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.

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.

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.

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.

Prolactin is a local growth factor in rat mammary tumors.

PRL is a mitogenic hormone that shares many characteristics with growth factors. The recent demonstration that rat mammary tissue expresses PRL messenger RNA (mRNA) led us to hypothesize that PRL may act as an autocrine/paracrine growth factor in the mammary gland and may be a determinant in mammary carcinogenesis. To examine this, mammary tumors were induced in rats by injection of the carcinogen nitrosomethylurea (NMU). In vitro studies used a cell line derived from NMU-induced mammary tumors. Expression of PRL and PRL receptor was assessed by reverse transcriptase-polymerase chain reaction. The NMU-induced mammary tumors and the cell line express mRNA for both PRL and PRL receptor (the long and short isoforms); additional hybridizing polymerase chain reaction products were seen in the tumors, but not in lactating mammary tissue. Immunoreactive PRL was detected in the NMU-induced tumors. The effect of PRL on cell proliferation was assessed by culturing NMU cells with PRL antiserum. The PRL antiserum inhibited cell proliferation by up to 70% compared to the effect of normal rabbit serum or GH antiserum. In summary, we showed that NMU-induced mammary tumors express mRNA for PRL and PRL receptor. Addition of PRL antiserum to cultured NMU cells significantly inhibited their growth. We propose that PRL may be acting as a local growth factor that stimulates the proliferation of mammary tumors.

Analysis of androgen receptor DNA reveals the independent clonal origins of uterine leiomyomata and the secondary nature of cytogenetic aberrations in the development of leiomyomata.

Uterine leiomyomata are thought to be monoclonal neoplasms. Accordingly, investigations of clonality with G6PD isoforms used as a marker for X chromosome inactivation have suggested independent origins for multiple tumors within individual uteri. However, results from a recent study assessing methylation differences between DNA of active and inactive X chromosomes have been interpreted to suggest that multiple tumors may arise from a common precursor. We have examined the clonality of 36 leiomyomata from 16 patients by analyzing X chromosome inactivation as indicated by the methylation status of the X-linked androgen receptor gene. As shown by this assay, all informative leiomyomata were monoclonal in origin. In patients with multiple leiomyomata, a random distribution of inactivation between the X homologs was noted, consistent with an independent origin of each tumor. Cytogenetic analysis was also performed on short-term cell cultures of 27 of the 36 tumors. In each of two tumors that had both cells with a clonal karyotypic abnormality and karyotypically normal cells, DNA prepared from short-term cultures showed a monoclonal pattern of X inactivation identical to that of the leiomyoma from which they were derived. These data suggest that karyotypically normal cells present in short-term cultures of uterine leiomyomata are part of the tumor clone, and that clonal expansion of tumor cells precedes the development of cytogenetic aberrations.