Expression and therapeutic targeting of dopamine receptor-1 (D1R) in breast cancer.

Patients with advanced breast cancer often fail to respond to treatment, creating a need to develop novel biomarkers and effective therapeutics. Dopamine (DA) is a catecholamine that binds to five G protein-coupled receptors. We discovered expression of DA type-1 receptors (D1Rs) in breast cancer, thereby identifying these receptors as novel therapeutic targets in this disease. Strong to moderate immunoreactive D1R expression was found in 30% of 751 primary breast carcinomas, and was associated with larger tumors, higher tumor grades, node metastasis and shorter patient survival. DA and D1R agonists, signaling through the cGMP/protein kinase G (PKG) pathway, suppressed cell viability, inhibited invasion and induced apoptosis in multiple breast cancer cell lines. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed tumor growth in two mouse models with D1R-expressing xenografts by increasing both necrosis and apoptosis. D1R-expressing primary tumors and metastases in mice were detected by fluorescence imaging. In conclusion, D1R overexpression is associated with advanced breast cancer and poor prognosis. Activation of the D1R/cGMP/PKG pathway induces apoptosis in vitro and causes tumor shrinkage in vivo. Fenoldopam, which is FDA (Food and Drug Administration) approved to treat renal hypertension, could be repurposed as a novel therapeutic agent for patients with D1R-expressing tumors.

Adipocyte prolactin: regulation of release and putative functions.

Pituitary-derived prolactin (PRL) is a well-known regulator of the lactating mammary gland. However, the recent discovery that human adipose tissue produces PRL as well as expresses the PRL receptor (PRLR) highlights a previously unappreciated action of PRL as a cytokine involved in adipose tissue function. Biologically active PRL is secreted by all adipose tissue depots examined: breast, visceral and subcutaneous. The expression of adipose PRL is regulated by a non-pituitary, alternative superdistal promoter. PRL expression and release increases during early pre-adipocyte differentiation and is stimulated by cyclic AMP activators, including beta adrenergic receptor agonists. PRL release from subcutaneous adipose explants is attenuated during obesity, suggesting that adipose PRL production is altered by the metabolic state. Several lines of evidence indicate that PRL suppresses lipid storage as well as the release of adipokines such as adiponectin, interleukin-6 and possibly leptin. PRL has also been implicated in the regulation of adipogenesis. A newly developed PRL-secreting human adipocyte cell line, LS14, should allow comprehensive examination of the regulation and function of adipocyte-derived PRL. Collectively, these studies raise the prospect that PRL affects energy homeostasis through its action as an adipokine and is involved in the manifestation of insulin resistance.

Distribution and characterization of plasmalemma vesicle protein-1 in rat endocrine glands.

Plasmalemma vesicle protein-1 (PV-1) is an integral membrane protein associated with endothelial cell caveolae and fenestrae. Since endocrine glands are enriched with fenestrated endothelium, we examined the distribution of PV-1 mRNA and protein in endocrine glands and determined its cellular localization. A single transcript was detected by RT-PCR in all endocrine glands examined. A synthetic peptide was used to generate antibodies for Western blotting and immunohistochemistry (IHC). Western blotting of membrane fractions from lung, pituitary, adrenal, testis and PV-1-transfected Cos-1 cells revealed a major 65 kDa protein. This protein binds to heparin with high affinity. Using IHC, PV-1 was localized to both endothelial cells of the adrenal zona reticularis and chromaffin cells of the medulla. In the pancreas, PV-1 expression was restricted to a few cells in the islets of Langerhans that partially overlap with somatostatin-positive delta-cells. In both neonatal and adult pituitaries, strong PV-1 immunoreactivity was detected in neural lobe pituicytes in a pattern similar to that of glial fibrillary acidic protein (GFAP). PV-1 and GFAP expression was seen in the adult, but not neonatal, intermediate lobe. Endothelial cells throughout the neonatal anterior lobe were PV-1 positive, but PV-1 in the adult was restricted to some endothelial and endocrine cells localized near the margins of lobe. In the adult testis, strong PV-1 expression was seen in germ cells within the seminiferous tubules that varied with the stage of spermatogenesis. In contrast, PV-1 in the neonatal testis was localized to the interstitial cells but not seminiferous tubules. In the ovary, PV-1 was expressed in stromal endothelial cells as well as the thecal layer of developing follicles. Over half the corpus luteal cells were positive for PV-1. Our data have shown that PV-1 is not restricted to endothelial cells but is localized in many types of endocrine and non-endocrine cells. Furthermore, PV-1 expression in the pituitary and testis is developmentally regulated.

Dopamine as a prolactin (PRL) inhibitor.

Dopamine is a small and relatively simple molecule that fulfills diverse functions. Within the brain, it acts as a classical neurotransmitter whose attenuation or overactivity can result in disorders such as Parkinson's disease and schizophrenia. Major advances in the cloning and characterization of biosynthetic enzymes, transporters, and receptors have increased our knowledge regarding the metabolism, release, reuptake, and mechanism of action of dopamine. Dopamine reaches the pituitary via hypophysial portal blood from several hypothalamic nerve tracts that are regulated by PRL itself, estrogens, and several neuropeptides and neurotransmitters. Dopamine binds to type-2 dopamine receptors that are functionally linked to membrane channels and G proteins and suppresses the high intrinsic secretory activity of the pituitary lactotrophs. In addition to inhibiting PRL release by controlling calcium fluxes, dopamine activates several interacting intracellular signaling pathways and suppresses PRL gene expression and lactotroph proliferation. Thus, PRL homeostasis should be viewed in the context of a fine balance between the action of dopamine as an inhibitor and the many hypothalamic, systemic, and local factors acting as stimulators, none of which has yet emerged as a primary PRL releasing factor. The generation of transgenic animals with overexpressed or mutated genes expanded our understanding of dopamine-PRL interactions and the physiological consequences of their perturbations. PRL release in humans, which differs in many respects from that in laboratory animals, is affected by several drugs used in clinical practice. Hyperprolactinemia is a major neuroendocrine-related cause of reproductive disturbances in both men and women. The treatment of hyperprolactinemia has greatly benefited from the generation of progressively more effective and selective dopaminergic drugs.

Exposure of newborn male and female rats to environmental estrogens: delayed and sustained hyperprolactinemia and alterations in estrogen receptor expression.

Environmental estrogens (xenoestrogens) are synthetic compounds that are abundant in the environment and mimic natural estrogens. The estrogenicity of two such compounds, bisphenol A (BPA) and octylphenol (OP), during development of the neuroendocrine system was investigated. The objective was to compare the effects of neonatal exposure to BPA, OP, and diethylstilbestrol (DES), a potent synthetic estrogen, on prepubertal serum PRL levels and estrogen receptor (ER) expression in the anterior pituitary and medial basal hypothalamus. Receptor expression in the uterus and prostate, two peripheral estrogen-responsive tissues, was also examined. Newborn male and female Fischer 344 rats were s.c. injected on days 1-5 after birth with corn oil (control), BPA and OP (100 or 500 microg/day), or DES (5 microg/day). Rats were bled on days 15, 20, and 25 and on the day of death (day 30), and serum PRL was analyzed by RIA. Relative expressions of ERalpha and ERbeta were determined by RT-PCR. BPA and OP induced delayed, but progressive, increases in serum PRL levels, up to 3-fold above control levels, in both males and females. The low dose of either compound was equally or more effective as the high dose in eliciting and sustaining elevated serum PRL levels, namely hyperprolactinemia. In contrast, the DES treatment resulted in a transient rise in serum PRL levels. BPA, OP, and, to a lesser extent, DES increased the expression of both ERalpha and ERbeta in the anterior pituitary of males, but not females, whereas the hypothalamic ERs were less responsive to these compounds. DES treatment caused down-regulation of ERalpha expression in the uterus and up-regulation of ERbeta in the prostate, whereas BPA or OP was without effect. In conclusion, exposure of newborn rats of either sex to environmental estrogens results in delayed and sustained hyperprolactinemia and differential alterations in ER expression in the hypothalamus and pituitary. DES appears to target the lower reproductive tract more effectively than the neuroendocrine system.

Vascular endothelial growth factor in the rat pituitary: differential distribution and regulation by estrogen.

UNLABELLED: Vascular endothelial growth factor (VEGF), an endothelial cell mitogen and permeability factor, participates in tumor angiogenesis, but less is known about its regulation or function in normal vascular homeostasis. In the uterus, which undergoes cyclic changes in its vasculature, VEGF is induced by estrogen. Since the pituitary gland contains highly permeable capillaries and is estrogen-responsive, our objectives were to localize VEGF expression within the pituitary and to determine whether it is regulated by estrogen in both the pituitary and the somatolactotrope cell line, GH(3). Ovariectomized rats were injected with estradiol, and pituitaries and uteri were subjected to in situ hybridization or quantitative reverse transcription-polymerase chain reaction (RT-PCR). VEGF expression was strong and punctate in the neural lobe, weaker and diffuse in the anterior lobe and undetectable in the intermediate lobe. Two VEGF isoforms, 164 and 120, were detected in all tissues. In the posterior pituitary, VEGF expression was 3- to 6-fold higher than in the anterior pituitary or uterus and was unaltered by estrogen. In contrast, anterior pituitary VEGF was induced by estrogen within 1 h, peaked at 3 h, and returned to basal levels by 24 h. Similar dynamics, albeit 10-fold higher, were seen in the uterus. Translated VEGF proteins were detected by Western blot in both the anterior pituitary and uterus. GH(3) cells also showed a dose- and time-dependent induction of VEGF expression by estrogen. IN CONCLUSION: (1) VEGF expression is higher in the neural lobe than in the anterior lobe and is undetectable in the intermediate lobe, (2) the expression of VEGF164 and VEGF120 is rapidly upregulated by estrogen in the anterior pituitary but is unchanged in the posterior pituitary, and (3) the pituitary lactotrope cell line, GH(3), also increases VEGF expression in response to estradiol.

Strain differences in vaginal responses to the xenoestrogen bisphenol A.

Bisphenol A (BPA) is the monomer component of polycarbonate plastics and epoxy resins; human exposure derives from leachate in foodstuffs packaged in certain plastics or from epoxy-based dental appliances. BPA stimulates prolactin secretion in Fischer 344 (F344) rats but not in Sprague-Dawley (S-D) rats. The present studies were performed to determine if another classic estrogen target tissue, the rat vagina, responds to BPA in a strain-specific manner. In F344 rats BPA increased DNA synthesis in vaginal epithelium with a median effective dose (ED(50)) of 37.5 mg/kg body weight; DNA synthesis was not stimulated in S-D rats by any dose tested. Clearance of (3)H-BPA from blood followed the same time course in both strains of rats, with a half-life of 90 min. Scatchard analysis of [(3)H]estradiol binding showed no strain differences in concentration or affinity of the vaginal estrogen receptor. BPA increased the level of mRNA for the immediate early gene, c-fos, with similar dose-response curves in both rat strains. Thus, F344 and S-D rats exhibit differences in sensitivity to BPA at the level of cell proliferation in the vaginal epithelium. However, metabolic clearance of BPA and the early events that lead to the proliferative response, receptor-ligand interaction and induction of immediate early genes, show no strain differences. These observations suggest that differences in intermediate effects must account for the difference in sensitivity of the proliferative response to the xenoestrogen. Furthermore, these results point to the need for caution in choosing a suitable end point and animal model when seeking to test the estrogenic effects of xenobiotics.

Proteolysis of human prolactin: resistance to cathepsin D and formation of a nonangiostatic, C-terminal 16K fragment by thrombin.

The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10- to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30+/-15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.

An approach to the development of quantitative models to assess the effects of exposure to environmentally relevant levels of endocrine disruptors on homeostasis in adults.

The workshop "Characterizing the Effects of Endocrine Disruptors on Human Health at Environmental Exposure Levels" was held to provide a forum for discussions and recommendations of methods and data needed to improve risk assessments of endocrine disruptors. This article was produced by a working group charged with determining the basic mechanistic information that should be considered when designing models to quantitatively assess potential risks of environmental endocrine disruptors in adults. To reach this goal, we initially identified a set of potential organ system toxicities in males and females on the basis of known and/or suspected effects of endocrine disruptors on estrogen, androgen, and thyroid hormone systems. We used this integrated, systems-level approach because endocrine disruptors have the potential to exert toxicities at many levels and by many molecular mechanisms. Because a detailed analysis of all these untoward effects was beyond the scope of this workshop, we selected the specific end point of testicular function for a more detailed analysis. The goal was to identify the information required to develop a quantitative model(s) of the effects of endocrine disruptors on this system while focusing on spermatogenesis, sperm characteristics, and testicular steroidogenesis as specific markers. Testicular function was selected because it is a prototypical integrated end point that can be affected adversely by individual endocrine disruptors or chemical mixtures acting at one specific site or at multiple sites. Our specific objective was to gather the information needed to develop models in the adult organism containing functional homeostatic mechanisms, and for this reason we did not consider possible developmental toxicities. Homeostatic mechanisms have the potential to ameliorate or lessen the effects of endocrine disruptors, but these pathways are also potential target sites for the actions of these chemicals.

Differential regulation and action of estrogen receptors alpha and beta in GH3 cells.

The pituitary lactotroph, a well established target for estrogens, expresses estrogen receptor-alpha (ER alpha) and -beta (ER beta). A truncated isoform of ER alpha, named TERP, is expressed in the pituitary, but not in the uterus. In this study we used the somatolactotroph cell line, GH3 cells, to examine 1) the expression of ER alpha, TERP, or ER beta and their regulation by estradiol; 2) the presence of receptor proteins; and 3) the effects of overexpressing ER beta or TERP on estrogen induction of the PRL gene and activation of the estrogen response element (ERE). Incubation of GH3 cells with estradiol (0.1-10 nM) produced dose-dependent increases in messenger RNA levels of ER beta and TERP, but not ER alpha, as determined by quantitative RT-PCR. Cell incubation with 1 nM estradiol resulted in a time-dependent biphasic increase in TERP and a delayed rise in ER beta, suggesting activation by both direct and indirect mechanisms. A polyclonal ER beta antibody directed against an N-terminal synthetic peptide was generated. This antibody detected ER beta-positive cells in ovarian granulosa cells and in many cells throughout the pituitary; its specificity was demonstrated by preabsorption with the synthetic peptide. The antibody detected a 58- to 60-kDa protein by Western blotting of ovarian, pituitary, and GH3 cell extracts. Cotransfection of ER beta and reporter genes (PRL promoter/luciferase or ERE/luciferase) into GH3 cells resulted in a dose-dependent increase in estrogen-induced PRL gene expression, with a lesser activation of the ERE. A 20-kDa TERP protein was undetectable in untreated GH3 cells and was weakly induced by estradiol. Overexpression of TERP had no effect on estrogen induction of either PRL or ERE. We conclude that 1) both ER beta and TERP messenger RNAs in GH3 cells are increased by estradiol in a dose- and time-dependent manner, whereas ER alpha is not altered; 2) a 58-kDa ER beta protein is expressed in both the pituitary and GH3 cells; and 3) overexpression of ER beta increases estrogen-induced PRL gene expression.

Heparin-binding property of human prolactin: a novel aspect of prolactin biology.

Prolactin (PRL) shares several characteristics with growth factors and cytokines, many of which are known to bind to heparan sulfate proteoglycans. In this study we examined the heparin-binding properties of selected members of the PRL/GH family, using heparin affinity columns followed by gel electrophoresis/Western blotting. Purified human PRL and its cleaved 16K fragment, but not human GH or placental lactogen, were retained on the heparin column and were displaced by 0.5 M NaCl. Native PRL in human pituitary extracts and amniotic fluid showed a similar binding affinity to heparin as the purified hormone. None of the other hormones tested, e.g., rat, ovine and bovine PRL, glycosylated ovine PRL or rat GH, bound to heparin. Two consensus heparin-binding sequences are present in human PRL but not in the other hormones included in this study. We postulate that the heparin-binding capability of PRL affects its biological activity as a growth factor and the angiostatic actions of its 16K fragment.

Cellular distribution and gene regulation of estrogen receptors alpha and beta in the rat pituitary gland.

The pituitary gland is a heterogeneous tissue comprised of several hormone secreting and supporting cells, most of which are targeted by estrogens. Estrogen-induced changes in the pituitary are presumably mediated via the classical estrogen receptor, ER alpha. However, a novel receptor, ER beta, and pituitary-specific truncated estrogen receptor products (TERPs) were recently identified. The objectives of this study were to examine the distribution of these receptors in the rat pituitary and compare their regulation by estradiol in Sprague-Dawley and the estrogen-sensitive Fischer 344 rats. Pituitary cryosections were subjected to immunocytochemistry for specific cell types, followed by in situ hybridization for ER alpha or ER beta. ER alpha was expressed by approximately 45% of the lactotrophs and melanotrophs, 35% of the corticotrophs and folliculo-stellate cells, and 25% of the gonadotrophs. The expression of ER beta showed a similar pattern but was generally lower than ER alpha. In two cell types, melanotrophs and gonadotrophs, ER beta expression was significantly lower than ER alpha. In the second experiment, pituitary sections were immunostained for ER alpha, followed by in situ hybridization for ER beta. Only a minute population (6-10%) of either anterior or intermediate lobe cells coexpressed ER alpha and ER beta. In the next experiment, Fischer 344 and Sprague-Dawley rats were injected with oil or estradiol for 24 h. Total RNA from dissected anterior and posterior (neurointermediate) pituitaries was subjected to RT-PCR for ER alpha, ER beta, or TERPs. Interestingly, ER alpha and ER beta were unchanged by estradiol in either lobe of the pituitary. In contrast, estradiol increased pituitary TERP messenger RNA levels 4- to 7-fold. A 20-kDa TERP protein was detected by Western blots in the pituitary but not the uterus. There were no differences in the estradiol-induced expression of any of the receptors between the two strains of rats. We conclude that: 1) ER beta is expressed in all anterior and intermediate lobe cell types examined, albeit at a lower level than ER alpha; 2) no more than 10% of pituitary cells coexpress ER alpha and ER beta; and 3) estradiol markedly increases TERP messenger RNA levels but does not alter the expression of ER alpha or ER beta. We propose that estrogen receptor heterogeneity contributes to the diversity of pituitary cell responsiveness to estrogens.

The xenoestrogen bisphenol A induces growth, differentiation, and c-fos gene expression in the female reproductive tract.

The xenoestrogen bisphenol A (BPA) has been shown to mimic estrogen both in vivo and in vitro. BPA stimulates PRL secretion and the expression of a PRL regulating factor from the posterior pituitary in the estrogen-sensitive Fischer 344 rat (F344), but not in Sprague-Dawley (SD) rats. The goal of the present studies was to examine the in vivo actions of BPA on the reproductive tract. The specific objectives were 1) to characterize the short term effects of BPA on cell proliferation and c-fos expression in the uterus and vagina, and 2) to compare the effects of prolonged exposure to low doses of BPA on the reproductive tract of F344 and SD rats. Treatment with single high doses of BPA induced cell proliferation in the uterus and vagina of ovariectomized F344 rats, as determined by bromodeoxyuridine immunostaining. This proliferation was dose dependent (from 37.5-150 mg/kg) and followed a time course similar to that of estradiol (E2). Quantitative RT-PCR revealed that both BPA and E2 increased c-fos messenger RNA levels in the uterus 14- to 16-fold within 2 h, which returned to basal levels after 6 h. In the vagina, BPA-induced c-fos expression remained elevated for up to 6 h, compared with the transient increase caused by E2. Treatment of F344 rats for 3 days with continuous release capsules that supplied a much lower dose of BPA (approximately 0.3 mg/kg x day) resulted in hypertrophy, hyperplasia, and mucus secretion in the uterus and hyperplasia and cornification of the vaginal epithelium. The reproductive tract of SD rats did not respond to this treatment paradigm with BPA. These studies demonstrate that 1) the molecular and morphological alterations induced by BPA in the uterus and vagina are nearly identical to those induced by estradiol; 2) the vagina appears to be especially sensitive to the estrogenic actions of BPA; 3) the reproductive tract of the inbred F344 rat appears more sensitive to BPA than that of the outbred SD rat; and 4) continuous exposure to microgram levels of BPA is sufficient for exerting estrogenic actions.

Xenoestrogens: the emerging story of bisphenol a.

Xenoestrogens are chemicals with diverse structure that mimic estrogen. Bisphenol A (BPA), a monomer of polycarbonate and epoxy resins, has been detected in canned food and human saliva. BPA stimulates cell proliferation and induces expression of estrogen-responsive genes in vitro, albeit with a relatively low potency. In vivo, BPA increases prolactin release and stimulates uterine, vaginal and mammary growth and differentiation. BPA shares similarities in structure, metabolism and action with diethylstilbestrol (DES), a known human teratogen and carcinogen. This review considers the hypothesis that BPA is converted in vivo to hydroxylated metabolite(s) with enhanced estrogenicity and genotoxicity.

Two distinct pituitary cell lines from mouse intermediate lobe tumors: a cell that produces prolactin-regulating factor and a melanotroph [seecomments].

The intermediate lobe (IL) of the pituitary produces a PRL-regulating factor (PRF). Targeted tumorigenesis, using the POMC promoter ligated to SV40 large T antigen (Tag), generated transgenic mice that develop IL tumors with PRF activity. Our goal was to establish and characterize a PRF-producing cell line. Two cell lines, which differ markedly in size and morphology, were independently developed from IL tumors and designated mIL5 and mIL39. These cells are transformed, as judged by rapid proliferation, low serum requirements, and generation of secondary tumors in nude mice. RT-PCR revealed that mIL39, but not mIL5 cells, express POMC and dopamine D2 receptors, typical of a melanotroph phenotype. Although mIL5 cells originated from an IL tumor, they do not express messenger RNA for SV40 Tag. The bioassay for PRF used GH3 cells stably transfected with the PRL promoter ligated to a luciferase reporter gene (GH3/luc). Coculture of mIL5 with GH3/luc cells induced cell-density dependent increases in PRL gene expression and release, whereas mIL39 cells showed negligible PRF activity. Incubation of GH3/luc cells with conditioned media from mIL5, but not mIL39 cells, stimulated PRL gene expression and release up to 10-fold. Coculture of mIL5 cells with primary rat anterior pituitary cells stimulated PRL, but not GH, release. Fractionation of mIL5 cell extracts by reverse phase HPLC resolved PRF activity into one major and one minor peak. In conclusion, we have developed two novel and distinct cell lines from mouse intermediate lobe tumors. The first reported melanotroph cell line, mIL39, could provide a valuable model for studying dopaminergic regulation of POMC gene expression and release. In contrast, the mIL5 cells do not express POMC, D2 receptors, or SV40 Tag and appear to have been immortalized by a spontaneous mutation(s). These cells produce and secrete a potent PRF and could be used for the purification and biochemical characterization of PRF.

Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice.

Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of pituitary hormone secretion. Mice with a disrupted D2 dopamine receptor gene had chronic hyperprolactinemia and developed anterior lobe lactotroph hyperplasia without evidence of adenomatous transformation. Unexpectedly, the mutant mice had no hyperplasia of the intermediate lobe melanotrophs. Aged female D2 receptor -/- mice developed uterine adenomyosis in response to prolonged prolactin exposure. These data reveal a critical role of hypothalamic dopamine in controlling pituitary growth and support a multistep mechanism for the induction and perpetuation of lactotroph hyperplasia, involving the lack of dopamine signaling, a low androgen/estrogen ratio, and a final autocrine or paracrine "feed-forward" stimulation of mitogenesis, probably by prolactin itself.

The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo.

Environmental estrogens (xenoestrogens) are a diverse group of chemicals that mimic estrogenic actions. Bisphenol A (BPA), a monomer of plastics used in many consumer products, has estrogenic activity in vitro. The pituitary lactotroph is a well established estrogen-responsive cell. The overall objective was to examine the effects of BPA on PRL release and explore its mechanism of action. The specific aims were to: 1) compare the potency of estradiol and BPA in stimulating PRL gene expression and release in vitro; 2) determine whether BPA increases PRL release in vivo; 3) examine if the in vivo estrogenic effects are mediated by PRL regulating factor from the posterior pituitary; and 4) examine if BPA regulates transcription through the estrogen response element (ERE). BPA increased PRL gene expression, release, and cell proliferation in anterior pituitary cells albeit at a 1000- to 5000-fold lower potency than estradiol. On the other hand, BPA had similar efficacy to estradiol in inducing hyperprolactinemia in estrogen-sensitive Fischer 344 (F344) rats; Sprague Dawley (SD) rats did not respond to BPA. Posterior pituitary cells from estradiol- or BPA-treated F344 rats strongly increased PRL gene expression upon coculture with GH3 cells stably transfected with a reporter gene. Similar to estradiol, BPA induced ERE activation in transiently transfected anterior and posterior pituitary cells. We conclude that: a) BPA mimics estradiol in inducing hyperprolactinemia in genetically predisposed rats; b) the in vivo action of estradiol and BPA in F344 rats is mediated, at least in part, by increasing PRL regulating factor activity in the posterior pituitary; c) BPA appears to regulate transcription through an ERE, suggesting that it binds to estrogen receptors in both the anterior and posterior pituitaries. The possibility that BPA and other xenoestrogens have adverse effects on the neuroendocrine axis in susceptible human subpopulations is discussed.

Cell-specific induction of c-fos expression in the pituitary gland by estrogen.

Estrogens regulate many functions of pituitary lactotrophs, including PRL gene expression, release, storage, and cellular proliferation. The mechanism by which estrogens exert such a variety of functions is poorly understood. In the uterus, estrogens rapidly and transiently induce the expression of the immediate early genes c-fos and c-jun in specific cell types. The Fos/Jun proteins form the activating protein-1 (AP1) transcription factor that mediates ligand-activated cell proliferation, differentiation, and secretion. Here we used Fischer 344 (F344) rats that develop hyperprolactinemia and prolactinomas in response to estrogens. The objectives were to: 1) determine whether estrogen induces c-fos expression in the pituitary gland and identify the responsive cells; 2) compare the dynamics of c-fos induction in the pituitary and uterus; and 3) examine the temporal relationship between c-fos expression and PRL release. Ovariectomized F344 rats were injected with 1 microg estradiol and killed at different times thereafter. Pituitaries were subjected to in situ hybridization for c-fos and immunostaining for selected pituitary cells. Estradiol stimulated c-fos expression in lactotrophs and folliculo-stellate cells within the anterior lobe without affecting either the intermediate or neural lobes. In a second experiment, c-fos messenger RNA levels were measured by solution hybridization in anterior pituitaries and uteri from estradiol-treated rats. Trunk blood was analyzed for PRL by RIA. The estrogen-induced c-fos rise in the uterus was rapid, robust, and transient, whereas that in the anterior pituitary was delayed, lower, and sustained. The profile of serum PRL levels resembles that of c-fos induction in the anterior pituitary. We conclude that: 1) both lactotrophs and folliculo-stellate cells increase c-fos expression in response to estrogens; 2) induction of c-fos expression may mediate some estrogenic effects on PRL synthesis and release and lactotroph proliferation in F344 rats; and 3) the atypical dynamics of c-fos induction in the pituitary could be due to indirect effects of estrogens on PRL-regulating factors within the hypothalamo-pituitary complex as well as to pituitary-specific estrogen receptor isoforms, coactivators, or repressors.

Novel estrogenic action of the pesticide residue beta-hexachlorocyclohexane in human breast cancer cells.

The estrogenic action of some persistent organochlorine pesticide residues may play a role in the progression of hormonally responsive tumors of the breast and uterus. The prototypical xenoestrogen o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) acts by binding and activating the estrogen receptor (ER). The present study focuses attention on the mechanisms through which another organochlorine compound, beta-hexachlorocyclohexane (beta-HCH), exerts estrogen-like effects in human breast cancer cells. Both o,p'DDT and beta-HCH stimulated proliferation in a dose-dependent manner in the ER-positive cell lines MCF-7 and T47D but not in the ER-negative lines MDA-MB231, MDA-MB468, and HS578T. Both compounds produced an increase in the steady state level of pS2 mRNA in MCF-7 cells. These responses were equal in magnitude to the maximal effect of estradiol, and they were inhibited by inclusion of the antiestrogen ICI164384. On the other hand, when tested in a competitive binding assay, beta-HCH did not displace 17beta-[3H]estradiol from the ER even at a concentration that was 40,000-fold higher than the tracer steroid. Furthermore, nuclear retention of the ER during homogenization procedures was induced by a 2- or 24-h treatment of MCF-7 cells with o,p'-DDT and 17beta-estradiol but not by treatment with beta-HCH; this indicates that beta-HCH nether activates the ER, nor is it converted intracellularly to an ER ligand. Transcriptional activation by beta-HCH occurs in estrogen-responsive GH3 rat pituitary tumor cells transfected with a luciferase reporter construct driven by a complex 2500-bp portion of the PRL gene promoter; this trans-activation response is inhibited by inclusion of ICI164384. However, beta-HCH is ineffective in stimulating a reporter construct driven only by a consensus estrogen response element and a minimal promoter derived from the herpes simplex virus thymidine kinase gene. Thus, beta-HCH cannot act on a simple, single estrogen response element; rather, it requires the combinatorial regulation found in a complex promoter. These data are consistent with the notion that beta-HCH stimulation of cell proliferation and gene expression is ER dependent, but its action is not through the classic pathway of binding and activating the ER. beta-HCH may represent a new class of xenobiotic that produces estrogen-like effects through nonclassic mechanisms and, therefore, may be of concern with regard to breast and uterine cancer risk.