Prolactin

Prolactin Hallgeir Rui* Department of Pathology, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, MD 20814, USA * corresponding author tel: 301-295-3801, fax: 301-295-1640, e-mail: [email protected] DOI: 10.1006/rcwy.2000.03013.

SUMMARY Prolactin is a tetrahelical cytokine most closely related to growth hormone and placental lactogens. It binds to specific prolactin receptors that belong to the WS-motif cytokine receptor family. Prolactin is secreted in a highly regulated manner into the circulation by the anterior pituitary, and acts on peripheral target tissues as a hormone. In addition, prolactin is expressed at many extrapituitary sites, particularly within the female and male reproductive organs and the cells of the immune system, acting locally as an autocrine or paracrine cytokine. Because of the ubiquitous expression of prolactin receptors, prolactin has a wide range of cellular and physiological effects. In mammals, prolactin is particularly critical for the differentiation of the mammary gland and for lactation. Hyperprolactinemia, the most common pituitary disorder, causes infertility and decreased libido in both men and women. Prolactin may also influence the progression of certain autoimmune diseases, and has been implicated as a promoter of neoplastic growth.

BACKGROUND

Discovery A distinct anterior pituitary hormone with `lactogenic' activity was originally discovered by Stricker and Grueter (1928), the factor being purified and given the name prolactin shortly thereafter (Riddle and Braucher, 1931; Riddle et al., 1932, 1933). Several sources provide reviews and historical references concerning this landmark discovery and the ensuing research on the actions of prolactin in mammals, birds, reptiles, amphibians, and fish (Riddle, 1963; Bern and Nicoll, 1968; Li, 1978; Nicoll, 1980). Because human

growth hormone, in contrast to growth hormone from nonprimate mammals, binds to prolactin receptors and has a potent lactogenic activity, it was not until the early 1970s that human prolactin was definitively identified as a separate entity (Lewis et al., 1971; Hwang et al., 1972). An essentially complete peptide sequence of human prolactin was first derived by the Edman degradation of tryptic fragments (Shome and Parlow, 1977), and the entire amino acid sequence, including a 28 residue signal peptide, was deduced from the nucleotide sequence of human cDNA cloned by Cooke and colleagues (1981). Furthermore, the extrapituitary production of prolactin was first detected in decidualized endometrial cells (Riddick et al., 1978) and subsequently in many other reproductive tissues, immune cells and brain. Because of its widespread expression and the realization that prolactin belongs to a family of tetrahelical polypeptides that activate a corresponding family of cytokine receptors, prolactin too is now regarded as a ubiquitous cytokine with autocrine and paracrine roles.

Alternative names Prolactin has been referred to as luteotropic hormone, luteotropin, mammotropic hormone, and mammotropin. To better reflect the diverse and pleiotropic effects of prolactin, the alternative names versatilin and omnipotin also have been suggested (Bern and Nicoll, 1968).

Structure A comparison of the amino acid sequences of prolactin from human and mouse is presented in Figure 1. After the processing of a 28 residue signal peptide, the mature human prolactin molecule is secreted as a

268 Hallgeir Rui Figure 1 Comparison of the amino acid sequences of human and mouse prolactin.

Figure 2 Putative threedimensional structure of prolactin based on that of growth hormone.

D B

A C

Prolactin polypeptide with 199 amino acid residues, whereas mouse prolactin is two residues shorter (Cooke, 1989). In both species, six cysteines form three intramolecular disulfide bridges. The molecular weight of human prolactin is approximately 23 kDa, but a 26 kDa glycosylated form is also produced. A three-dimensional structure of prolactin has not been reported, but an extensive modeling and structural analysis of prolactin has been provided by Goffin and colleagues (1996). A similar model of the prolactin molecule based on the three-dimensional structure of growth hormone is shown in Figure 2. Prolactin is expected to conform structurally to the tetrahelical cytokine fold, with a molecular core consisting of four antiparallel  helices, A, B, C, and D, in an up-up-down-down arrangement (Goffin et al., 1996). Mutational analyses support this three-dimensional structure and have revealed putative interaction sites with prolactin receptors (for a review, see Goffin et al., 1996). Cysteine bonds formed by the pairwise coupling of residues Cys4-Cys11, Cys58Cys174, and Cys191-Cys199 are not shown in Figure 2. Prolactin circulates in blood as monomers of 23± 26 kDa (Lewis et al., 1985). In addition, larger `macroprolactins' (big-prolactin and big-big prolactin) represent both homo-oligomeric aggregates and immunoglobulin-complexed prolactin (reviewed by Sinha, 1995). Furthermore, the physiological proteolysis of prolactin to a C-terminally truncated 16 kDa variant results in a molecule with distinct biological activities that may activate unique receptors (Mittra, 1980; Clapp and Weiner, 1992; Clapp et al., 1993; D'Angelo et al., 1999).

Main activities and pathophysiological roles Prolactin is expressed in mammals, birds, reptiles, amphibians, and fish, and has a wide spectrum of effects (Nicoll, 1980). In fact, more than 300 distinct biological activities of prolactin have been recorded, in large part because of the ubiquitous expression of prolactin receptors (Bole-Feysot et al., 1998). In addition to endocrine effects mediated by pituitary prolactin secretion, it has become increasingly evident that prolactin is synthesized at many extrapituitary sites, particularly in reproductive organs, immune cells, and brain (for a review, see Ben-Jonathan et al., 1996). It is therefore clear that prolactin can act as a local paracrine and autocrine factor in diverse tissues and cells. At the cellular level, prolactin regulates the growth, survival, differentiation, and activation state of target cells. At the physiological level, the effects of prolactin

269

may be divided into seven partially overlapping areas: (1) reproduction, (2) immune function, (3) water/ electrolyte balance (osmoregulation), (4) stress adaption, (5) behavior/brain/psychology, (6) metabolism, and (7) skin function. Of these, the behavioral and psychologic effects of prolactin, and its effects on skin, may be coupled to reproduction and reproductive cycles. The biological effects of prolactin are reviewed in more detail below. In humans, prolactin is important for both the physiological and psychological aspects of reproductive function, and the hormone also affects certain aspects of immune cell function. Evidence for this involvement of prolactin in humans has to a large extent been disclosed by symptoms and signs associated with hyperprolactinemia, the most frequent endocrine disturbance of the pituitary. Galactorrhea, anovulation and consequent amenorrhea, decreased libido, and impotence are typical effects of chronic hyperprolactinemia (Thorner et al., 1998). On the other hand, as illustrated by hypophysectomy or isolated idiopathic prolactin deficiency, a lack of pituitary prolactin in adults is not associated with vital deficiencies beyond fertility problems (Turkington, 1972; Kauppila et al., 1987).

GENE AND GENE REGULATION

Accession numbers Mouse prolactin gene: K03236, X02892, X04418 Human prolactin gene: E02152, D00411, S74425, X00368, L33865, M58594, M31661, M29386

Chromosome location The human prolactin gene is present as a single copy on chromosome 6 (Owerbach et al., 1981). By the use of somatic cell hybridization, the assignment of the prolactin gene was narrowed to 6pter-p21.1 (Taggart et al., 1987). Evans and colleagues went on to locate prolactin in the interval 6p22.2-p21.3, distal to HLA-C (Evans et al., 1989). The mouse prolactin gene maps to chromosome 13, clustered with genes encoding mouse placental lactogens and other prolactin-like genes (Jackson-Grusby et al., 1988).

Relevant linkages The human prolactin gene is located in close proximity to the HLA complex. This colocalization is of interest because of a possible association between prolactin-secreting adenomas and specific HLA alleles

270 Hallgeir Rui (Farid et al., 1980), as well as a proposed genetic linkage between prolactin and rheumatoid arthritis (Brennan et al., 1996). Furthermore, a woman with recurrent spontaneous abortions was found to have idiopathic hypoprolactinemia and mosaicism for a partial deletion of chromosome 6p, the breakpoint being located at 6p23 (D'Alessandro et al., 1992).

Regulatory sites and corresponding transcription factors The human prolactin gene is more than 15 kb long and contains six exons (Truong et al., 1984). The transcription of the gene is driven by two tissuespecific promoters, a proximal promoter that is used in the pituitary and a very distal promoter that is used in extrapituitary cells and tissues, such as decidua, myometrium, and lymphoid cells (DiMattia et al., 1990; Berwaer et al., 1994; Gellersen et al., 1994). A noncoding exon 1a is only expressed in extrapituitary tissues and has a transcriptional start site 5.8 kb upstream of the pituitary start site (Berwaer et al., 1994). In extrapituitary sites, exon 1a is spliced to the first pituitary exon 1b, generating a transcript that is approximately 150 bp larger than its pituitary counterpart (Gellersen et al., 1989), differing only in the 50 untranslated region. The downstream promoter that directs transcription in pituitary lactotrophs is under the control of the POU homeodomain transcription factor Pit-1. There are two clusters of three and eight Pit-1-binding sites within the pituitary promoter of the human prolactin gene. In addition, there is an AP-1 site (Peers et al., 1990) and a degenerate ERE sequence (Gellersen et al., 1995; reviewed by Ben-Jonathan et al., 1996). Transcriptional control of the distal, nonpituitary start site in endometrial stromal cells is linked to decidual differentiation during the secretory phase of the ovulatory cycle (DiMattia et al., 1990; Gellersen et al., 1994). Two consensus binding sites for CCAAT/enhancer-binding proteins (C/EBP) mediate the cAMP/PKA-induced activation of this nonpituitary prolactin gene promoter in human decidual cells (Pohnke et al., 1999). Cyclic AMP, alone or in synergy with PHA, also stimulated the activation of this upstream prolactin gene promoter in Jurkat T cells, possibly through the activation of C/ EBP proteins (Reem et al., 1999).

Cells and tissues that express the gene See Table 2.

PROTEIN

Accession numbers Mouse prolactin protein: P06879 (SwissProt) Human prolactin protein: 4506105, 4234766, 385771, 1658518, 1620399, 385771, 758096, 531103, 531101, 190356

Sequence See Figure 3.

Description of protein The amino acid sequences of prolactin from human and mouse are aligned in Figure 1. As reported above, prolactin circulates in blood as monomers and as larger `macroprolactins' (big-prolactin, big-big prolactin), representing both homo-oligomeric aggregates and immunoglobulincomplexed prolactin. In certain asymptomatic subjects with hyperprolactinemia, stable circulating complexes of immunoglobulin and prolactin have been identified (Bonhoff et al., 1995; Hattori and Inagaki, 1998), suggesting that antiprolactin autoantibodies may occasionally neutralize the activity of the hormone. Furthermore, the proteolysis of prolactin to its 16 kDa variant (see above) results in a molecule with distinct activities that may activate unique receptors.

Discussion of crystal structure As discussed in the section on Structure, a threedimensional structure of prolactin has not been reported although models have been proposed based on growth hormone. See Figure 2.

Important homologies Human prolactin is most homologous to growth hormone (16% amino acid identity) and placental lactogen (13% amino acid identity). In rodents, prolactin is more closely related to a series of prolactinlike genes that are expressed in placenta in temporally defined patterns during the course of pregnancy (for reviews, see Forsyth, 1994; Soares et al., 1998; see also Table 1).

Prolactin

271

Figure 3 Amino acid sequence of human and mouse prolactin protein (signal peptide underlined).

Table 1 Mouse prolactin family Prolactin Placental lactogen I Placental lactogen II Prolactin-like protein-A Prolactin-like protein-B Prolactin-like protein-C variant Prolactin-related protein Proliferin Proliferin-related protein Prolactin-like protein-F Prolactin-like protein-G Adapted from Soares et al. (1998).

Posttranslational modifications Oxidation The six cysteine residues in prolactin undergo oxidation and form stable, successive intramolecular

disulfide bonds. C-terminal, central, and N-terminal bridges are formed by the pairwise coupling of cysteine residues Cys4-Cys11, Cys58-Cys174, and Cys191Cys199. Proteolysis Mature prolactin, formed by proteolytic removal of a 28 kDa signal peptide, can be further modified by proteases (see the review by Sinha, 1995). Cathepsin-D proteolysis at position 133 generates two fragments of 16 and 8 kDa respectively, which may exist as both disulfide-linked heterodimers and monomers (Mittra, 1980; Cole et al., 1991; Baldocchi et al., 1993). Kallikrein, a trypsin-like protease, cleaves prolactin at position 173 to remove the C-terminal disulfide loop and give rise to a 22 kDa fragment (Anthony et al., 1993). Particular interest has arisen with respect to the anti-angiogenic effect of the 16 kDa prolactin fragment (Clapp et al., 1993; Clapp and Weiner, 1992; D'Angelo et al., 1999). Glycosylation A proportion of pituitary and circulating prolactin is glycosylated in most species. Approximately 20% of circulating human prolactin is glycosylated through

272 Hallgeir Rui N-linkage at position 31 (Lewis et al., 1985; Champier et al., 1987). Mouse prolactin is also glycosylated, but the extent and linkage site remain to be determined (Sinha, 1995). The physiological function of glycosylation of prolactin may be to reduce biological potency, while extending the half-life of the molecule (Hoffman et al., 1993).

Table 2 Cells and tissue that express prolactin

Brain

Tissue

Cell types

Pituitary

Lactotrophs Somatolactotrophs

Hypothalamus Pons-medulla Other regions

Phosphorylation A significant proportion of prolactin molecules are phosphorylated on their serine and threonine residues. In bovine pitiutary, between 20% and 80% of the prolactin was phosphorylated, particularly on Ser90, with Ser26 and Ser34 constituting minor sites (Kim and Brooks, 1993). In rat prolactin, Ser177 was found to be the major site of phosphorylation (Wang et al., 1996), and this site is positionally conserved in prolactin from all species. In general, the phosphorylation of prolactin is associated with reduced bioactivity (Wang and Walker, 1993; Brooks and Saiduddin, 1998), but does not affect the biological half-life (Brooks and Saiduddin, 1998). Furthermore, substitution of the positionally conserved serine residue Ser179 in human prolactin with either Asp or Glu, which are structural mimics of phosphoserine, led to a loss of bioactivity in both mutants. Indeed, the Ser179Asp mutant acted as a potent prolactin antagonist (Chen et al., 1998). However, antagonist activity was not detected in phosphorylated bovine prolactin (Brooks and Saiduddin, 1998). Because prolactin is phosphorylated in secretory granules during its release from pituitary lactotrophs, it is possible that phosphorylation serves to reduce local bioactivity during secretion. Further work is needed to establish the physiological role of prolactin phosphorylation.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Prolactin is produced by lactotrophs and somatolactotrophs of the anterior pituitary. In addition, local production occurs in a broad range of cell types, particularly in uterus, mammary gland, prostate, cells of the immune system and certain brain regions (see Table 2). Immortalized pituitary cell lines that secrete prolactin include the highly studied rodent GH3, GH4C1, and MMQ cell lines (Gautvik et al., 1983; Kineman and Frawley, 1994). In addition, the human

Reproductive system

Uterus

Decidualized endometrial stromal cells

Ovary Testis

Leydig cells

Mammary

Epithelial cells

Prostate

Epithelial cells

Immune/ hematopoietic system

T lymphocytes B lymphocytes NK cells Mononuclear cells Thymic epithelial cells

Other organs

Skin

Dermal fibroblasts

Kidney

Parietal cells

Adrenal Adapted from Ben-Jonathan et al. (1996).

B-lymphoblastoid IM-9-P3 cell line secretes considerable amounts of prolactin (DiMattia et al., 1988).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators A multitude of inhibitors and stimulators of pituitary prolactin secretion have been identified (for a review, see Thorner et al., 1998). The secretion of prolactin, unlike that of other pituitary hormones, is under tonic inhibition by hypothalamic dopamine. TRH, VIP, and prolactin-releasing peptide are potent stimulatory peptides (Hinuma et al., 1998; Thorner et al., 1998). In addition, estrogens are strong stimulators of pituitary prolactin secretion (Day et al., 1990; Murdoch et al., 1995). Table 3 summarizes the most established regulators of pituitary prolactin secretion (adapted from Thorner et al., 1998).

Prolactin Steroid hormones frequently modulate the effects of prolactin. For example, estrogen and progesterone from the placenta suppress the lactogenic effect of pituitary prolactin in the mammary gland, whereas glucocorticoids exert a synergistic effect. Thus, for milk production to begin, the reduction in estrogen and progesterone levels associated with the shedding of the placenta at parturition is needed. However, interactions between prolactin and steroids are highly dependent on cell type and hormonal milieu. Whereas prolactin is antiapoptotic in lymphoid cells, and glucocorticoids are proapoptotic, prolactin and glucocorticoids appear to have a synergistic antiapoptotic effect in differentiated mammary gland. In addition, ergot-derivatives (e.g. bromocriptine) act as dopamine agonists and inhibit pituitary prolactin release, and many psychopharmaceutical drugs stimulate prolactin secretion (see Table 3).

RECEPTOR UTILIZATION Prolactin could be considered to be a monogamous cytokine in that it binds exclusively to prolactin receptors. In contrast, human growth hormone binds to growth hormone receptors as well as prolactin receptors (Kossiakoff et al., 1994). However, the prolactin receptor gene gives rise to alternatively spliced variants with identical extracellular binding domains but with long or short cytoplasmic domains (reviewed by Bole-Feysot et al., 1998). This is of functional importance because of the different signaling capacities of the receptor splice variants. Furthermore, the proteolytic 16 kDa prolactin variant may interact with a unique, yet-to-be identified receptor (Clapp and Weiner, 1992).

IN VITRO ACTIVITIES

In vitro findings Because prolactin receptors are expressed ubiquitously, and couple to several parallel signal transduction pathways downstream of the JAK2 tyrosine kinase (Lebrun et al., 1994; Rui et al., 1994), prolactin affects the function of a variety of cells. The effects are cell and context dependent, and include the regulation of growth, survival, differentiation, and activation state types, particularly on cells of the mammary gland and other reproductive organs (mammary and prostate epithelial cells, Leydig cells, granulosa cells), and in the immune system (T cells, B cells, NK cells)

273

(see the reviews by Topper et al., 1986; Groner and Gouilleux, 1995; Ben-Jonathan et al., 1996; Ferrag et al., 1997; Yu-Lee, 1997; Clevenger et al., 1998; Yu-Lee et al., 1998).

Regulatory molecules: Inhibitors and enhancers Steroid hormones are frequently potent modulators of prolactin effects, and may be inhibitory or synergistic depending on cell type or hormonal milieu (Topper et al., 1986; Groner and Gouilleux, 1995). In addition, interactions have been described with polypeptide growth factors such as EGF, IGF-1, and insulin.

Bioassays used The Nb2 lymphoma (Gout et al., 1980) and pigeon cropsac (Riddle et al., 1933; Horseman and Buntin, 1995) assays are used.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The physiological effects of prolactin on the seven areas previously mentioned are described below. Reproduction In the mammary gland, prolactin plays a critical role in stimulating mammary gland differentiation in pregnant mammals, and is important for the initiation and maintenance of milk production (Topper et al., 1986; Rosen et al., 1994; Groner and Gouilleux, 1995). An interesting comparative function of prolactin in a submammalian species is the induction of cropmilk in the cropsac of nursing pigeons (reviewed by Horseman and Buntin, 1995). In mammals, prolactin-induced mammary differentiation occurs in synergy with insulin and corticosteroids, and only after circulating estrogens and progesterone levels fall at parturition will prolactin induce milk secretion (for extensive reviews, see Hennighausen et al., 1991; Rosen et al., 1994; Groner and Gouilleux, 1995). Gene knockout studies in mice have verified the critical roles that prolactin and prolactin receptors play

274 Hallgeir Rui in mammary differentiation and lactation (Ormandy et al., 1997; Goffin et al., 1998; Steger et al., 1998; Horseman, 1999). Specifically, prolactin was found to affect mammary gland morphogenesis by controlling ductal side branching and terminal end bud regression in virgin mice through indirect mechanisms, but it acted directly on the mammary epithelium to induce lobuloalveolar development during pregnancy (Brisken et al., 1999). Furthermore, gene knockout studies have also demonstrated that transcription factor STAT5a is a central downstream mediator of prolactin action in mammary glands (Hennighausen et al., 1997; Liu et al., 1997). In the gonads, prolactin may regulate ovarian and testicular function both directly through gonadal prolactin receptors and indirectly by modulating gonadotropin secretion (Smith, 1980; Ben-Jonathan et al., 1996). Prolactin stimulates dopamine release in the hypothalamus and influences gonadotropin secretion, and physiological hyperprolactinemia during pregnancy and lactation, as well as pathological hyperprolactinemia, are associated with suppression of the hypothalamic-pituitary-gonadal axis. The inhibition of the pulsatile secretion of GnRH from the pituitary by prolactin results in impaired gonadotropin secretion and the inhibition of gonadal function (Thorner et al., 1998). However, fertility and circulating testosterone levels were normal in male prolactin knockout mice, whereas ovarian steroid production was insufficient for fertility in females (Steger et al., 1998). The direct effect of prolactin on testicular function appears to be particularly pronounced in seasonally breeding animals, in which prolactin may act directly on the testes as a gonadotropin (Jabbour et al., 1998). Prolactin receptors are expressed in Leydig cells as well as in steroid-producing cells in the ovary (Rolland and Hammond, 1975; Charreau et al., 1977), and evidence suggests direct effects of prolactin on rodent ovarian function, particularly in the regulation of progesterone metabolism (Martel et al., 1994; Zhong et al., 1997). In the uterus and placenta, prolactin is produced in myometrial cells and decidualized endometrial cells (Riddick et al., 1978; DiMattia et al., 1990; Gellersen et al., 1991, 1994; Stewart et al., 1995). Prolactin produced locally in the uterus is thought to be important for the establishment and progression of pregnancy (for a review, see Soares et al., 1998), although the exact roles of decidual prolactin in this process remain to be established. Two proposed functions involve a regulation of the water/electrolyte balance of the amnion and local immune reactions to prevent the rejection of the implant. Furthermore, another specific role that has been proposed for

decidual prolactin is to induce the extensive angiogenesis of the placenta associated with trophoblast invasion, based on the observation that prolactin markedly stimulated the local expression of the angiogenic factor bFGF (Srivastava et al., 1998). In the prostate, a number of stimulatory effects of prolactin have been reported on prostate growth and differentiation in rodents (for reviews, see Costello and Franklin, 1994; Reiter et al., 1999). These effects can, however, to a large extent be compensated for in mice defective in either prolactin or prolactin receptor expression (Bole-Feysot et al., 1998; Steger et al., 1998). Only a moderate size reduction of the ventral prostate was observed in prolactin-null mice (Steger et al., 1998), whereas male accessory organs appeared normal in prolactin receptor knockout mice. On the other hand, mice rendered chronically hyperprolactinemic by the overexpression of prolactin had dramatically enlarged prostate glands, although this effect might in part be mediated via a moderately elevated testosterone level (Wennbo et al., 1997a). The parallel observation of the local production of prolactin within the epithelial compartment of rodent and human prostate has further suggested the physiological involvement of prolactin in prostate growth and differentiation (Nevalainen et al., 1997a, 1997b). In fact, certain data point to prolactin as a tumor promoter in rodent prostate (Nakamura et al., 1990; Reiter et al., 1999). Immune Function An extensive literature has documented effects of prolactin on various functions of immune cells, including lymphoid cells (T cells, B cells, and NK cells), monocytes, macrophages, and thymic epithelial cells (for extensive reviews, see Russell, 1989; Berczi, 1994; Dardenne and Savino, 1994; Ferrag et al., 1994; Leite-de-Moraes et al., 1995; Murphy et al., 1995; Weigent, 1996; Ferrag et al., 1997; Yu-Lee, 1997; Clevenger et al., 1998; De Mello-Coelho et al., 1998; Velkeniers et al., 1998; Yu-Lee et al., 1998). Recent work involving gene targeting in mice has, however, suggested that the ancestral and fundamental roles of prolactin as a regulator of hematopoiesis and immune function have been effectively shared by other and more specialized tetrahelical cytokines. In knockout mice lacking either the gene for prolactin or the prolactin receptor, a redundancy of prolactin function in the immune system was revealed by the finding of normal immunity and hematopoietic parameters (Ormandy et al., 1997; Goffin et al., 1998; Horseman et al., 1998; Bouchard et al., 1999). Although prolactin was not found to be critical for immune function in mice, redundant functions of

Prolactin prolactin on immune cells will be more difficult to uncover and may require combination knockout strategies. Nonetheless, a series of research documents that hyperactivity of the prolactin-prolactin receptor axis affects immune function, particularly autoimmunity, hematopoietic cell growth promotion, and leukemogenesis (for a review, see Hooghe et al., 1998; Neidhart 1998). Osmoregulation The regulation of water/electrolyte balance seems to be the most prominent role of prolactin in fish and reptiles, and might represent the most ancient function of prolactin (for a review, Bern and Nicoll, 1968; Nicoll, 1980; Ben-Jonathan et al., 1996). In mammals, a role for prolactin in osmoregulation is perhaps reflected in the local production of prolactin and expression of prolactin receptors in the kidney (Sakai et al., 1999) and in secretory organs such as the mammary, prostate, and lacrimal glands (Shennan, 1994). Furthermore, decidual prolactin present in amniotic fluid has been suggested to regulate fluid balance (Handwerger and Freemark, 1987). Stress Prolactin is a stress hormome (Drago et al., 1989). The circulating prolactin level rises rapidly in response to emotional and physical stress, both in humans and other mammals (Meites and Clemens, 1972; Noel et al., 1972; Siegel et al., 1980). In soldiers undergoing an extensive combat course, prolactin levels were elevated at the start of the course, presumably in anticipation of stress, but underwent a decrease over the 3 days of continuous physical strain (Aakvaag et al., 1978). Consistent with these and other observations, it has been postulated that stress situations associated with passive coping are accompanied by an increased plasma prolactin level, whereas stress situations associated with active coping are associated with an unchanged or even lowered level (Theorell, 1992). Furthermore, data indicate that prolactin affects neuroendocrine, behavioral, and autonomic responses to stress, such as changes in the motility of the gastric musculature and gastric acid secretion, and in thermoregulation (Drago et al., 1990, 1993; Drago and Amir, 1984). In the rat, hyperprolactinemia has been demonstrated to protect against stressinduced ulcers (Drago et al., 1985). These and other data suggest that stress-induced hyperprolactinemia is not a mere consequence of stress activation, but may play a role in physiological mechanisms leading to the restoration of body homeostasis. Decreased libido in

275

stressed and hyperprolactinemic subjects is perhaps an endurance-preserving stress adaption response. Behavior and Psychological Effects The behavioral effects of prolactin in vertebrates are primarily linked to reproduction. Prolactin stimulates parental behavior such as nesting and egg hatching in birds, and nest building and the nursing of pups in mice, rats, and rabbits (for reviews, see Scapagnini et al., 1985; Dutt et al., 1994; Horseman and Buntin, 1995). Confirming this long-standing notion of the behavioral effects of prolactin, which were first observed by Riddle and colleagues (1935), elegant studies of prolactin receptor knockout mice demonstrated a reduction in maternal behavior in these animals (Lucas et al., 1998). Metabolism The liver is involved in the clearance of circulating prolactin, and the expression on liver cells of disproportionally high levels of the putative `nonsignaling' short prolactin isoform may facilitate this function (Jahn et al., 1997; Perrot-Applanat et al., 1997). Prolactin, however, also has some growth hormone-like effects on the liver, and stimulates IGF-1 production (Murphy et al., 1988; Strain and Ingleton, 1990). Independently, prolactin and growth hormone stimulate cell proliferation and insulin production in Langerhans islets (Billestrup and Nielsen, 1991; Sorenson and Brelje, 1997; Nielsen et al., 1999). Furthermore, softened bones in prolactin receptor null mice, combined with the detection of prolactin receptors on osteoblasts but not osteoclasts, suggested an involvement of prolactin in bone metabolism (Bouchard et al., 1999). In light of previously reported effects of prolactin on cholecalciferol secretion by the kidney (MacIntyre et al., 1978; Haug et al., 1982), an indirect effect of prolactin on calcium metabolism also might be involved in this phenotype. Skin In seasonal animals such as blue fox and red deer, evidence indicates that prolactin stimulates the transition from the winter to the summer coat (for a review, see Curlewis, 1992). Furthermore, prolactin may promote the proliferation of skin epithelial cells and support hair growth (Paus, 1991; Stenn and Paus, 1999). These findings are relevant for the occasional finding of hirsutism in cases of hyperprolactinemia (Thorner et al., 1998).

276 Hallgeir Rui

Species differences The predominant functions of prolactin differ between vertebrates: 1. 2. 3. 4.

Mammals ± milk production Birds ± cropmilk production, maternal behavior Amphibians and reptiles ± osmoregulation Fish ± osmoregulation.

Knockout mouse phenotypes The prolactin gene was disrupted by homologous recombination in mice by Horseman and colleagues (1997). The following phenotypic observations were made: 1. Female null mice were infertile. 2. The mammary glands of mutant female mice developed a normal ductal tree, but ducts failed to develop lobular decorations, which is a characteristic of the normal virgin adult mammary gland. 3. Male prolactin knockout mice showed normal fertility. 4. Male prolactin knockout mice had a significantly reduced median eminence dopamine content, plasma LH level, and LH and FSH secretion in vitro. 5. Plasma testosterone levels were normal in male prolactin knockout mice. 6. The weights of the seminal vesicles and ventral prostate were moderately reduced in prolactin knockout mice. 7. These results indicate that absence of prolactin reduces pituitary LH release, attenuates median eminence dopaminergic activity, and affects the growth of the seminal vesicles and ventral prostate. 8. Prolactin knockout mice demonstrated normal myelopoiesis and primary lymphopoiesis. 9. Pituitary weight in prolactin knockout mice was doubled, presumably as a result of reduced feedback inhibition and hypertrophy and/or hyperplasia of nonfunctional lactotrophs. In conclusion, the data suggested that prolactin is critical for fertility and mammary development in female mice, whereas prolactin deficiency can be compensated with regard to male fertility and general hematopoiesis and immune system development. These observations in prolactin knockout mice harmonize with the extended analysis of prolactin receptor-null mice developed in Kelly's laboratory (Ormandy et al., 1997).

Briefly, prolactin receptor knockout mice presented multiple reproductive defects in female mice, including: 1. sterility caused by a complete failure of embryonic implantation; 2. irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnancy; 3. in heterozygous female mice, an almost complete loss of lactation after the first but not subsequent pregnancies. Furthermore, half of the male prolactin receptor knockout mice were infertile or showed reduced fertility. When transplanted into the fat pads of prolactin receptor‡/‡ mice, mammary gland tissue from receptor-negative mice showed normal side branching and the formation of alveolar buds, but no lobuloalveolar development during pregnancy. Thus, prolactin affects mammary morphogenesis in two different ways: by controlling ductal side branching and terminal end bud regression in virgin animals via indirect mechanisms, and by acting directly on the mammary epithelium to produce lobuloalveolar development during pregnancy (Briskin et al., 1999). Homozygous mutant and heterozygous mutant nulliparous females showed a deficiency in pupinduced maternal behavior. Moreover, primiparous heterozygous females exhibit a profound deficit in maternal care when challenged with foster pups. These studies clearly establish the prolactin receptor as a regulator of maternal behavior (Lucas et al., 1998). Finally, prolactin receptor-negative embryos of mice showed a reduced degree of bone development of their calvaria. In adults, the absence of prolactin receptors was associated with a decrease in the bone formation rate and a reduction of bone mineral density. This suggests that an effect of prolactin on osteoblasts could be required for normal bone formation and the maintenance of bone mass (Bouchard et al., 1999).

Transgenic overexpression The introduction of a prolactin transgene into mice under the control of a constitutive metallothionine promoter led to chronic hyperprolactinemia, leading to an increased rate of mammary tumor formation in females (Wennbo et al., 1997b). In addition, advanced prostate hyperplasia was observed in male mice, an effect that was associated with a moderate elevation of circulating testosterone level (Wennbo et al., 1997a).

Prolactin

Pharmacological effects There has been no reported clinical use of prolactin in humans. However, based on animal studies, the application of prolactin and growth hormone as lowtoxicity adjuvants to aid bone marrow recovery has been proposed (Murphy et al., 1999).

Interactions with cytokine network In general, because prolactin activates the transcription factor STAT5 in most target cells, prolactin tends to interact positively and in a redundant manner with other cytokines that also activate STAT5 (e.g. IL-2, IL-3, IL-5, IL-7, IL-9, IL-15, GM-CSF, erythropoietin, thrombopoietin, and growth hormone). For example, prolactin and IL-2 stimulate growth in an additive fashion in Nb2 lymphocytes (Kirken et al., 1994).

Endogenous inhibitors and enhancers See Table 3.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Basal levels of prolactin in adults vary considerably, with levels of 4.0±25.0 mg/L in nonpregnant women (median 10.0 mg/L), and levels of 0.5±19.0 mg/L in men (median 8.5 mg/L) (Le Moli et al., 1999). Serum prolactin, which increases throughout pregnancy, falls with the onset of labor and then exhibits variable patterns of secretion, depending on whether breastfeeding occurs.

Role in experiments of nature and disease states Reproduction Among pituitary tumors, 60% secrete prolactin and cause a state of chronic hyperprolactinemia. Pituitary

277

adenomas reveal pathophysiologic effects of prolactin in men by inducing decreased libido, impotence, gynecomastia, galactorrhea, hypospermia, and occasionally reduced beard growth (Thorner et al., 1998). In premenopausal women, the cardinal effects of hyperprolactinemia are amenorrhea, a cessation of the normal cyclic ovarian function, galactorrhea, decreased libido, occasional hirsutism, and an increased long-term risk of osteoporosis (Palermo et al., 1994; Thorner et al., 1998). Specifically, pathologic milk discharge, galactorrhea, is a result of hyperprolactinemia in both men and women, and attests to the importance of prolactin in lactation. A few cases of isolated, idiopathic prolactin deficiency in women have been reported, fertility problems and alactogenesis being the most significant consequences (Turkington, 1972; Falk, 1992; Kauppila, 1997). In addition, one case of prolactin deficiency has been reported that was associated with alactogenesis in a woman who had two normal pregnancies that were conceived without the use of ovulationinducing drugs (Kauppila et al., 1987). Furthermore, in rodent models, a chronically elevated prolactin level is associated with an increased formation of mammary tumors (Wennbo et al., 1997b), and there is renewed interest in prolactin as a breast cancerpromoting factor in humans (Reynolds et al., 1997; Vonderhaar, 1998; Goffin et al., 1999). Prolactin is also produced by the myometrium (Riddick et al., 1978), and is expressed in leiomyomas, possibly acting as a growth factor (Nowak et al., 1999).

Autoimmune Diseases Accumulating data suggest that an elevated prolactin level represents a risk factor for certain autoimmune diseases in humans and rodents. These include adjuvant arthritis in rats, collagen type II-induced arthritis in rats and mice, type I diabetes in mice, and systemic lupus erythematosus (SLE) in mice and humans (for reviews, see McMurray, 1996; Neidhart, 1998; Walker et al., 1998; Ostensen, 1999). Furthermore, a connection between an elevated prolactin level and rheumatoid arthritis (RA) also has been suggested, but this correlation is less clear. Interestingly, there is a genetic association between RA and genes encoded in the HLA complex, particularly HLA DR4, and the human prolactin gene is located in close proximity to the HLA region on the short arm of chromosome 6. The hypothesis has been put forth that associations between HLA DR4 and reproductive risk factors in RA are caused by a linkage disequilibrium between DR4 and an abnormally regulated prolactin gene polymorphism (Brennan et al., 1996).

278 Hallgeir Rui Table 3 Stimuli that regulate pituitary prolactin secretion Enhancers

Inhibitors

Physiological Pregnancy (estrogen effect) Nursing/suckling Neurogenic: Nipple stimulation/chest wall trauma/ breast manipulation /spinal cord lesions Sleep Stress (hypoglycemia) Exercise Seizures Hypothyroidism (elevated TRH) Pituitary stalk lesions Pituitary tumors Renal failure Liver failure Pharmacologic Estrogen

Dopamine agonists (bromocriptine, levodopa)

TRH

GABA

Prolactin-releasing hormone/peptide VIP Dopamine antagonists (phenothiazines, haloperidol, metoclopramide, reserpine, methyldopa, amoxapine, opiates) -Endorphin Opioids Monamine oxidase inhibitors Cimetidine (intravenous) Verapamil Pathologic Pituitary tumors

Pseudohypoparathyroidism

Hypothalamic/pituitary stalk lesions

Pituitary destruction/removal

Neuraxis irradiation

Lymphocytic hypophysitis

Chest wall lesions Spinal cord lesions Hypothyroidism Chronic renal failure Severe liver disease Adapted from Thorner et al. (1998).

Prolactin Hematopoietic Cancer An involvement of prolactin in the development and progression of leukemia and lymphoma has also been suggested (for a review, see Hooghe et al., 1998). For example, a hyperactive, mutant prolactin receptor has been described in the prolactin-dependent Nb2 lymphoma (Ali et al., 1991). Furthermore, the autocrine production of prolactin is detectable in several transformed human lymphocytic lines, including IM-9-P3, Jurkat, Hut-78, U937, and YT (DiMattia et al., 1988; Pellegrini et al., 1992). The fact that tyrosine kinase JAK2, the principal downstream mediator of prolactin action (Lebrun et al., 1994; Rui et al., 1994), is oncogenic in cells of both lymphoid and myeloid origin (Peeters et al., 1997), lends support to the notion that prolactin may promote the growth of hematopoietic cancers. In fact, elevated serum prolactin was detected in more that 50% of patients with acute myeloid leukemia (Hatfill et al., 1990), although this observation might be the result of an associated stress response. Osmoregulation An excessive levels of amniotic fluid, polyhydramnios, is often associated with diabetes mellitus, multiple pregnancies, or fetal malformation. Interestingly, amniotic fluid in polyhydramnios is characterized by a selectively reduced level of prolactin but not of other hormones (Luciano and Varner, 1984; Saandakou et al., 1992). At the same time, patients with chronic polyhydramnios showed a reduced number of prolactin receptors within the chorion laeve (Healy et al., 1985).

IN THERAPY

Preclinical ± How does it affect disease models in animals? Prolactin treatment aids bone marrow recovery in transplanted mice (Woody et al., 1999). Prolactin treatment in mice worsens the symptoms of certain experimental autoimmune diseases (e.g. adjuvant arthritis; Neidhart, 1998).

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. There are no reported results of clinical trials with human prolactin.

279

Pharmacokinetics The pituitary prolactin secretory rate is approximately 18.6 nmol per day (400 mg per day). The hormone is cleared by the liver (75%) and the kidney (25%), and its half-life in plasma is approximately 50 minutes (Thorner et al., 1998).

Toxicity Not reported. Iatrogenic hyperprolactinemia may be expected to mimic symptoms of chronic pituitary hyperprolactinemia, although the effects of acute hyperprolactinemia are hard to predict.

References Aakvaag, A., Sand, T., Opstad, P. K., and Fonnum, F. (1978). Hormonal changes in serum in young men during prolonged physical strain. Eur. J. Appl. Physiol. 39, 283±291. Ali, S., Pellegrini, I., and Kelly, P. A. (1991). A prolactindependent immune cell line (Nb2) expresses a mutant form of prolactin receptor. J. Biol. Chem. 266, 20110±20117. Anthony, P. K., Stoltz, R. A., Pucci, M. L., and Powers, C. A. (1993). The 22K variant of rat prolactin: evidence for identity to prolactin-(1-173), storage in secretory granules, and regulated release. Endocrinology 132, 806±814. Ben-Jonathan, N., Mershon, J. L., Allen, D. L., and Steinmetz, R. W. (1996). Extrapituitary prolactin: distribution, regulation, functions, and clinical aspects. Endocr. Rev. 17, 639± 669. Berczi, I. (1994). The role of the growth and lactogenic hormone family in immune function. Neuroimmunomodulation 1, 201±216. Bern, H. A., and Nicoll, C. S. (1968). The comparative endocrinology of prolactin. Recent. Prog. Horm. Res. 24, 681±720. Berwaer, M., Martial, J. A., and Davis, J. R. (1994). Characterization of an up-stream promoter directing extrapituitary expression of the human prolactin gene. Mol. Endocrinol. 8, 635±642. Billestrup, N., and Nielsen, J. H. (1991). The stimulatory effect of growth hormone, prolactin, and placental lactogen on beta-cell proliferation is not mediated by insulin-like growth factor-I. Endocrinology 129, 883±888. Bole-Feysot, C., Goffin, V., Edery, M., Binart, N., and Kelly, P. A. (1998). Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 19, 225±268. Bonhoff, A., Vuille, J. C., Gomez, F., and Gellersen, B. (1995). Identification of macroprolactin in a patient with asymptomatic hyperprolactinemia as a stable PRL-IgG complex. Exp. Clin. Endocrinol. Diabetes 103, 252±255. Bouchard, B., Ormandy, C. J., Di Santo, J. P., and Kelly, P. A. (1999). Immune system development and function in prolactin receptor-deficient mice. J. Immunol. 163, 576±582. Brennan, P., Ollier, B., Worthington, J., Hajeer, A., and Silman, A. (1996). Are both genetic and reproductive associations with rheumatoid arthritis linked to prolactin? Lancet 348, 106±109. Brisken, C., Kaur, S., Chavarria, T. E., Binart, N., Sutherland, R. L., Weinberg, R. A., Kelly, P. A., and

280 Hallgeir Rui Ormandy, C. J. (1999). Prolactin controls mammary gland development via direct and indirect mechanisms. Dev. Biol. 210, 96±106. Brooks, C. L., and Saiduddin, S. (1998). Phosphorylation of bovine prolactin eliminates luteotropic activity in the rat. Life Sci. 63, 1281±1287. Champier, J., Claustrat, B., Sassolas, G., and Berger, M. (1987). Detection and enzymatic deglycosylation of a glycosylated variant of prolactin in human plasma. FEBS Lett. 212, 220±224. Charreau, E. H., Attramadal, A., Torjesen, P. A., Purvis, K., Calandra, R., and Hansson, V. (1977). Prolactin binding in rat testis: specific receptors in interstitial cells. Mol. Cell. Endocrinol. 6, 303±307. Chen, T. J., Kuo, C. B., Tsai, K. F., Liu, J. W., Chen, D. Y., and Walker, A. M. (1998). Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone. Endocrinology 139, 609±616. Clapp, C., and Weiner, R. I. (1992). A specific, high affinity, saturable binding site for the 16-kilodalton fragment of prolactin on capillary endothelial cells. Endocrinology 130, 1380±1386. Clapp, C., Martial, J. A., Guzman, R. C., Rentier-Delure, F., and Weiner, R. I. (1993). The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology 133, 1292±1299. Clevenger, C. V., Freier, D. O., and Kline, J. B. (1998). Prolactin receptor signal transduction in cells of the immune system. J. Endocrinol. 157, 187±197. Cole, E. S., Nichols, E. H., Lauziere, K., Edmunds, T., and McPherson, J. M. (1991). Characterization of the microheterogeneity of recombinant primate prolactin: implications for posttranslational modifications of the hormone in vivo. Endocrinology 129, 2639±2646. Cooke, N. E. (1989). In ``Endocrinology'' (ed L. J. De-Groot., G. M. Besser, G. F. Cahill Jr, et al.), Prolactin: normal synthesis, regulation, and actions, pp. 384±407. W. B. Saunders, Philadelphia. Cooke, N. E., Coit, D., Shine, J., Baxter, J. D., and Martial, J. A. (1981). Human prolactin: cDNA structural analysis and evolutionary comparisons. J. Biol. Chem. 256, 4007±4016. Costello, L. C., and Franklin, R. B. (1994). Effect of prolactin on the prostate. Prostate 24, 162±166. Curlewis, J. D. (1992). Seasonal prolactin secretion and its role in seasonal reproduction: a review. Reprod. Fertil. Dev. 4, 1±23 Cytokines Cell. Mol. Ther. 3, 197±213. D'Alessandro, E., Santiemma, V., Lo Re, M. L., Ligas, C., and Del Porto, G. (1992). 6p23 deletion mosaicism in a woman with recurrent abortions and idiopathic hypoprolactinemia. Am. J. Med. Genet. 44, 220±222. D'Angelo, G., Martini, J. F., Iiri, T., Fantl, W. J., Martial, J., and Weiner, R. I. (1999). 16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells. Mol. Endocrinol. 13, 692±704. Dardenne, M., and Savino, W. (1994). Prolactin-mediated cellular interactions in the thymus. Ann. NY Acad. Sci. 741, 100±107. Day, R. N., Koike, S., Sakai, M., Muramatsu, M., and Maurer, R. A. (1990). Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol. Endocrinol. 4, 1964±1971. De Mello-Coelho,V., Savino, W., Postel-Vinay, M. C., and Dardenne, M. (1998). Role of prolactin and growth hormone on thymus physiology. Dev. Immunol. 6, 317±323. De Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992). Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306±312. DiMattia, G. E., Gellersen, B., Duckworth, M. L., and Friesen, H. G. (1990). Human prolactin gene expression: the use of an alternative noncoding exon in decidua and

the IM-9-P3 lymphoblast cell line. J. Biol. Chem. 265, 16412± 16421. DiMattia, G. E., Gellersen, B., Bohnet, H. G., and Friesen, H. G. (1988). A human B-lymphoblastoid cell line produces prolactin. Endocrinology 122, 2508±2517. Drago, F., and Amir, S. (1984). Effects of hyperprolactinaemia on core temperature of the rat. Brain. Res. Bull. 12, 355±358. Drago, F., Continella, G., Conforto, G., and Scapagnini, U. (1985). Prolactin inhibits the development of stress-induced ulcers in the rat. Life Sci. 36, 191±197. Drago, F., D'Agata, V., Iacona, T., Spadaro, F., Grassi, M., Valerio, C., Raffaele, R., Astuto, C., Lauria, N., and Vitetta, M. (1989). Prolactin as a protective factor in stressinduced biological changes. J. Clin. Lab. Anal. 3, 340±344. Drago, F., D'Agata, V., Spadaro, F., Grassi, M., and Valerio, C. (1990). Prolactin as a protective factor in stress-induced gastric ulcers. Ann. NY Acad. Sci. 597, 248±251. Drago, F., Grassi, M., and Genazzani, A. A. (1993). Neuromediators in aging and gastric mucosal injury. J. Physiol. Paris 87, 379±383. Dutt, A., Kaplitt, M. G., Kow, L. M., and Pfaff, D. W. (1994). Prolactin, central nervous system and behavior: a critical review. Neuroendocrinology 59, 413±419. Evans, A. M., Petersen, J. W., Sekhon, G. S., and DeMars, R. (1989). Mapping of prolactin and tumor necrosis factor-beta genes on human chromosome 6p using lymphoblastoid cell deletion mutants. Somat. Cell Mol. Genet. 15, 203±213. Falk, R. J. (1992). Isolated prolactin deficiency: a case report. Fertil. Steril. 58, 1060±1062. Farid, N. R., Noel, E. P., Sampson, L., and Russell, N. A. (1980). Prolactin-secreting adenomata are possibly associated with HLA-B8. Tissue Antigens 15, 333±335. Ferrag, F., Lebrun, J. J., Touraine, P., Nagano, M., Dardenne, M., and Kelly, P. A. (1994). Prolactin and the immune system. Immunomethods 5, 21±30. Ferrag, F., Goffin, V., Buteau, H., and Kelly, P. A. (1997). Immune function of prolactin (PRL) and signal transduction by PRL/GH/cytokine receptors: specificity, redundancy and lessons from chimaeras. Cytokines Cell Mol. Ther. 3, 197±213. Forsyth, I. A. (1994). Comparative aspects of placental lactogens: structure and function. Exp. Clin. Endocrinol. 102, 244±251. Gautvik, K. M., Gordeladze, J. O., Jahnsen, T., Haug, E., Hansson, V., and Lystad, E. (1983). Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactinproducing rat pituitary tumor cells (GH cells). J. Biol. Chem. 258, 10304±10311. Gellersen, B., DiMattia, G. E., Friesen, H. G., and Bohnet, H. G. (1989). Prolactin (PRL) mRNA from human decidua differs from pituitary PRL mRNA but resembles the IM-9-P3 lymphoblast PRL transcript. Mol. Cell. Endocrinol. 64, 127±130. Gellersen, B., Bonhoff, A., Hunt, N., and Bohnet, H. G. (1991). Decidual-type prolactin expression by the human myometrium. Endocrinology 129, 158±168. Gellersen, B., Kempf, R., Telgmann, R., and Di Mattia, G. E. (1994). Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma. Mol. Endocrinol. 8, 356± 373. Gellersen, B., Kempf, R., Telgmann, R., and Di Mattia, G. E. (1995). Pituitary-type transcription of the human prolactin gene in the absence of Pit-1. Mol. Endocrinol. 9, 887±901. Goffin, V., Shiverick, K. T., Kelly, P. A., and Martial, J. A. (1996). Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen, and related proteins in mammals. Endocr. Rev. 17, 385±410.

Prolactin Goffin, V., Bouchard, B., Ormandy, C. J., Weimann, E., Ferrag, F., Touraine, P., Bole-Feysot, C., Maaskant, R. A., Clement-Lacroix, P., Edery, M., Binart, N., and Kelly, P. A. (1998). Prolactin: a hormone at the crossroads of neuroimmunoendocrinology. Ann. NY Acad. Sci. 840, 498±509. Goffin, V., Touraine, P., Pichard, C., Bernichtein, S., and Kelly, P. A. (1999). Should prolactin be reconsidered as a therapeutic target in human breast cancer? Mol. Cell. Endocrinol. 151, 79±87. Gout, P. W., Beer, C. T., and Noble, R. L. (1980). Prolactinstimulated growth of cell cultures established from malignant Nb rat lymphomas. Cancer Res. 40, 2433±2436. Groner, B., and Gouilleux, F. (1995). Prolactin-mediated gene activation in mammary epithelial cells. Curr. Opin. Genet. Dev. 5, 587±594. Handwerger, S., and Freemark, M. (1987). Role of placental lactogen and prolactin in human pregnancy. Adv. Exp. Med. Biol. 219, 399±420. Hatfill, S. J., Kirby, R., Hanley, M., Rybicki, E., and Bohm, L. (1990). Hyperprolactinemia in acute myeloid leukemia and indication of ectopic expression of human prolactin in blast cells of a patient of subtype M4. Leuk. Res. 14, 57±62. Hattori, N., and Inagaki, C. (1998). Immunological aspects of human growth hormone and prolactin. Domest. Anim. Endocrinol. 15, 371±375. Haug, E., Pedersen, J. I., and Gautvik, K. M. (1982). Effects of vitamin D3 metabolites on production of prolactin and growth hormone in rat pituitary cells. Mol. Cell. Endocrinol. 28, 65±79. Healy, D. L., Herington, A. C., and O'Herlihy, C. (1985). Chronic polyhydramnios is a syndrome with a lactogen receptor defect in the chorion laeve. Br. J. Obstet. Gynaecol. 92, 461±467. Hennighausen, L., Westphal, C., Sankaran, L., and Pittius, C. W. (1991). Regulation of expression of genes for milk proteins. Biotechnology 16, 65±74. Hennighausen, L., Robinson, G. W., Wagner, K. U., and Liu, X. (1997). Prolactin signaling in mammary gland development. J. Biol. Chem. 272, 7567±7569. Hinuma, S., Habata, Y., Fujii, R., Kawamata, Y., Hosoya, M., Fukusumi, S., Kitada, C., Masuo, Y., Asano, T., Matsumoto, H., Sekiguchi, M., Kurokawa, T., Nishimura, O., Onda, H., and Fujino, M. (1998). A prolactin-releasing peptide in the brain. Nature 393, 272±276 [published erratum appears in Nature 1998, 394, 302]. Hoffmann, T., Penel, C., and Ronin, C. (1993). Glycosylation of human prolactin regulates hormone bioactivity and metabolic clearance. J. Endocrinol. Invest. 16, 807±816. Hooghe, R., Merchav, S., Gaidano, G., Naessens, F., and Matera, L. (1998). A role for growth hormone and prolactin in leukaemia and lymphoma? Cell. Mol. Life Sci. 54, 1095±1101. Horseman, N. D. (1999). Prolactin and mammary gland development. J. Mammary Gland Biol. Neoplasia 4, 79±88. Horseman, N. D., and Buntin, J. D. (1995). Regulation of pigeon cropmilk secretion and parental behaviors by prolactin. Annu. Rev. Nutr. 15, 213±238. Horseman, N. D., Zhao, W., Montecino-Rodriguez, E., Tanaka, M., Nakashima, K., Engle, S. J., Smith, F., Markoff, E., and Dorshkind, K. (1997). Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J. 16, 6926±6935. Hwang, P., Guyda, H., and Friesen, H. (1972). Purification of human prolactin. J. Biol. Chem. 247, 1955±1958. Jabbour, H. N., Clarke, L. A., McNeilly, A. S., Edery, M., and Kelly, P. A. (1998). Is prolactin a gonadotrophic hormone in red deer (Cervus elaphus)? Pattern of expression of the prolactin receptor gene in the testis and epididymis. J. Mol. Endocrinol. 20, 175±182.

281

Jackson-Grusby, L. L., Pravtcheva, D., Ruddle, F. H., and Linzer, D. I. (1988). Chromosomal mapping of the prolactin/ growth hormone gene family in the mouse. Endocrinology (United States) 122, 2462±2466. Jahn, G. A., Daniel, N., Jolivet, G., Belair, L., Bole-Feysot, C., Kelly, P. A., and Djiane, J. (1997). In vivo study of prolactin (PRL) intracellular signalling during lactogenesis in the rat: JAK/STAT pathway is activated by PRL in the mammary gland but not in the liver. Biol. Reprod. 57, 894±900. Kauppila, A. (1997). Isolated prolactin deficiency. Curr. Ther. Endocrinol. Metab. 6, 31±33. Kauppila, A., Chatelain, P., Kirkinen, P., Kivinen, S., and Ruokonen, A. (1987). Isolated prolactin deficiency in a woman with puerperal alactogenesis. J. Clin. Endocr. Metab. 64, 309±312. Kim, B. G., and Brooks, C. L. (1993). Isolation and characterization of phosphorylated bovine prolactin. Biochem. J. 296, 41±47. Kineman, R. D., and Frawley, L. S. (1994). Secretory characteristics and phenotypic plasticity of growth hormone- and prolactin-producing cell lines. J. Endocrinol. 140, 455±463. Kirken, R. A., Rui, H., Malabarba, M. G., and Farrar, W. L. (1994). Identification of interleukin-2 receptor-associated tyrosine kinase p116 as novel leukocyte-specific Janus kinase. J. Biol. Chem. 269, 19136±19141. Kossiakoff, A. A., Somers, W., Ultsch, M., Andow, K., Muller, Y. A., and De Vos, A. M. (1994). Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors. Protein Sci. 3, 1697±1705. Lebrun, J. J., Ali, S., Sofer, L., Ullrich, A., and Kelly, P. A. (1994). Prolactin-induced proliferation of Nb2 cells involves tyrosine phosphorylation of the prolactin receptor and its associated tyrosine kinase JAK2. J. Biol. Chem. 269, 14021±14026. Leite-de-Moraes, M. C., Touraine, P., Kelly, P. A., Kuttenn, F., and Dardenne, M. (1995). Prolactin receptor expression in lymphocytes from patients with hyperprolactinemia or acromegaly. J. Endocrinol. 147, 353±359. Le Moli, R., Endert, E., Fliers, E., Mulder, T., Prummel, M. F., Romijn, J. A., and Wiersinga, W. M. (1999). Establishment of reference values for endocrine tests. II: Hyperprolactinemia. Neth. J. Med. 55, 71±75. Lewis, U. J., Singh, R. N., and Seavey, B. K. (1971). Human prolactin: isolation and some properties. Biochem. Biophys. Res. Commun. 44, 1169±1176. Lewis, U. J., Singh, R. N., Sinha, Y. N., and Vanderlaan, W. P. (1985). Glycosylated human prolactin. Endocrinology 116, 359±363. Li, C. H. (1978). Prolactin. Trends Biochem. Sci. 3, 284. Liu, X., Robinson, G. W., Wagner, K. U., Garrett, L., WynshawBoris, A., and Hennighausen, L. (1997). Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11, 179±186. Lucas, B. K., Ormandy, C. J., Binart, N., Bridges, R. S., and Kelly, P. A. (1998). Null mutation of the prolactin receptor gene produces a defect in maternal behavior. Endocrinology 139, 4102±410. Luciano, A. A., and Varner, M. W. (1984). Decidual, amniotic fluid, maternal and fetal prolactin in normal and abnormal pregnancies. Obstet. Gynecol. 63, 384±388. MacIntyre, I., Colston, K. W., Szelke, M., and Spanos, E. (1978). A survey of the hormonal factors that control calcium metabolism. Ann. NY Acad. Sci. 307, 345±355. McMurray, R. W. (1996). Prolactin and systemic lupus erythematosus. Ann. Med. Interne (Paris) 147, 253±258. Martel, C., Gagne, D., Couet, J., Labrie, Y., Simard, J., and Labrie, F. (1994). Rapid modulation of ovarian

282 Hallgeir Rui 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase gene expression by prolactin and human chorionic gonadotropin in the hypophysectomized rat. Mol. Cell. Endocrinol. 99, 63±71. Meites, J., and Clemens, J. A. (1972). Hypothalamic control of prolactin secretion. Vitam. Horm. 30, 165±221. Mittra, I. (1980). A novel `cleaved prolactin' in the rat pituitary. I: Biosynthesis, characterization and regulatory control. Biochem. Biophys. Res. Commun. 95, 1750±1759. Murdoch, F. E., Byrne, L. M., Ariazi, E. A., Furlow, J. D., Meier, D. A., and Gorski, J. (1995). Estrogen receptor binding to DNA: affinity for nonpalindromic elements from the rat prolactin gene. Biochemistry 34, 9144±9145. Murphy, L. J., Tachibana, K., and Friesen, H. G. (1988). Stimulation of hepatic insulin-like growth factor-I gene expression by ovine prolactin: evidence for intrinsic somatogenic activity in the rat. Endocrinology 122, 2027±2033. Murphy, W. J., Rui, H., and Longo, D. L. (1995). Effects of growth hormone and prolactin immune development and function. Life Sci. 57, 1±14. Nakamura, A., Shirai, T., Ogawa. K., Wada, S., Fujimoto, N. A., Ito, A., and Ito, N. (1990). Promoting action of prolactin released from a grafted transplantable pituitary tumor (MtT/F84) on rat prostate carcinogenesis. Cancer Lett. 53, 151±157. Neidhart, M. (1998). Prolactin in autoimmune diseases. Proc. Soc. Exp. Biol. Med. 217, 408±419. Nevalainen, M. T., Valve, E. M., Ingleton, P. M., Nurmi, M., Martikainen, P. M., and Harkonen, P. L. (1997a). Prolactin and prolactin receptors are expressed and functioning in human prostate. J. Clin. Invest. 99, 618±627. Nevalainen, M. T., Valve, E. M., Ahonen, T., Yagi, A., Paranko, J., and Harkonen, P. L. (1997b). Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture. FASEB J. 11, 1297. Nicoll, C. S. (1980). Ontogeny and evolution of prolactin's functions. Fed. Proc. 39, 2563±2566. Nielsen, J. H., Svensson, C., Galsgaard, E. D., Moldrup, A., and Billestrup, N. (1999). Beta cell proliferation and growth factors. J. Mol. Med. 77, 62±66. Noel, G. L., Suh, H. K., Stone, J. G., and Frantz, A. G. (1972). Human prolactin and growth hormone release during surgery and other conditions of stress. J. Clin. Endocrinol. Metab. 35, 840±851. Nowak, R. A., Mora, S., Diehl, T., Rhoades, A. R., and Stewart, E. A. (1999). Prolactin is an autocrine or paracrine growth factor for human myometrial and leiomyoma cells. Gynecol. Obstet. Invest. 48, 127±132. Ormandy, C. J., Camus, A., Barra, J., Damotte, D., Lucas, B., Buteau, H., Edery, M., Brousse, N., Babinet, C., Binart, N., and Kelly, P. A. (1997). Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev. 11, 167±178. Ostensen, M. (1999). Sex hormones and pregnancy in rheumatoid arthritis and systemic lupus erythematosus. Ann. NY Acad. Sci. 876, 131±144. Owerbach, D., Rutter, W. J., Cooke, N. E., Martial, J. A., and Shows, T. B. (1981). The prolactin gene is located on chromosome 6 in humans. Science 212, 815±816. Palermo, R., Albano, C., Mangione, D., and Napoli, P. (1994). Chronic anovulation due to prolactin hypersecretion. Acta Eur. Fertil. 25, 161±172. Paus, R. (1991). Does prolactin play a role in skin biology and pathology? Med. Hypotheses. 36, 33±42. Peers, B., Voz, M. L., Monget, P., Mathy-Hartert, M., Berwaer, M., Belayew, A., and Martial, J. A. (1990). Regu-

latory elements controlling pituitary-specific expression of the human prolactin gene. Mol. Cell. Biol. 10, 4690±4700. Peeters, P., Raynaud, S. D., Cools, J., Wlodarska, I., Grosgeorge, J., Philip, P., Monpoux, F., Van Rompaey, L., Baens, M., Van den Berghe, H., and Marynen, P. (1997). Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9,12) in a lymphoid and t(9,15,12) in a myeloid leukemia. Blood 90, 2535±250. Pellegrini, I., Lebrun, J. J., Ali, S., and Kelly, P. A. (1992). Expression of prolactin and its receptor in human lymphoid cells. Mol. Endocrinol. 6, 1023±1031. Perrot-Applanat, M., Gualillo, O., Pezet, A., Vincent, V., Edery, M., and Kelly, P. A. (1997). Published erratum appears in Mol Endocrinol 1997 Nov, 11(12): 1781 Dominant negative and cooperative effects of mutant forms of prolactin receptor. Mol. Endocrinol. 11, 1020±1032. Pohnke, Y., Kempf, R., and Gellersen, B. (1999). CCAAT/enhancer-binding proteins are mediators in the protein kinase A-dependent activation of the decidual prolactin promoter. J. Biol. Chem. 274, 24808±24818. Reem, G. H., Ray, D. W., and Davis, J. R. (1999). The human prolactin gene upstream promoter is regulated in lymphoid cells by activators of T-cells and by cAMP. J. Mol. Endocrinol. 22, 285±292. Reiter, E., Hennuy, B., Bruyninx, M., Cornet, A., Klug, M., McNamara, M., Closset. J., and Hennen, G. (1999). Effects of pituitary hormones on the prostate. Prostate 38, 159±165. Reynolds, C., Montone, K. T., Powell, C. M., Tomaszewski, J. E., and Clevenger, C. V. (1997). Expression of prolactin and its receptor in human breast carcinoma. Endocrinology 138, 5555±5560. Riddick, D. H., Luciano, A. A., Kusmik, W. F., and Maslar, I. A. (1978). De novo synthesis of prolactin by human decidua. Life Sci. 3, 1913±1921. Riddle, O. (1963). Prolactin in vertebrate function and organization J. Natl Cancer Inst. 31, 1039±1109. Riddle, O., and Braucher, P. F. (1931). Studies on the physiology of reproduction in birds. Control of the special secretion of the crop-gland in pigeons by anterior pituitary hormones. Am. J. Physiol. 97, 617±627. Riddle, O., Bates, R. W., and Dykshorn, S. W. (1932). A new hormone of the anterior pituitary. Proc. Soc. Exp. Biol. Med. 29, 1211±1215. Riddle, O., Bates, R. W., and Dykshorn, S. W. (1933). The preparation, identification and assay of prolactin ± a hormone of the anterior pituitary. Am. J. Physiol. 105, 191±216. Riddle, O., Lahr, E. L., and Bern, H. A. (1935). Maternal behaviour induced in virgin rats by prolactin. Proc. Soc. Exp. Biol. Med. 32, 730±734. Rolland, R., and Hammond, J. M. (1975). Demonstration of a specific receptor for prolactin in porcine granulosa cells. Endocr. Res. Commun. 2, 281±298. Rosen, J. M., Humphreys, R., Krnacik, S., Juo, P., and Raught, B. (1994). The regulation of mammary gland development by hormones, growth factors, and oncogenes. Prog. Clin. Biol. Res. 387, 95±111. Rui, H., Kirken, R. A., and Farrar, W. L. (1994). Activation of receptor-associated tyrosine kinase JAK2 by prolactin. J. Biol. Chem. 269, 5364±5368. Russell, D. H. (1989). New aspects of prolactin and immunity: a lymphocyte-derived prolactin-like product and nuclear protein kinase C activation. Trends Pharmacol. Sci. 10, 40±44. Saandakou, A., Kassanos, D., Phocas, I., Kontoravdis, A., Chryssicopoulos, A., and Zourlas, P. A. (1992). Amniotic fluid hormone profiles during normal and abnormal pregnancy. Clin. Exp. Obstet. Gynecol. 19, 180±188.

Prolactin Sakai, Y., Hiraoka, Y., Ogawa, M., Takeuchi, Y., and Aiso, S. (1999). The prolactin gene is expressed in the mouse kidney. Kidney Int. 55, 833±840. Scapagnini, U., Drago, F., Continella, G., Spadaro, F., Pennisi, G., and Gerendai, I. (1985). In ``Prolactin. Basic and Clinical Correlates'' (ed M. MacLeod, M. O. Thorner, and I. Scapagnini), Experimental and clinical effects of prolactin on behavior, pp. 583±590. Liviana Press, Podova. Shennan, D. B. (1994). Regulation of water and solute transport across mammalian plasma cell membranes by prolactin. J. Dairy Res. 61, 155±166. Shome, B., and Parlow, A. F. (1977). Human pituitary prolactin (hPRL): the entire linear amino acid sequence. Clin. Endocrinol. Metab. 45, 1112±1115. Siegel, R. A., Conforti, N., and Chowers, I. (1980). Neural pathways mediating the prolactin secretory response to acute neurogenic stress in the male rat. Brain. Res. 198, 43±53. Sinha, Y. N. (1995). Structural variants of prolactin: occurrence and physiological significance. Endocr. Rev. 16, 354± 169. Smith, M. S. (1980). Role of prolactin in regulating gonadotropin secretion and gonad function in female rats. Fed. Proc. 39, 2571±2576. Soares, M. J., Muller, H., Orwig, K. E., Peters, T. J., and Dai, G. (1998). The uteroplacental prolactin family and pregnancy. Biol. Reprod. 58, 273±284. Sorenson, R. L., and Brelje, T. C. (1997). Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm. Metab. Res. 29, 301±307. Srivastava, R. K., Gu, Y., Ayloo, S., Zilberstein, M., and Gibori, G. (1998). Developmental expression and regulation of basic fibroblast growth factor and vascular endothelial growth factor in rat decidua and in a decidual cell line. J. Mol. Endocrinol. 21, 355±362. Steger, R. W., Chandrashekar, V., Zhao, W., Bartke, A., and Horseman, N. D. (1998). Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 139, 3691±3695. Stenn, K. S., and Paus, R. (1999). What controls hair follicle cycling? Exp. Dermatol. 8, 229±236. Stewart, E. A., Jain, P., Penglase, M. D., Friedman, A. J., and Nowak, R. A. (1995). The myometrium of postmenopausal women produces prolactin in response to human chorionic gonadotropin and alpha-subunit in vitro. Fertil. Steril. 64, 97297±6. Strain, A. J., and Ingleton, P. M. (1990). Growth hormone- and prolactin-induced release of insulin-like growth factor by isolated rat hepatocytes. Biochem. Soc. Trans. 18, 1206. Stricker, P., and Grueter, R. (1928). Action du lobe anterieur de l'hypophyse sur la montee laiteuse. Compt. Rend. Soc. Biol. (Paris) 99, 1978±1980. Taggart, R. T., Mohandas, T. K., and Bell, G. I. (1987). Assignment of the human preprogastricsin (PGC). to chromosome 6 and regional localization of PGC (6pter-p21.1), prolactin PRL (6pter-p21.1). Cytogenet. Cell Genet. 46, 701±702. Theorell, T. (1992). Prolactin ± a hormone that mirrors passiveness in crisis situations. Integr. Physiol. Behav. Sci. 27, 32±38. Thorner, M. O., Vance, M. L., Laws, E.R. Jr, Horvath, E., and Kovacs, K. (1998). In ``Williams Textbook of Endocrinology, 9th edn'' (ed J. D. Wilson, D. W. Foster, H. M. Kronenberg, and P. R. Larsen), The anterior pituitary, pp. 249±326. W. B. Saunders, Philadelphia. Topper, Y. J., Sankaran, L., Chomczynski, P., Prosser, C., and Qasba, P. (1986). Three stages of responsiveness to hormones in the mammary cell. Ann. NY Acad. Sci. 464, 1±10.

283

Truong, A. T., Duez, C., Belayew, A., Renard, A., Pictet, R., Bell, G. I., and Martial, J. A. (1984). Isolation and characterization of the human prolactin gene. EMBO J. 3, 429±437. Turkington, R. W. (1972). Phenothiazine stimulation test for prolactin reserve: the syndrome of isolated prolactin deficiency. J. Clin. Endocrinol. 34, 247±249. Velkeniers, B., Dogusan, Z., Naessens, F., Hooghe, R., and Hooghe-Peters, E. L. (1998). Prolactin, growth hormone and the immune system in humans. Cell. Mol. Life Sci. 54, 1102±1108. Vonderhaar, B. K. (1998). Prolactin: the forgotten hormone of human breast cancer. Pharmacol. Ther. 79, 169±178. Walker, S. E., Miller, D., Hill, D. L., and Komatireddy, G. R. (1998). Prolactin, a pituitary hormone that modifies immune responses. Proceedings of the Mini-symposium on Prolactin and SLE, held at the 5th International Conference on Systemic Lupus Erythematosus, Cancun, Mexico. Lupus 7, 371±375. Wang, Y. F., and Walker, A. M. (1993). Dephosphorylation of standard prolactin produces a more biologically active molecule: evidence for antagonism between nonphosphorylated and phosphorylated prolactin in the stimulation of Nb2 cell proliferation. Endocrinology 133, 2156±2160. Wang, Y. F., Liu, J. W., Mamidi, M., and Walker, A. M. (1996). Identification of the major site of rat prolactin phosphorylation as serine 177. J. Biol. Chem. 271, 2462±2469. Weigent, D. A. (1996). Immunoregulatory properties of growth hormone and prolactin. Pharmacol. Ther. 69, 237±257. Wennbo, H., Kindblom, J., Isaksson, O. G., and Tornell, J. (1997a). Transgenic mice overexpressing the prolactin gene develop dramatic enlargement of the prostate gland. Endocrinology 138, 4410±4415. Wennbo, H., Gebre-Medhin, M., Gritli-Linde, A., Ohlsson, C., Isaksson, O. G., and Tornell, J. (1997b). Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J. Clin. Invest. 100, 2744±2751. Woody, M. A., Welniak, L. A., Richards, S., Taub, D. D., Tian, Z., Sun, R., Longo, D. L., and Murphy, W. J. (1999). Use of neuroendocrine hormones to promote reconstitution after bone marrow transplantation. Neuroimmunomodulation 6, 69±80. Yu-Lee, L. Y. (1997). Molecular actions of prolactin in the immune system. Proc. Soc. Exp. Biol. Med. 215, 35±52. Yu-Lee, L., Luo, G., Moutoussamy, S., and Finidori, J. (1998). Prolactin and growth hormone signal transduction in lymphohaemopoietic cells. Cell. Mol. Life Sci. 54, 1067±1075. Zhong, L., Parmer, T. G., Robertson, M. C., and Gibori, G. (1997). Prolactin-mediated inhibition of 20alpha-hydroxysteroid dehydrogenase gene expression and the tyrosine kinase system. Biochem. Biophys. Res. Commun. 235, 587±592.

LICENSED PRODUCTS Genzyme, Inc. Product Code: 80-3910-01 Vial size: 50 mg Expression system: C127 mammalian cell line Bioassay: Nb2 (rat T cell line) proliferation assay Cross-reactivity: Active in human, mouse and rat systems Physical state: Lyophilized Shelf life: One year at 2±8 C