Duplication Of A Common Ancestral Gene Biology Essay

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PRL is part of a family of lactogenic hormones that include GH and placental lactogen. Lactogens have similar gene and amino acid sequences, protein structure and biological functions (87). The gene for hPRL is located on chromosome 6, while the genes for hGH and hPL are located on chromosome 17. The three hormones are derived from duplication of a common ancestral gene about 400 million years ago (88). The fish somatolactin gene possesses 24% homology with PRL and GH and may be the ancestral gene for GH and PRL (89). Human PRL shares 40% homology with GH and PL, and homology varies across species. The PRL gene is expressed in all vertebrates and occurs as one copy per haploid genome (90). The sequence similarity is higher within a single class of vertebrates, and diverges between distantly related species. For example, there is 97% homology between PRL in primates and 99% homology between ovine and bovine PRL (88), while human and carp only share 36% homology (91).

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PRL is primarily expressed by the anterior pituitary gland, from which it is secreted into circulation and functions as a hormone. However, in humans it is also produced by extrapituitary sites, including the decidua, myometrium, brain, breast, prostate, lymphocytes, and adipocytes, where it can function as a cytokine (28;92). Notably, PRL synthesis in other species is largely confined to the pituitary gland. The rat PRL gene contains five exons and four introns and is about 10 kb long. However, the human PRL gene contains six exons, including the additional noncoding exon 1a located 5.8 kb upstream of exon 1b, and is approximately 18 kb long (93). The transcription of extrapituitary PRL is regulated differently from that of pituitary PRL, although the mature PRL proteins produced by all sites are identical (94).

2. Proximal PRL Promoter

In the pituitary, transcription begins at exon 1b and is driven by a 2.5 kb proximal promoter (Fig. 4), which consists of a 0.25 kb proximal region, located at +33 and -250 bp relative to the pituitary start site, and a 2.25 kb distal enhancer, located at -1,750 to 1,320 bp (95). Binding of the transcription factor Pit-1 to its consensus sequences in the proximal promoter is required for pituitary PRL transcription. Pit-1 is a POU-homeodomain transcription factor specific to the anterior pituitary that is critical in the development of lactotrophs, somatotrophs and thyrotrophs as well as the regulation of the GH, PRL, GH releasing hormone (GHRH) receptor, the β subunit of thyroid stimulating hormone (TSH), and Pit-1 gene expression (96-98). Pit-1 knockout mice do not develop lactotrophs, somatotrophs, and thyrotrophs, resulting in hypoplasia of the anterior pituitary as well as dwarfism (96). Within the proximal region there are three binding sites for Pit-1 (P1 - P3), while the distal enhancer contains eight binding sites for Pit-1 (D1-6, D9, D10). However, only the D2 and D6 sites contain classical Pit-1 binding sequences. In contrast, the rat PRL promoter contains only four Pit-1 binding sites. Pit-1 can regulate PRL alone or with other transcription factors including ETS-1 and C/EBPα (92). Thus, PRL and GH, which both depend on Pit-1 for transcription, are differently regulated due to these cofactors. While mainly expressed in the pituitary, some recent studies have reported expression of Pit-1 and other POU-domain factors in normal human breast tissue, breast tumors, and MCF-7 breast cancer cells as well as in human placenta (99-101).

Pituitary PRL expression is stimulated by many ligands that cause an increase in cAMP levels, including vasoactive intestinal peptide (VIP), thyrotropin releasing hormone (TRH), and pituitary adenylate cyclase activating peptide (PACAP). However, the human proximal promoter does not contain an exact cAMP response element (CRE) and therefore does not bind the CRE binding (CREB) transcription factor, which is downstream of the cAMP/PKA pathway. Instead, the effect of cAMP is mediated by an asymmetric CRE-like sequence (CLE) located at -99 to -92 in the proximal promoter (102). Dopamine, which decreases intracellular cAMP levels, is the primary physiological inhibitor of PRL gene transcription in the pituitary (103).

In addition to Pit-1, the proximal promoter is regulated by several other transcription factors. Other consensus sequences within the proximal promoter for transcription factors include an activator protein-1 (AP-1) binding site within the proximal region (104). A nonpalidromic estrogen response element (ERE) is located 1.7 to 1.8 kb upstream of exon 1b, within the distal region, and estrogen induces PRL transcription by causing looping of the DNA to bring the enhancer and promoter in close proximity (105). The proximal promoter also contains binding sequences for calcium and TRH.

Superdistal PRL Promoter

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The existence of an alternative promoter for PRL, the superdistal promoter, was discovered in decidual cells. The superdistal promoter is about 3.0 kb in length and controls PRL transcription from extrapituitary sites, including the decidua, endometrium, breast, prostate, lymphocytes, and adipose tissue. In non-pituitary cells, exon 1a, which is located 5.8 kb upstream of the pituitary start site, serves as the transcriptional start site for PRL (Fig. 4). Following transcription, exon 1a is spliced into exon 1b, giving extrapituitary PRL an identical coding region but with a longer 5' untranslated region (UTR) than pituitary PRL (106). However, the PRL protein produced in the pituitary and extrapituitary sites is identical (92).

There are many predicted binding sites for transcription factors in the superdistal promoter, including two AP-1 sites, one CREB site, two hepatocyte nuclear factor-1 (HNF-1) sites, an imperfect ERE, eight C/EBP sites and an ETS site (Fig. 5) (94;107). CREB has a binding site located at the -12 position of the PRL superdistal promoter, and C/EBP has a binds between -332 and -270 (108). In addition, cAMP-response element modulator (CREM) can bind to CRE sites either as homodimers with itself or as heterodimers with CREB (109). MAPK and estrogen signaling can activate AP-1, which binds to a site located in the enhancer region of the superdistal promoter (107). The regulation of the superdistal promoter can vary markedly between different types of cells and tissue (108).

The expression of PRL in non-pituitary sites is independent of Pit-1 (110). While there are two Pit-1 consensus sequences, located at -2186 and -2800 bp within the superdistal promoter, Pit-1 has not been shown to activate the superdistal promoter. Pit-1 expression was originally thought to be limited to the pituitary, but some recent studies have shown Pit-1 expression in normal and cancerous human breast tissue and cells as well as in the placenta (101). However, the PRL promoter utilized for PRL transcription in these cells tissues has not been determined. Recent studies suggest that some breast cancer cell-lines can utilize both the proximal and superdistal promoters (111).

Protein Structure

The PRL protein was first purified from the anterior pituitary in 1933 by Dr. Riddle as a factor that induced mammary gland development and lactation in rabbits and stimulated crop milk production in pigeons, and PRL was named for its role in lactation (112). PRL currently has over 300 identified functions, and has been found in a variety of cell and tissue types in all vertebrates. In most mammals, PRL is a 23-kDa protein consisting of a single polypeptide chain of 197-199 amino acid residues, with variable sequence homology between species (90). Mature PRL is formed by proteolytic cleavage of the PRL precursor molecule, which is 227 amino acids long, to remove a 28 residue signal peptide from the N-terminus end (113). PRL is arranged into four anti-parallel α-helices with up-up-down-down topology, as confirmed by NMR spectroscopy (Fig. 6) (114;115). This helix-bundle motif is similar to the structures of interferon, several interleukins and GH. The hPRL protein has three highly conserved disulfide bonds between the cysteine residues 4-11, 58-174, and 191-199, which stabilize the protein's conformation (87).

Variants

In humans, PRL has multiple structural variants other than 23 kDa PRL, including big PRL (40-60 kDa), macroprolactin (100 kDa), and 16K PRL (16 kDa). Macroprolactin, also called big-big PRL or oligomeric PRL, is a polymeric complex that is formed by monomeric PRL binding to IgG. Since macroprolactin is detected in immunologically based assays and has a long half-life, patients with high macroprolactin levels are often diagnosed with hyperprolactinemia. However, macroprolactin can be separated from lower molecular weight forms of PRL by polyethylene glycol precipitation (116). This is clinically useful since macroprolactin has little or no biological activity in vivo, and hyperprolactinemia due to macroprolactinemia is usually idiopathic. Macroprolactin is too large to easily diffuse across capillary walls, which may account for its low biological activity (117).

Several variants of PRL, including the N-terminal 14, 16, and 22 kDa isoforms, are the result of proteolytic cleavage of 23 kDa PRL. Unlike normal PRL, 16 kDa PRL (16K PRL) is reported to have anti-angiogenic activity. While the receptor for 16K PRL has yet to be identified, its anti-angiogenic actions appear to be mediated by inhibiting proliferation and inducing apoptosis in endothelial cells, thus reducing tumor growth (118). In addition, 16K PRL also decreases cell-to-cell and cell-to-matrix interactions as well as the degradation of the extracellular matrix, which all combine to inhibit angiogenesis (119). In the B16-F10 mouse model of melanoma, 16K PRL reduces tumor growth and tumor blood vessel size as well as metastasis to the lung (120). 16K PRL also decreases growth of HCT116 human colon cancer cells and microvascular density in a xenograft mouse model (121). 16K PRL retains much of the biological activity of normal PRL, such as stimulating the proliferation of Nb2 cells. However, 16K PRL only has approximately 65% and 10% of the mitogenic and lactogenic activities of 23 kDa PRL, respectively (122).

2. Posttranslational Modifications

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PRL is a heterogeneous protein that is subjected to multiple post-translational modifications such as proteolytic cleavage, polymerization, glycosylation, and phosphorylation (113). PRL is glycosylated in many species, including mammalian, reptile, and avian, and the amount of glycosylation varies from 1% to 60% between species and reproductive state (115). In mammals, the consensus sequence for attachment of the carbohydrate moiety to PRL is at residue 31 through nitrogen (N-linked). Glycosylation has multiple effects on PRL, such as reducing biological activity, receptor binding affinity and immunological reactivity as well as increasing the metabolic clearance rate (123). In lactotrophs, glycosylated PRL is secreted constitutively instead of stored in secretory vescicles (124).

Phosphorylation of PRL can occur at multiple sites, and phosphorylated PRL has been found in bovine and murine pituitary glands. As with glycosylation, the rate of phosphorylation of PRL varies with reproductive state. While phosphorylated PRL isoforms are secreted in vitro, it is unknown whether phospho-PRL is secreted into circulation in vivo (115). Phosphorylation reduces the biological activity of PRL, and phospho-PRL inhibits the secretion of nonphosphorylated isoforms from GH3 cells (125). In addition, phosphorylated PRL antagonizes the signaling and mitogenic actions of normal PRL in the Nb2 bioassay (126). Recent in vitro studies have also shown that S179D PRL, a model of phospho-PRL, is anti-angiogenic and decreases cell proliferation, invasion and migration (127). S179D PRL also inhibits proliferation of MCF-7 breast cancer cells and reduces tumor incidence and size in a DU145 prostate cancer cell xenograft mouse model (128;129). These studies suggest that the synthetic S179D PRL may be useful in cancer therapy.

Regulation of PRL

PRL in humans is produced both by the pituitary and by multiple extrapituitary sites, and PRL transcription is controlled by two distinct promoters. Initial studies with endometrial cells and lymphocytes revealed cell type-specific regulation of PRL expression and release. However, the regulation of extrapituitary PRL has not been fully characterized. More recently, our laboratory has found PRL synthesis in adipose tissue. As more metabolic functions of PRL are discovered, elucidating the regulation of PRL in adipose tissue becomes increasingly important to our understanding of the metabolic syndrome.

1. Pituitary Lactotrophs

The human pituitary is located behind the sinus cavity at the base of the brain, directly below the hypothalamus. The pituitary is divided into two lobes, the anterior and posterior lobes. In adult humans, these two lobes are separated by a thin layer of cells called the intermediate lobe, which is only well-defined in embryonic development. The intermediate lobe produces peptide products of the pro-opiomelancortin (POMC) gene, including melanocyte-stimulating hormone (MSH) and β-endorphin. The posterior lobe stores and secretes vasopressin and oxytocin, which are produced in the hypothalamus. The five major cell-types of the anterior lobe are lactotrophs, somatotrophs, thyrotrophs, corticotrophs, and gonadotrophs, which synthesize and release the hormones PRL, GH, TSH, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH)/ follicle stimulating hormone (FSH), respectively (88). Lactotrophs comprise 10-20% of anterior pituitary cells, while about 50% of the cells are somatotrophs (130).

Dopamine has been identified as the primary physiological inhibitor of PRL gene expression and release in lactotrophs, which will be discussed in detail in a later chapter (103). In the presence of estrogen, dopamine also induces the apoptosis of lactotrophs (131). Estrogen binds to estrogen receptor-α (ERα) to mediate PRL expression and release in lactotrophs. This activation of PRL expression is enhanced by interaction with Pit-1 (132). In addition, pituitary PRL production is stimulated by insulin, glucocorticoids, epidermal growth factor (EGF) and activators of cAMP, including VIP, TRH, and PACAP. TNF-α stimulates PRL secretion, while IL-1 inhibits TRF-stimulated PRL release from rat anterior pituitary cells. A PRL releasing factor (PRF) has been suspected for a long time but its identity remains elusive (103).

2. Decidual and Myometrial Cells

The decidua develops from the uterine lining, or endometrium, during the late luteal phase of the menstrual cycle in preparation for pregnancy. Eventually, the decidua forms the border between the fetal chorion and amnion during pregnancy. While the PRL protein produced by decidual cells is identical to pituitary PRL, transcription of decidual PRL is controlled by an alternative, superdistal promoter, which is responsible for the tissue-specific regulation of PRL expression. During pregnancy, PRL gene expression steadily increases due to high progesterone levels, and PRL is a marker of decidualization (133). Activation of the cAMP/PKA pathway is known to induce the superdistal PRL promoter in human decidual cells and lymphocytes through CREB and C/EBP (108). Annexin-1 is reported to suppress PRL transcription and subsequent release in decidual tissue by inhibiting the cAMP pathway at a point after cAMP activation (134). Insulin and insulin-like growth factor-1 (IGF-1) stimulate PRL synthesis and release from decidual cells (135;136). Many inflammatory cytokines, including TNF-α, IL-1α, IL-1β, IL-2, IL-8, and transforming growth factor-β (TGF-β), inhibit the gene expression and release of decidual PRL (137;138).

In the non-pregnant uterus, the myometrium forms the middle layer of the uterine lining. In contrast to the decidua, progesterone inhibits PRL production in the myometrium. Endothelin-3 stimulates, while EGF, VIP, and interferon-α (IFN-α) inhibit, PRL release from the myometrium. In addition, IL-4 decreases both PRL mRNA levels and release from myometrial tissue, however, IL-6 has no effect on PRL release (139).

3. Lymphocytes

The endocrine, nervous, and immune systems interact through the actions of catecholamines, hormones and cytokines. Immune cells produce both PRL and GH and express the PRLR, and have recently been found to express dopamine receptors and the dopamine transporter (DAT) (140). Many inflammatory cytokines are also produced by endocrine organs and nervous tissue. The inflammatory state observed in obesity appears to be associated with cardiovascular disease and thus is of great concern with rising obesity rates (21). Given the expression of PRL and its receptor in immune cells, preadipocytes and adipocytes and that PRL can function as an autocrine/paracrine factor, PRL is likely involved in the communication between the immune cells and preadipocytes/ adipocytes within adipose tissue. As discussed in later sections, PRL is also associated with the pathogenesis of multiple autoimmune diseases, in which the cells of the immune system, normally responsible for protecting the body from invasion by infectious diseases and foreign bodies, becomes deregulated and starts to attack the body's own cells (141).

The regulation and functions of PRL in immune cells have been most extensively studied in lymphocytes. The many types of cells that make up the immune system develop from progenitor cells in the bone marrow and thymus. Lymphocytes mature and differentiate in several tissues and organs, including the spleen, lymph nodes, thymus, and bone marrow. Lymphocytes can be further classified as T-cells, B-cells, and natural killer cells (92). PRL is an immunomodulator that induces the release of several cytokines, including IL-2 and IFNγ, in lymphocytes (142). In addition, PRL reduces IL-6 secretion from neighboring preadipocytes (28). In lymphocytes, PRL is regulated by multiple inflammatory cytokines. Several interleukins, including IL-2, IL-4, and IL-1β, decrease PRL mRNA levels from T-lymphocytes (143). Bromocriptine treatment in both in vitro and in vivo experiments in rats demonstrated that PRL affects the immunocompetence of lymphocytes by competing with the immunosuppressant cyclosporine for binding on T lymphocytes (144).

4. Adipocytes

Our laboratory recently discovered that the majority of local PRL production in the breast occurs in adipose rather than glandular tissue (145). Furthermore, we found that the main source of PRL in breast adipose tissue is mature adipocytes, and that both the subcutaneous and visceral adipose depots also produce PRL (28). In addition, two human adipocyte cell-lines, LS14 and SW872, express and release PRL (28;146). Many in vitro studies on adipocytes utilize rodent adipocyte cell-lines. However, despite expression of the PRLR, PRL is not produced in rodent adipocytes. There are also differences in the effect of PRL between species, as PRL treatment does not inhibit lipolysis in mouse adipose tissue as it does in human adipose tissue. Thus, rodent primary adipocytes and adipocyte cell-lines are not suitable models for the study of PRL in human adipocytes (147).

Our laboratory also found that PRL release from primary human breast preadipocytes is stimulated by activators of cAMP, such as IBMX, isoproterenol, and PACAP, as in the decidua. Primary breast preadipocytes contain two positive regulatory domains in the PRL superdistal promoter, one of which overlaps with the CREB and C/EBP binding regions, and an inhibitory region between -317 and -1556 (148). While PRL has many metabolic actions, the regulation of PRL gene expression and release has not been studied in other adipose depots.

PRL release from adipose explants begins to rise only after three days in culture (145). A delayed rise in PRL has been observed in both the pituitary lactotrophs as well as other PRL-producing cells, including dermal fibroblasts, decidual and myometrial cells (103;149-151). This suggests that PRL expression and/or release in vivo is suppressed by tissue-specific inhibitory factor(s), and that the rise in PRL release in culture may be the result of removal from tonic inhibition (145). However, the endogenous inhibitor(s) of PRL production in adipocytes and adipose tissue has not yet been identified.

Receptors

1. Protein Structure and Variants

The PRLR is a member of the class 1 cytokine receptor superfamily, which includes the receptors for GH, several interleukins, and leptin (90). The PRLR gene is located on chromosome 5, and the gene contains at least 10 exons and is over 100 kb in length (152). The PRLR is a single-pass transmembrane protein with an extracellular domain (ECD), an intracellular domain (ICD), and a 24 amino acid transmembrane domain (153). The ICD couples to various signaling molecules to mediate the effects of its ligand, while the ECD dictates ligand binding specificity (154). The ECD is approximately 200 amino acids long and is divided into two fibronectin-like domains (S1 and S2). The S1 domain is where most of the PRL binding sites are located, and it contains two pairs of disulfide-linked cysteine residues and two glycosylation sites. In the S2 domain there is a conserved WS motif (Trp-Ser-X-Trp-Ser) that is required for folding and dimerization of PRLR (Fig. 7).

In humans, alternative splicing of the PRL-R transcript generates multiple isoforms, including the long isoform, intermediate isoform, two short isoforms and a soluble isoform that functions as a binding protein (155-157). The length of the ICD differs between PRLR isoforms, while the ECD is the same in all isoforms. All isoforms contain the conserved region called Box 1, a hydrophobic motif (Pro-X-Pro) that has the typical folding of Src-homology domain 3 (SH3)-binding domains and is required for the binding of Jak2 and the resulting activation of signaling. Another conserved region, Box 2, consists of a sequence of hydrophobic, then negatively charged, then positively charged residues. However, the short isoforms do not contain Box 2 because of its distance from the membrane (aa 288-298), thus, Jak2 is not phosphorylated by the short PRLR isoforms (90).

Activation and Signal Transduction

The PRL protein contains two sites, one consisting of helices 1 and 4 and the other consisting of 1 and 3, which are necessary for homodimerization of the receptor and the formation of a 1:2 complex between PRL and two PRLRs, thus activating the receptors (Fig. 8). The first site binds to a PRLR to form a dimer, then another PRLR is recruited to complete the active trimeric complex. However, the trimeric complex is unstable and quickly dissociates back into a dimeric complex of PRL and one PRLR (158). Some reports suggest that the short PRLR, which does not bind Jak2, acts as a dominant-negative by hetero-dimerizing with the long PRLR (159). In addition to PRL, the PRLR can be activated by GH and PL (160).

After activation by its ligands, the PRLR signals through multiple pathways. The Jak-Stat pathway is the best recognized signaling pathway for PRL and its receptor (Fig. 7). The PRLR lacks intrinsic tyrosine kinase activity, however, the activated PRLR causes phosphorylation of itself as well as various intracellular proteins through Jak family of kinases (161). Jak2 proteins are associated with the PRLR constitutively (162). Within one minute of PRL binding to its receptor, the dimerization of the receptors induces Jak2 transphosphorylation by bringing two Jak2 proteins in close proximity to one another (163). Jak2 phosphorylates specific tyrosine residues on Stat5a and Stat5b. Stat1 and Stat3 can also be phosphorylated by Jak2. The structure of Stats include an SH3 domain, a Src-homology domain 2 (SH2) binding domain, and a DNA binding domain, all of which are conserved (164). Jak2 also phosphorylates tyrosine residues on the PRLR. The activated Stats hetero- or homo-dimerize and are subsequently translocated into the nucleus, where they act as transcription factors for various genes (165). Stats bind to gamma-interferon-activated sequences (GAS), or Stat consensus sequences, located in the promoters of target genes (166).

In different cells, PRL can also signal through other intracellular pathways. The Ras/Raf/MAPK pathway is linked to the phosphotyrosine residues of the activated PRLR through the adapter proteins, Shc, Grb, and SOS (167). The PRLR have also been shown to mediate the activation of Src and Fyn, which are part of the Src family of kinases. PRL and its receptor can also signal through the IRS/PI3K/Akt pathways. Finally, the Jak2/Stat pathway is inhibited by several classes of proteins, including the SH2-containing protein tyrosine phosphotases (SHPs), protein inhibitors of Stats (PIAS), and SOCS (90).

Receptor Distribution and Regulation

The PRLR is expressed in a broad range of cell and tissue types, including the pituitary, CNS, lung, kidney, adrenals, spleen, heart, thymus, GI tract, and skin. The PRLR is also found in reproductive organs such as the breast, ovaries, uterus, and prostate (115;168). The relative levels of the different PRLR isoforms depend on the tissue type and reproductive state. PRLR expression is regulated by several hormones and thus displays sexual dimorphism. The long PRLR is the major isoform expressed the majority of tissue types, including the mammary gland, adrenals, kidney, and GI tract (90). PRLR expression is elevated during proestrus in the ovary and uterus (169). In mammary gland epithelial tissue, PRLR expression increases during pregnancy, spikes following birth, and then declines after weaning. However, PRLR expression does not change in the stromal tissue of the breast during pregnancy or lactation (170). The rise in PRLR levels during pregnancy may be due to elevated levels of PRL in serum since PRL can regulate its own receptor. However, the effects of PRL during pregnancy are counter-acted by increased circulating levels of progesterone, which inhibits PRLR expression. After parturition, progesterone levels fall while PRL levels remain high, which may explain the sharp elevation in PRLR expression (171). In contrast, progesterone stimulates PRLR expression in human endometrial tissue. Estrogen stimulates PRLR expression in kidney, liver, endometrium, and breast cancer cells (172), while glucocorticoids increase PRLR expression in both the liver and mammary glands (173).

Suggestive of PRL's involvement in cancer progression, expression of the PRLR is detected in multiple prostate, breast cancer, cervical, and GI cancer cell-lines, with expression highest in T47D breast cancer cells (174;175). In addition, overexpression of PRL in breast cancer cells increases PRLR expression and stimulates tumor growth when cells were injected into nude mice (176). Finally, the PRLR is produced in organs important to metabolism including the brain, liver, and pancreas. Unlike in other tissues, the short PRLR isoform is the predominant form expressed in the liver (90). Our laboratory also recently detected PRLR expression in white adipose tissue, primary adipocytes and adipocyte cell-lines (28;145). We found that PRL inhibits lipolysis, leptin release and IL-6 production in adipocytes, and other groups have found PRL involvement in the regulation of other adipokines (28;177). While many of the metabolic roles of PRL and its receptor have been elucidated, the functions of PRL and PRLR in adipose tissue are still under investigation.

Physiological Functions of PRL

PRL functions as both a circulating hormone of pituitary origin and as a locally-produced cytokine (92). In addition to being a circulating hormone, PRL is also defined as a cytokine, based on its multiple sites of production, ubiquitous receptor distribution and signaling through the Jak-Stat and MAP-kinase pathways (90). PRL has over 300 reported functions, more than any other pituitary hormone (92). Named for its role in lactation, PRL is involved in mammary gland development and lactation, osmoregulation, immune system regulation, and behavioral alterations. Under different physiological conditions, PRL stimulates cell proliferation and differentiation, increases cell survival, or acts as a secretagogue (92).

Mammary Gland Development and Lactation

One of the primary roles of PRL is the regulation of mammary gland development. The stages of development include organogenesis, lobuloalveolar differentiation, lactation, galactopoiesis, and involution. During organogenesis, or mammogenesis, PRL influences the development of the mammary glands from the terminal end buds of the primary ductal system into the fully mature non-pregnant mammary gland. At this stage, the mammary gland is a branched system of alveolar buds and lacks terminal end buds. During pregnancy, PRL, progesterone, and placental lactogen induce lobular buds to expand and differentiate into the mature lobuloalveolar system. After parturition, progesterone levels plummet and PRL induces milk protein gene expression and lactogenesis, or the initiation of milk production. PRL is also involved in galactopoiesis, or the maintenance of lactation. With each pregnancy, the mammary gland matures and undergoes functional changes. However, this process is reversible. After weaning, PRL levels drop and involution returns the mammary gland to the non-lactating condition (178).

Rodents have been the main model system for studying the involvement of PRL in mammary gland development. While PRLR knockout mice (PRLR-/-) initially have normal development of their mammary glands, after puberty there is minimal ductal arborization and terminal end buds do not differentiate into alveolar buds. However, when the mammary glands of PRLR-/- mice were transplanted into wild-type mice, the transplants matured normally during puberty. This suggests that PRL has indirect effects on the epithelial cells that are mediated through other tissues. Later, these transplanted mammary epithelial cells from PRLR-/- mice have typical side branching alveolar bud formation during pregnancy, but lack lobuloalveolar differentiation. Thus, PRL acts indirectly on the ductal system during mammogenesis, but during pregnancy directly controls lobuloalveolar development (179). Similar to the PRLR-/- mice, PRL knockout mice (PRL-/-) only exhibit mild ductal branching and lack lobular budding in virgin animals. Exogenous PRL therapy was able to cause a partial recovery of this stage of mammary maturation. Unlike in the mammary glands of PRLR-/- mice, development of the mammary glands in pregnant PRL-/- mice treated with progesterone is complete (180). Observations in humans also indicate the involvement of PRL in mammary development. Women with hyperprolactinemia can have galactorrhea, or improper lactation unrelated to childbirth (181), while in the rare cases of isolated PRL deficiency, (i.e. low or undetectable PRL levels in serum), women can reproduce but do not lactate (182-184).

In lactation, PRL stimulates milk production by increasing the expression of β-casein, whey acidic protein (WAP), and α-lactalbumin, and PRL is required for the production of milk (185). Unfortunately, studies on lactation are limited by the absence of an in vitro model of fully functional, lactating mammary glands as well as by the complexity of mammary gland development and lactation. In addition, the overlapping functions of PRL, GH, and PL make it difficult to determine the actions of each individual hormone (186). During lactation, lipid production is decreased in adipose tissue and increases rapidly in the mammary gland. This diversion of resources provides the extra energy to the mammary gland that it needs for the process of lactation. PRL stimulates the production of lipids in the mammary gland by increasing LPL, pyruvate dehydrogenase, ACC, and FAS. In humans, lactation commences two days after parturition. The suckling response, in which suckling induces PRL production and subsequent milk production, is also a well studied phenomenon.

Metabolic Functions

The role of PRL in lipid metabolism in the lactating mammary gland is well recognized. However, PRL has multiple metabolic roles outside of the mammary gland. PRL and its receptor are expressed in adipose tissue, preadipocytes, and mature adipocytes. In humans, increased weight is seen with sustained elevated PRL levels, or hyperprolactinemia. Subsequent reductions in PRL levels by treatment with bromocriptine are accompanied by reduction in BMI (187). Weight gain is also a side effect of many antipsychotic drugs that increase PRL production (188). In addition, the rise in PRL during pregnancy corresponds with increasing weight. Thus, the role of PRL in obesity warrants further investigation (189).

The PRLR is expressed in pancreatic islets during fetal development through adulthood. Islets contain β-cells, which produce insulin in response to dietary intake as well as hormonal signals. Treatment with exogenous PRL stimulates β-cell proliferation and production of insulin in the pancreas. PRLR-/- mice have decreased pancreatic islet size, density, β-cell mass, and insulin content (190). In β-cells, PRL also stimulates the glucose sensors glucokinase and GLUT2, which decreases the glucose requirement for induction of insulin release (191).

PRL is involved in a number of metabolic processes in adipocytes and adipose tissue, including regulation of adipogenesis, adipokine production and lipid metabolism. In mouse 3T3-L1 cells, PRL stimulates differentiation into mature adipocytes (192). PRL upregulates expression of the transcription factors C/EBPβ and PPARγ, which are important in adipogenesis (193). During lactation, PRL decreases the activities of LPL, FAS, and ACC, thus reducing lipogenesis in adipose tissue. PRL also inhibits lipolysis in rodent adipocytes (177). The production of adiponectin is suppressed by PRL both in human adipose tissue and in mice (194). PRL has also been reported to regulate leptin, but exact role of PRL in leptin regulation is controversial (195).

Other Functions

The function of PRL in reproduction in rodents is well recognized. PRL surges are required for maintenance of the corpus luteum during the initial 10-12 days of pregnancy (196). In contrast, PRL has not been found to have a direct role in reproduction outside of lactation in humans. However, hyperprolactinemia decreases fertility and libido in both men and women, indicating that PRL may have more moderate or indirect functions in human reproduction (181).

As described earlier, PRL is also an important regulator of the immune system. Lymphocytes produce PRL and the PRLR. In addition, immune cells are exposed to PRL in circulation as well as adipose-produced PRL acting as cytokine in an autocrine/paracrine factor. PRL induces proliferation of lymphocytes and acts as a survival factor by increasing expression of anti-apoptotic genes. In addition, PRL can stimulate the differentiation of immune cells (197). Finally, PRL is associated with many autoimmune disorders, as discussed in the next section, and contributes to the suppression of the immune system during pregnancy (198).

PRL-related Pathologies

In normal adults, PRL levels in circulation are typically below 25 ng/ml, but during pregnancy PRL concentrations can increase up to 10-fold higher than in non-pregnant women. Excessive PRL production, or hyperprolactinemia, is usually caused by a pituitary adenoma called a prolactinoma. Other conditions that may result in elevated PRL levels include hypothyroidism, cirrhosis, renal failure, chest wall injury and certain medications. Elevated PRL can be found in about 10% of the general population. However, approximately 10-40% of hyperprolactinemia cases appear to be idiopathic, with no apparent clinical symptoms. These patients may have high levels of macroprolactin, a large PRL polymer, which is not biologically active in vivo (181). In addition, a study involving MRI scans of healthy adults estimated that approximately 10% of the normal adult population have asymptomatic or non-secreting pituitary adenomas (199), while pituitary tumors have been found in up to 20% of adults in random autopsies (200). While most pituitary adenomas are sporadic, mutations in the MEN1, CDKN1B, PRKAR1A and AIP genes are associated with familial pituitary adenoma syndromes (201).

Prolactinomas tend to be slow growing, benign tumors that originate from a single cell. The most common type of pituitary adenoma, prolactinomas account for approximately one-third of all pituitary tumors (187). Characteristics of hyperprolactinemia include infertility, amenorrhea, galactorrhea, and increased weight in women and impotence, infertility and gynecomastia in men. Hypopituitarism, headaches, and visual problems may also result from the intracranial mass of large tumors. Standard first-line treatment is normally with the dopamine type-2 receptor (D2R) agonists bromocriptine or cabergoline. These medications reduce PRL secretion and tumor size in most patients, but require long-term administration and can have side-effects. However, in 10-20% of cases, prolactinomas fail to respond to drug therapy and adenomas are removed by surgery (181). Indicative of the role of PRL in metabolism, normalization of prolactin levels by treatment with bromocriptine or cabergoline is also associated with weight loss and a reduction in serum insulin and inflammatory cytokines (202;203).

There is considerable evidence suggesting that PRL is involved in the progression of several types of cancer, including breast, prostate, ovarian, and colorectal cancers. PRL stimulates proliferation and acts as a survival factor in breast cancer cells. Transgenic mice that overexpress the PRL gene have increased rates of mammary tumors and prostate hyperplasia (204;205). In humans, PRL levels are correlated with breast cancer risk in both premenopausal and postmenopausal women (206). In addition, patients with hyperprolactinemia have poorer prognosis in metastatic breast cancer and are resistant to chemotherapy treatment, while co-treatment with cabergoline improves response to chemotherapy (207). Knockdown of the PRLR decreases proliferation in T47D breast cancer cells, while growth of tumors injected into nude mice is inhibited by PRLR shRNA or by the PRL antagonist G129R (208;209). Our laboratory recently reported that PRL reverses the cytotoxic effects of several chemotherapeutic agents in multiple breast cancer cell-lines (210). In ovarian carcinoma cells, apoptosis induced by the chemotherapy drug cisplatin was blocked by treatment with PRL (211). PRL increases proliferation and survival of prostate cancer cells, while cell viability is decreased by the PRLR antagonist, Delta1-9G129R-hPRL, indicating that endogenously produced PRL increases proliferation and/or acts as an anti-apoptotic factor in these cells (212-214). Finally, higher PRL levels are associated with later stage of disease and poorer prognosis in colorectal cancer (215).

Autoimmune diseases are more prevalent in women, which is likely due to sexual dimorphism in expression of hormones such as estrogen and PRL (216). PRL activates the immune system, both through its proliferative and anti-apoptotic effects as well as its stimulation of immunoglobulin and autoantibody production. Hyperprolactinemia is associated with multi-organ and organ specific autoimmune diseases including multiple sclerosis (MS), rheumatoid arthritis (RA), type-1 diabetes mellitus, Sjogren's syndrome, Hashimoto's thyroiditis, celiac disease, and systemic lupus erythematosus (SLE) (141;217-219). The most studied is role of PRL in SLE. Elevated PRL levels in NZB/NZW mice, a model of SLE, accelerated SLE disease activity and mortality (220;221). However, in human patients the relationship between PRL and disease progression is not as clear, which may be explained by the increased prevalence of the biologically inactive macroprolactin in SLE patients with hyperprolactinemia. Patients with macroprolactinemia tend to have had a shorter duration of illness and less disease activity, while patients with high levels of free 23 kDa PRL have more symptoms of active SLE (222;223).