how they affect its activity and localization.

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We hypothesize, from the c u r r e n t n u m b e r o f s u b s t r a t e s a l r e a d y identified, t h a t ~/-PAK p h o s p h o r y l a t e s a large n u m b e r of s u b s t r a t e s . Identification of the s u b s t r a t e s i n v i v o is v e r y i m p o r t a n t . The effects of phosp h o r y l a t i o n on activity a n d localization is a n o p e n - e n d e d question. Are the s u b s t r a t e s a n d t a r g e t i n g the s a m e or different d u r i n g cytostasis a n d apoptosis? Finally, w h a t leads to different functions of ~- a n d ~/-PAK? Are t h e y r e g u l a t e d differently a t t r a n s c r i p t i o n a n d t r a n s l a t i o n ? Is t h e r e differential t a r g e t i n g or c o m p e t i t i o n for t a r g e t i n g to specific sites? Are t h e s e differences d e p e n d e n t on the cell type? M a n y questions r e m a i n . ACKNOWLEDGMENTS

Supported by N I H Grant GM26738. W e thank Dr. Polygena Tuazon for advice and assistance. We also thank Dr. Rolf Jakobi, Dr. Zhongdong Huang, and Barbara Waiter for criticallyreading the manuscript and Fabiola Gonzalez for preparing the figures and tables and processing the text.

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VITAMINSANDHORMONES,VOL.62

Androgen Receptors and Their Biology DOLORES J. LAMB,*,t NANCY L. WEIGEL,t AND MARCO MARCELLI t4 *Scott Department of Urology, tDepartment of Molecular and Cellular Biology, and $Department of Medicine, Baylor College of Medicine, Houston, Texas 77030 I. Mechanism of Androgen Action A. Steroid Hormone Action II. Sexual Development A. Androgen Receptor and Male Genitourinary Development B. Androgen Receptor Mutations and Androgen Insensitivity Syndrome III. Androgen Receptors and Disease A. Androgen Receptor Mutations and Prostate Cancer IV. Androgen Action and Other Diseases A. Skin Diseases B. Hair: Allopecia and Hirsutism C. Immune Function D. Androgen Polyglutamine Repeats and Disease V. Conclusion References

I. MECHANISM OF ANDROGEN ACTION A. STEROID HORMONE ACTION

Although androgens freely diffuse into all cells, their action is mediated by androgen receptors that are only present in androgen-responsive tissues, such as prostate, testis, and skin. The androgen receptor is a member of the steroid receptor superfamily of transcription factors. Jensen and Jacobson (1968) and Gorski and colleagues (1968) proposed nearly 25 years ago that steroid receptors mediate the effect of steroid on gene expression. Since this time, great advances in our understanding of the molecular basis of steroid action have occurred. In the presence of ligand, the androgen receptor undergoes a change in conformation resulting in activation to a form that interacts tightly as a dimer with specific DNA sequences, termed hormone responsive elements (HRE) or androgen responsive elements (ARE). Interaction of the receptor with these regions of DNA, in conjunction with other nuclear proteins ultimately results in the transcription Ofspecific 199

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hormone-responsive genes. The new messenger RNAs are subsequently translated into new proteins and ultimately cell function is altered. In general, this model was proposed about 30 years ago and although our understanding of the complexity of receptor regulation of specific gene expression has increased, there is still much to learn, as we have not yet identified the vast majority of genes whose expression is regulated by the androgen receptor. Prior to cloning of the androgen receptor gene in 1988 by Chang and co-workers (1988) and Lubhan et al. (1998a,b) and soon after by several others (Tilley et al., 1989; Trapman et al., 1988; Brinkmann et al., 1989), most of our understanding of androgen receptor structure and function came from the biochemical characterization of the protein. Like all steroid receptors, the androgen receptor binds androgen with high affinity and limited capacity. The development of radiolabeled androgens provided a powerful tool to study AR function. These steroids enabled investigators to investigate the cellular localization of the receptors under different hormonal milieus and to define the physicochemical properties of the protein. The protein has a molecular weight of 108 kDa and studies in the 1970s demonstrated that it is present in the unliganded state as a complex that migrates on a sucrose gradient at 8-10S. In the presence of high salt and/or receptor activation to the form that bound to DNA, the receptor was transformed into a smaller 4S protein. The androgen receptor (AR) gene is located in the pericentromeric region of the long arm of chromosome X at Xql 1-12 and contains eight exons (Fig. 1). Thus, men have only 1 copy of this gene. It encodes a protein with a length of 919 amino acids. There are three known functional domains: The amino (NH2) terminal domain is encoded by exon 1, the largest exon, and is critical to target gene transcriptional regulation. This region contains polymorphic glutamine (CAG) and glycine (GGC) repetitive sequences (the length of these repeats varies among individuals in the general population) as well as a third region of eight proline residues. CAG repeat length varies by racial group with Caucasians exhibiting an average of 21 glutamines and African-American men with 18 repeats. As is discussed, these polymorphisms may influence androgen receptor function resulting in disease or in an increased likelihood of disease development. The functional characteristics of exons 2 through 8 are somewhat better understood because mutations in these regions of the gene may profoundly influence receptor function leading to significant phenotypic alterations. Exons 2 and 3 encode the centrally located DNA-binding domain (comprising about 70 amino acids with two zinc containing

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Androgen Receptor Structure lm F u n c t i o n a l Domains A R Protein

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FIG. 1. The androgen receptor gene was clonedin 1998by Chang and co-workers(1988) and Lubhan et al. (1988a,b) and soon after by several others (Tilleyet al., 1989;Trapman et al., 1988; Brinkmann et al., 1989). There are eight exons encoding the receptor with a large exon 1 required for transactivation and exons 2-8 encoding the hormone and DNA-binding domains of the receptor. fingers) and the 5' region of exon 4 encodes the hinge region with the nuclear targeting signal. The 3' region of exon 4 and exons 5 through 8 encode the steroid-binding domain that confers ligand specificity. 1. L i g a n d B i n d i n g

The androgen receptor normally binds testosterone (T) or dihydrotestosterone (DHT) with high affinity (0.1 nM) and limited capacity. Until antibodies to the receptor were generated, the characteristic of steroid binding using a radiolabeled androgen provided the primary means to analyze the receptor. Other steroids m a y interact when present at extremely high concentrations, but under normal conditions, these are the two major androgens influencing development and cell function through interaction with the androgen receptor. Adrenal androgens and nonandrogenic steroids such as progesterone can also bind, but with much less affinity (Wilson and French, 1976). Synthetic steroids such as R1881 have been developed that bind with high affinity and do not undergo aromatization and these have provided useful tools for the study of androgen receptor structure. Techniques such as photoaffinity labeling using R1881 permitted analysis of the AR under denaturing conditions (Brinkmann et al., 1985, 1988). Male sexual development requires both T and DHT for full virilization. DHT binds to the androgen receptor with greater affinity than T (Wilbert et al., 1983). There is differential gene regulation depending upon whether T or DHT is the ligand bound to the AR. Thus, individuals

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with defects in the 5~-reductase gene (the enzyme responsible for the conversion ofT to DHT) exhibit deficits in virilization and male pseudohermaphadism, but the phenotype will be markedly different than the androgen insensitivity patients described below (Imperato-McGinley, 1974). In vitro ligand binding studies using fibroblasts have provided important information for the analysis of androgen insensitivity patients, although this approach provides little insight into the molecular basis of the defect. 2. Coactivators a n d Corepressors

Studies in the past 5 years have demonstrated that the transcriptional activity of the steroid receptors is stimulated by proteins termed coactivators (McKenna et al., 1999). Originally identified by their ligand-dependent interactions with the hormone-binding domains of steroid receptors, these proteins have no intrinsic DNA-binding activity. Rather, they bind to the receptors themselves, recruiting additional proteins such as histone acetyltransferases, and interact with the basal transcriptional machinery to enhance transcription of target genes (McKenna et al., 1999). Most of the steroid receptor coactivators identified to date stimulate the activity of all steroid receptors as well as selected other transcription factors. The list of proteins that enhance the activity of the androgen receptor as well as of other steroid receptors has been growing rapidly. The relative importance of each of these proteins and their precise functions are unknown. Among these coactivators are the p160 family that is composed of steroid receptor coactivator 1 (SRC-1), GRIP1 or TIF2 and RAC3 (also called pCIP, ACTR, AIB1, and TRAM1; reviewed in McKenna et al., 1999). Although these proteins were originally identified as proteins that interact with the AF2 region of the ligand-binding domain of the steroid receptors, it is clear that at least GRIP1 and SRC-1 interact with, and stimulate the activity of, AF1 of the androgen receptor (Ma et al.). These proteins are themselves histone acetyltransferases (HAT) and recruit another HAT, P/CAF, as well as the coregulators CBP and p300 (McKenna et al., 1999). Other proteins reported to interact directly with and enhance activity of steroid receptors include proteins as diverse as E6-AP, a ubiquitin ligase (McKenna et al., 1999), and Tip60, which was first identified as a coactivator of the human immunodeficiency virus TAT protein (Brady et al., 1999). Finally, one coactivator, SRA, functions at an RNA rather than at the protein level (Lanz et al., 1999). In addition to the proteins that coactivate virtually all steroid receptors, several coactivators have been identified by interaction with the

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androgen receptor. These include those identified by Changes group including ARA70 (Yeh and Chang, 1996). ARA55 (Fujimoto et al., 1999) ARA54 (Kang et al., 1999), and ARA 24 (Hsiao and Chang, 1999) as well as two proteins designated SNURF (Moilanen et al., 1998) and a kinase, ANPK (Moilanen et al., 1998). These proteins do not share homologies with the p160 family of coactivators and in some cases combinations of coactivators yields higher activities than either alone. Although ARA70 has been reported as a preferential activator of the androgen receptor (Yeh and Chang, 1996), others have found that it also activates other steroid receptors (Alen et al., 1999). One novel property of ARA70 is that its expression causes estradiol to act as an agonist for the androgen receptor (Yeh et al., 1998). Collectively, these proteins can profoundly influence the activity of the steroid receptors. 3. Transactivation a. The A n d r o g e n Receptor B i n d s to D N A to Activate Transcription as a Dimer. Upon introduction of ligand, the androgen receptor exhibits NH2-terminal and ligand~binding domain protein interactions (Langley et al., 1995). Based upon analysis of mutated androgen receptors, Wilson and colleagues have proposed that the receptor dimerization upon transactivation results in an antiparallel arrangement of the androgen receptor monomers (Langley et al., 1998). The dimerization reaction is probably stabilized by interaction with the hormone responsive element of the gene, or HRE. The receptor binds to HREs usually in the promoter regions of androgen responsive genes. There are at least four classes of consensus sequences of response elements for the steroid receptor superfamily and the HREs for androgen receptors are in Class 1 that includes the glucocorticoid receptor. Roche and co-workers (1992) have reported that this sequence consists of two 6-bp symmetrical elements separated by a 3-bp spacer (5'GGA/TACANNNTGTTCT). In addition, the sequences surrounding these half-sites influence the binding affinity of the receptor and the overall activity of the response element. b. The A n d r o g e n Receptor Contains at Least Two Regions Involved in Transcriptional Activation. One of these TAFs is located N terminally (Taf-1) and this is constitutively active. The C-terminall.y located TAF (TAF-2) is active only upon binding the ligand. The activity of TAF-1 is cell type and promoter specific and it is thought that this property may play a role in the partial agonistic effects of some antagonists. The N-terminal domain of the androgen receptor plays an important, although not fully understood, role in transactivation. There are two regions in the NH2-terminal domain that interact with the ligand-binding domain, especially in the region of residues 370-494 (Boerrevoets et al.,

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1998). Activation function 2 (AF2) domain is localized in the ligand binding domain of the androgen receptor forming a hydrophobic region that binds the LXXLL motif of p160 transcriptional coactivators (He et al., 1999). According to Wilson and colleagues, there is evidence that this region serves to stabilize helix 12 through NH(2)-/carboxylterminal interaction, resulting in a slower rate of androgen dissociation (He et al., 1999). The level of transcriptional response induced by TIF2 depends upon the promoter context (Boerrevoets et al., 1998). c. Androgens May Also Act to Inhibit Gene Transactivation. A physiologic example of this is the repression of the ~-subunit of the pituitary gonadotropins (LH and FSH). The structural properties of the AR required for repression appear to be distinct from those required for transactivation.(30;31) d. Crosstalk between Signal Transduction Pathways. Steroid receptors, such as the androgen receptor, are phosporylated, although the exact physiologic role is not clearly understood. Phosphorylation of the AR occurs in both an androgen-dependent and -independent manner. Steroids are not the only pathways to receptor activation. Nazareth and Weigel presented evidence that modulators of the protein kinase A can activate androgen receptor transcriptional function in the absence of ligand (Nazareth and Weigel, 1996). Similarly, growth factors such as epidermal growth factor, keratinocyte growth factor, interleukin-6, LHRH, and insulinlike growth factor 1 have been shown to activate the androgen receptor (Hobisch et al., 1998; Culig et al., 1994, 1995, 1997). These alternative pathways for androgen activation could have profound effects, especially for conditions such as prostate cancer, as this is one potential route to androgen-independent growth. II. SEXUALDEVELOPMENT A. ANDROGENRECEPTOR AND i ~ E

GENITOURINARYDEVELOPMENT

The androgen receptor is required for normal male genitourinary development. The androgen receptor binds both testosterone and its metabolite, 5a-dihydrotestosterone. Testosterone (T) is the principal androgen secreted by the testis. In circulation, it is mainly bound to two proteins: Sex hormone-binding globulin and albumin. Protein-bound testosterone is in dynamic equilibrium with free hormone, the latter comprising 1 to 3% of the total. Free testosterone enters the target cell through passive diffusion. Inside the cell, testosterone can be reduced to dihydrotestosterone (DHT) by the enzyme 5~-reductase or aromatized to estradiol by aromatase.

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The Biology of the Androgen Receptor

FIG. 2. The androgen receptor is required for the normal development, growth, and function of the male. Tissues containing the androgen receptor are targets of androgen action. Testosterone and dihydrotestosterone affect different tissues during development as well as after puberty. In addition to regulation of the reproductive tract function, the androgen receptor is involved in immune function, skin, hair, and muscle function.

DHT and T exert their effects in mediating the development of the normal male phenotype via a single receptor protein, the androgen receptor (AR), which is encoded on the X chromosome. Although dihydrotestosterone and testosterone bind to the same receptor protein, they perform distinct physiological roles. The receptor-T complex is responsible for virilization of the Wolffian ducts during male phenotypic sex differentiation and probably for the regulation of spermatogenesis. The receptor-DHT complex promotes development of the male external genitalia and prostate during embryogenesis and most of the events associated with sexual maturation at the time of male puberty. The role of the androgen receptor in the development and function of normal individuals is shown in Fig. 2. B. ANDROGEN RECEPTOR MUTATIONSAND ANDROGEN INSENSITIVITYSYNDROME

A functional androgen receptor is required for the development of the male genital tract as demonstrated by studies of the tfm mouse (tfm, testicular feminization) and androgen insensitivity patients. Thus, much of our knowledge of androgen receptor function comes from studies of patients and animal models with various mutations in the androgen receptor gene. In the absence of a functioning androgen receptor, the testes will develop; however, the female internal genitalia do not develop due to the secretion of Mullerian inhibiting hormone (MIS or antimullerian hormone, AMH) by the testis. Male internal genitalia

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initially develop from the Wolffian ducts, but since there is no androgen effect on these tissues, the epididymis, vas deferens, and prostate in the adult are absent or rudimentary (reviewed in Wiener et al., 1997). Androgen insensitivity patients with a mutation of the androgen receptor leading to total loss of function have a shortened vagina with no uterus or fallopian tubes. The etiology of androgen resistance can be attributed to defects in the androgen receptor gene. Mutations have been identified that result in termination codons, frameshifts, alterations of mRNA splicing, deletions, and point mutations. Mutations leading to varying degrees of androgen insensitivity have been reported in each of the eight exons of the androgen receptor gene. Most defects identified, however, occur in the two regions that encode the DNA and hormone binding domains of the receptor.

1. Functional and Developmental Consequences of Androgen Insensitivity in Humans Testicular feminization or androgen insensitivity syndrome (AIS) was reported by Morris in 1953 who described 82 patients with a completely female phenotype in the presence of testes. This syndrome is linked to mutations in the androgen receptor gene. Depending upon the location and nature of the mutation, the phenotypic expression of the mutant androgen receptor in XY individuals ranges from normal-appearing males to phenotypic females with complete AIS. Quigley et al. (1995) proposed a grading scheme from 1 to 7 depending on the degree of virilization. Grades 6 and 7 constitute complete AIS with unambiguously female external genitalia, a blind-ending vagina, and bilateral testes. Female breast development and a female body contour occur in response to estrogen at puberty (from aromatization of circulating testosterone), but there is a lack of androgen-mediated features normally seen in XX individuals, such as pubic hair and acne. The testes continue to develop, but are located in the inguinal canal, consistent with the hypothesis that transinguinal, but not transabdominal testicular migration, is androgen- dependent (Hutson, 1986). Germ cells are present, but normally decline in number, probably due to cryptorchidism. Incomplete androgen insensitivity is less common. At one time, partial AIS was referred to as Reifenstein, Gilbert-Dreyfus, or Lubs syndromes. However, since the molecular basis of these syndromes is now understood, all are now referred to as partial AIS. These individuals will have male or female phenotypes, depending upon the functional consequences of the AR mutation present. Individuals with partial AIS

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(grades 3-5) may have ambiguous genitalia with severe hypospadias (a congenital anomaly of the penis and urethra where the urethra opens on the ventral portion of the penis or in the perineum), chordee (a curvature of the penis with concavity downward that may occur with severe hypospadias), and cryptorchidism (a developmental defect where the testes fail to descend into the scrotum and are localized in the abdomen or inguinal canal). The external genitalia of some children with AR mutations may be predominantly female, while some individuals may have rudimentary Wolffian duct structures, such as the vas deferens due to partial androgen response. Others, such as those with Reifenstein's syndrome, are genotypic males. Near the normal end of the spectrum are individuals with the infertile male syndrome (Aiman et al., 1979), and the undervirilized fertile male syndrome (Grino et al., 1988). Patients with the infertile male syndrome have azoospermia or severe oligospermia (Aimen et al., 1997) whereas those with undervirilized male syndrome have normal sperm counts, but may have gynecomastia, a small phallus, and decreased beard and body hair (Grino et al., 1988). Three oligospermic patients were identified who exhibited a mutation changing methionine (886) valine (Ghadessy et al., 1999). The functional transactivation studies did not demonstrate any alteration of ligand binding specificity but the mutant AR consistently exhibited diminished activity by 50% (Ghadessy et al., 1999). It was suggested that this mutation altered AR protein:protein and TIF2 coactivator interactions. The role of androgen receptor mutations in common male congenital anomalies, such as isolated hypospadias and cryptorchidism, is less clear. Severe hypospadias and cryptorchidism are found in conditions such as Reifenstein's syndrome that are androgen-receptor related. However, attempts to demonstrate a gross anomaly of the androgen receptor in patients with isolated genital anomalies such as hypospadias and cryptorchidism have been largely unsuccessful, although it was causative in a few isolated cases (Sutherland et al., 1996; Wiener et al., 1998; Nordenskjold et al., 1999; Albers et al., 1997; Rodien et al., 1996; Allera et al., 1995; Klocker et al., 1992, 1995; Batch et al., 1992, 1993a,b).

III. ANDROGENRECEPTORSAND DISEASE

Androgen receptors are involved in both health (Fig. 2) and disease in additions to congenital defects (Fig. 3). In this section, the role of this important receptor in disease is summarized.

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Androgen Receptors and Disease

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FIG. 3. The androgen receptor has been implicated in congenital syndromes affecting genitourinary and male sexual development; in the development of ache, allopecia, and hirsitism; in prostate cancer; in male infertility; and in Kennedy's disease, a neurodegenerative disease.

A. ANDROGEN RECEPTOR MUTATIONS AND PROSTATE CANCER

Investigators have proposed a number of different androgen receptor related mechanisms that may provide a growth advantage to the tumor. Research has focused on the possibility that receptor mutations may occur in advanced prostate cancer altering function and cell proliferation. The basis of this hypothesis came from the observation that tumors forming in steroid responsive tissues such as breast or prostate frequently contain steroid receptors. The growth of these tumors, like the natural growth of the organ, is regulated by steroid hormones. Hormone ablation therapy is the main treatment for metastatic prostate cancer. Unfortunately, although the results are initially quite positive (tumor regression), within a short time the tumor progresses to a rapidly proliferating, hormone-independent state, resulting in significant morbidity and mortality for the patient. "Outlaw receptors" or receptor variants that would exhibit aberrant behavior were first identified for breast cancers (McGuire et al., 1991).

1. Frequency of Receptor Mutations in Advanced Prostate Cancer A number of researchers, including our own laboratory are currently testing the hypothesis that androgen receptor mutations occur in advanced prostate cancer and provide a growth advantage to the tumor, perhaps especially under conditions of androgen ablation therapy (Table I). Some reports suggest a high level of androgen receptor

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mutations (approaching 50% of tumors analyzed) in advanced prostate cancer (Tilley et al., 1990, 1996; Taplin et al., 1995, 1999; Fenton et al., 1997; Gaddipati et al., 1994), while others report a lower frequency (with some labs reporting no mutations present (Newmarket al., 1992; Culig et al., 1997a,b; Wang and Uchida, 1997; Shimazaki et al., 1997; Tan et al., 1997; Suzuki et al., 1993, 1996; Peterziel et al., 1995; Ruizeveld de Winter et al., 1994; Ris-Stalpers et al., 1993; Veldscholte et al., 1990). At least some of this controversy is due to patient selection bias. In general, all laboratories agree that mutations are rare in early stage disease with just five mutations identified in 231 patients analyzed (Newmark et al., 1992; Suzuki et al., 1993, 1996; Ruizeveld de Winter et al., 1994; Schoenberg et al., 1994; Marcelli et al., 1999; Castagnaro et al., 1993; Elo et al., 1995; Evans et al., 1996; Paz et al., 1997; Watanabe et al., 1997). Our study of nearly 100 stage B specimens (84 micro dissected) representing 20% of the total published specimens analyzed supports this observation. Just one laboratory has evaluated the incidence of mutations in latent prostate cancer (Table I) (Takahashi et al., 1995). According to these authors, inactivating mutations of the AR may play a role in preventing the development of prostate cancer in Japanese men. These authors hypothesize that these inactivating mutations in latent cancer of Japanese men, but not in American men, would prevent the progression of the disease to a clinically relevant entity. Thus, this difference in the prevalence of AR mutations may account for the different prevalence of prostate cancer among Americans and Japanese men (Takahashi et al., 1995). The primary lesions from patients with stage C and D disease have an overall incidence of 12% (29 in 238 cases) (Tilley et al., 1996; Gaddipati et al., 1994; Newmarket al., 1992; Tan et al., 1997; Suzuki et al., 1993, 1996; Ruizeveld de Winter et al., 1994; Evans et al., 1996; Watanabe et al., 1997; de vere White et al., 1999). The incidence of AR mutations is higher in metastatic disease (7 of 26 cases with a frequency of 22%) (Culig et al., 1995, 1997a,b, 1998, 1999; Tilley et al., 1990, 1996; Taplin et al., 1995, 1999; Gaddipati et al., 1994; Peterziel et al., 1995; Klocker et al., 1994). 2. Factors Influencing the A n a l y s i s o f A R Mutations in Prostate Cancer The small amounts of DNA available precluded the analysis ofexon 1 for many of the published studies as well as some of our own studies of micro-dissected metastatic tumor. This may influence the overall rate of mutations reported. The studies of Tilley et al., (1996) exemplify this observation with nearly 50% of the mutations reported in their series in exon 1.

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Patient selection criteria likely also influence the observed frequency of AR mutations (i.e., stages A-B, C, and D before and after androgen ablation therapy and antiandrogen therapy). Our results further show that AR mutations can exist prior to androgen-ablative treatments and some of these are "loss of function" mutations. Recently, Joyce et al. (1998), suggested that flutamide treatment can elicit an increase in AR mutations (5/16) in micro metastases based upon their studies of patients undergoing treatment with androgen ablation and flutamide. Methodological differences may influence the ability to detect an AR mutation in prostate cancer specimens. Our studies suggest that poorquality tissue fixation or processing can result in irreproducible mutations upon SSCP and DNA sequence analysis (Lamb et al., unpublished). Some studies did not use micro dissected tumor specimens and the contribution of normal tissue to the analysis is unknown. Our studies demonstrated that a mutation could be observed by SSCP only if present in at least 10-15% of the genomic DNA. We used microdissected tissue from patients unexpectedly found to have non-organconfined disease at the time of radical prostatectomy (metastatic disease present in frozen sections of the regional lymph nodes). We found 11 mutations in six patients and one patient with 4 different mutations (a frequency of 15.7%).

3. Types of A R Mutations in Prostate Cancer: Functional Consequences Our results show that AR mutations can exist prior to androgenablative treatments and some of these are "loss of function" mutations (Nazareth et al., 1999). Recently, Taplin et al., suggested that flutamide treatment can elicit an increase in AR mutations (5/16) in micro metastases based upon their studies of patients undergoing treatment with androgen ablation and flutamide (Joyce et al., 1998). The AR mutations in patients receiving androgen ablation monotherapy (1/17, 5.9%) were of the "gain of function" type. Further analysis of a larger number of patients is required to see if this trend is consistent, as our results do not support this hypothesis. Our studies of autopsy samples from just eight patients who died of their disease demonstrated a "gain of function" mutation in patients with androgen ablation monotherapy. In short, additional studies are required to demonstrate whether androgen ablation therapy induces a specific type of mutation (loss or gain of function) and whether these mutations can be used to predict disease progression and outcomes. Some of these "gain-of-function" mutations may lead to a superactive receptor that may be activated at low ligand concentrations (Tilley

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et al., 1999). Other mutations may influence the steroid specificity of the receptor (Fenton et al., 1997; Tan et al., 1997; Veldscholte et al., 1990, 1992; Culig et al., 1996). Our laboratory has recently identified "loss-of-function" inactivating mutations of the androgen receptor in metastatic prostate cancer (Marcelli et al., 1999). We have identified a cysteine to tyrosine mutation at amino acid 619 that results in the loss of DNA binding activity and the hormone-dependent formation of protein aggregates in the cell. This mutation occurred just outside of the zinc fingers of the DNA-binding domain. 4. Alternative Pathways for Androgen Receptor Modulation in Prostate Cancer

Additional alternative pathways of androgen receptor action influencing function have been proposed. Amplification of the androgen receptor gene has been reported to occur in about 30% of all advanced prostate cancers (Koivisto and Rantala, 1999; Koivisto et al., 1995, 1996, 1997, 1998; Koivisto and Helin, 1999; Bubendorf et al., 1999; Nupponen et al., 1998; Palmberg et al., 1997; Visakorpi et al., 1995). Ligand-independent activation of the androgen receptor resulting from cross talk with other signal transduction pathways may result in receptor activation in the absence of androgen (Nazareth and Weigel, 1996; Culig et al., 1994, 1995a,b; Klocker et al., 1994). Alterations in the size of the polyglutamine repeat may influence androgen action in prostate cancer and this is discussed in more detail below. As there are multiple pathways for growth stimulation of cells, it has been proposed that some signal transduction pathways may bypass the need for androgen action (Papandreou et al., 1998; Craft et al., 1999; Makridakis et al., 1997). Bioavailability of androgen may influence prostate cancer development or progression (Makridakis et al., 1997; Rois et al., 1998), as may agents that modulate androgen receptor action such as coactivators and corepressor as well as agents such as caveolin (Nasu et al., 1998).

IV. ANDROGENACTIONAND OTHERDISEASES A. SKIN DISEASES

Androgen receptors are present in the skin and genital skin fibroblasts have provided an important source of androgen responsive cells for investigators to study. These cells were frequently obtained from patients to study androgen insensitivity prior to the availability of

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molecular probes to measure androgen receptor defects. Differential display PCR has been used with genital skin fibroblasts to identify new androgen regulated genes (Nitsche et al., 1996). Although the androgen receptor concentration in the epithelial cells themselves is rather low, the concentration in the sebocytes is similar to prostatic cells. The receptor is not present in the undifferentiated prepubertal sebocytes, but is induced at puberty apparently due to circulating androgens (Miyake et al., 1994). Not surprisingly, acne is an androgen-mediated disease. Androgen receptor is even expressed in periodontal and gingival tissue (Parkar et al., 1996). B. HAm: ALLOPECIAAND HIRSUTISM Hirsutism, or increased facial hair in women, is frequently due to elevated circulating androgen levels. However, in idiopathic hirsutism, there appears to be an association with alterations in the length of the polyglutamine repeat altering androgen receptor function (described below) (Vottero et al., 1999). In addition, it is possible that alterations in normal coactivator or corepressor functions could also influence relative receptor reactivity leading to altered receptor function in skin and hair follicles as well as other androgen-responsive tissues. The actions of androgens on hair are location dependent, as androgen exerts an inhibitory action in male pattern baldness, suppressing the growth of the hair (Keller et al., 1996). C. IMMUNE FUNCTION

Androgens may play a role in the regulation of immune function. Olsen and Viselli and colleagues have reported interesting studies in a rat model. They demonstrated that castration results in impaired immune function due to induction of immature thymocyte proliferation and increased apoptosis of B lymphocytes as well as an alteration in B cell development (Viselli et al., 1995a,b, 1997; Olsen et al., 1994, 1998). Perhaps these androgen related alterations contribute in part to the gender differences in incidence of autoimmune related illnesses between males and females. D. ANDROGENPOLYGLUTAMINEREPEATS AND DISEASE Exon 1 contains a micro-satellite region encoding a series of CAG repeats encoding a stretch of polyglutamines. This region has been the focus of a number of studies demonstrating that length, as expansion

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of the polyglutamine region can be associated with a number of different pathologies. Within the normally observed spectrum of repeats (

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