Inhibition of MAPK by prolactin signaling through the short form of its receptor in the ovary and decidua: involvement of a novel phosphatase.

Prolactin (PRL) is essential for normal reproduction and signals through two types of receptors, the short (PRL-RS) and long (PRL-RL) form. We have previously shown that transgenic mice expressing only PRL-RS (PRLR(-/-)RS) display abnormal follicular development and premature ovarian failure. Here, we report that MAPK, essential for normal follicular development, is critically inhibited by PRL in reproductive tissues of PRLR(-/-)RS mice. Consequently, the phosphorylation of MAPK downstream targets are also markedly inhibited by PRL without affecting immediate upstream kinases, suggesting involvement of MAPK specific phosphatase(s) in this inhibition. Similar results are obtained in a PRL-responsive ovary-derived cell line (GG-CL) that expresses only PRL-RS. However, we found the expression/activation of several known MAPK phosphatases not to be affected by PRL, suggesting a role of unidentified phosphatase(s). We detected a 27-kDa protein that binds to the intracellular domain of PRL-RS and identified it as dual specific phosphatase DUPD1. PRL does not induce expression of DUDP1 but represses its phosphorylation on Thr-155. We also show a physical association of this phosphatase with ERK1/2 and p38 MAPK. Using an in vitro phosphatase assay and overexpression studies, we established that DUPD1 is a MAPK phosphatase. Dual specific phosphatase inhibitors as well as siRNA to DUPD1, completely prevent PRL-mediated MAPK inhibition in ovarian cells. Our results strongly suggest that deactivation of MAPK by PRL/PRL-RS contributes to the severe ovarian defect in PRLR(-/-)RS mice and demonstrate the novel association of PRL-RS with DUPD1 and a role for this phosphatase in MAPK deactivation.

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function.

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.

The molecular control of corpus luteum formation, function, and regression.

The corpus luteum (CL) is one of the few endocrine glands that forms from the remains of another organ and whose function and survival are limited in scope and time. The CL is the site of rapid remodeling, growth, differentiation, and death of cells originating from granulosa, theca, capillaries, and fibroblasts. The apparent raison d'etre of the CL is the production of progesterone, and all the structural and functional features of this gland are geared toward this end. Because of its unique importance for successful pregnancies, the mammals have evolved a complex series of checks and balances that maintains progesterone at appropriate levels throughout gestation. The formation, maintenance, regression, and steroidogenesis of the CL are among the most significant and closely regulated events in mammalian reproduction. During pregnancy, the fate of the CL depends on the interplay of ovarian, pituitary, and placental regulators. At the end of its life span, the CL undergoes a process of regression leading to its disappearance from the ovary and allowing the initiation of a new cycle. The generation of transgenic, knockout and knockin mice and the development of innovative technologies have revealed a novel role of several molecules in the reprogramming of granulosa cells into luteal cells and in the hormonal and molecular control of the function and demise of the CL. The current review highlights our knowledge on these key molecular events in rodents.

Prolactin independent rescue of mouse corpus luteum life span: identification of prolactin and luteinizing hormone target genes.

The corpus luteum (CL) plays a central role in the maintenance of pregnancy in rodents, mainly by secreting progesterone. Female mice lacking prolactin (PRL) receptor (R) are sterile due to a failure of embryo implantation, which is a consequence of decreased luteinizing hormone (LH) receptor expression in the CL and inadequate levels of progesterone. We attempted to treat PRLR(-/-) females with human chorionic gonadotropin (hCG) and showed a de novo expression of LHR mRNA in the corpora lutea. Binding analysis confirmed that the LHR in hCG-treated PRLR(-/-) animals was functional. This was accompanied with increased expression of steroidogenic enzymes involved in progesterone synthesis. Despite these effects, no embryo implantation was observed because of high expression of 20alpha-hydroxysteroid dehydrogenase. To better appreciate the molecular mechanisms underlying maintenance of the CL, a series of mRNA expression-profiling experiments was performed on isolated corpora lutea of PRLR(-/-) and hCG-treated PRLR(-/-) mice. This approach revealed several novel candidate genes with potentially pivotal roles in ovarian function, among them, p27, VE-cadherin, Pten, and sFRP-4, a member of the Wnt/frizzled family. This study showed the differential role of PRL and LH in CL function and identified new targets of these hormones in luteal cells.

Prolactin signaling through the short isoform of the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues.

BACKGROUND: It has been well established that prolactin (PRL) signals through the long form of its receptor (PRL-RL) and activates the Jak/Stat pathway for transcription of PRL target genes. However, signaling pathways mediated through the short PRL-R isoform (PRL-RS) remains controversial. Our recent finding that PRL signaling through PRL-RS represses two transcription factors critical for follicular development lead us to examine other putative PRL/PRL-RS target transcription factors in the decidua and ovary, two well-known target tissues of PRL action in reproduction. METHODS: In this investigation we used mice expressing PRL-RS on a PRL-R knockout background and a combo protein/DNA array to study the transcription factors regulated by PRL through PRL-RS only. RESULTS: We show that PRL activation of the PRL-RS receptor either stimulates or inhibits the DNA binding activity of a substantial number of transcription factors in the decidua as well as ovary. We found few transcription factors to be similarly regulated in both tissues, while most transcription factors are oppositely regulated by PRL in the decidua and ovary. In addition, some transcription factors are regulated by PRL only in the ovary or only in the decidua. Several of these transcription factors are involved in physiological pathways known to be regulated by PRL while others are novel. CONCLUSION: Our results clearly indicate that PRL does signal through PRL-RS in the decidua as well as the ovary, independently of PRL-RL, and activates/represses transcription factors in a tissue specific manner. This is the first report showing PRL/PRL-RS regulation of specific transcription factors. Many of these transcription factors were not previously known to be PRL targets, suggesting novel physiological roles for this hormone.

Prolactin receptor-associated protein/17beta-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival.

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.

Prolactin signaling through the short form of its receptor represses forkhead transcription factor FOXO3 and its target gene galt causing a severe ovarian defect.

Prolactin (PRL) is a hormone with over 300 biological activities. Although the signaling pathway downstream of the long form of its receptor (RL) has been well characterized, little is known about PRL actions upon activation of the short form (RS). Here, we show that mice expressing only RS exhibit an ovarian phenotype of accelerated follicular recruitment followed by massive follicular death leading to premature ovarian failure. Consequently, RS-expressing ovaries of young adults are depleted of functional follicles and formed mostly by interstitium. We also show that activation of RS represses the expression of the transcription factor Forkhead box O3 (FOXO3) and that of the enzyme galactose-1-phosphate uridyltransferase (Galt), two proteins known to be essential for normal follicular development. Our finding that FOXO3 regulates the expression of Galt and enhances its transcriptional activity indicates that it is the repression of FOXO3 by PRL acting through RS that prevents Galt expression in the ovary and causes follicular death. Coexpression of RL with RS prevents PRL inhibition of Galt, and the ovarian defect is no longer seen in RS transgenic mice that coexpress RL, suggesting that RL prevents RS-induced ovarian impairment. In summary, we show that PRL signals through RS and causes, in the absence of RL, a severe ovarian pathology by repressing the expression of FOXO3 and that of its target gene Galt. We also provide evidence of a link between the premature ovarian failure seen in mice expressing RS and in mice with FOXO3 gene deletion as well as in human with Galt mutation.

Decidual prolactin silences the expression of genes detrimental to pregnancy.

Although the main role of prolactin (PRL) in pregnant rodents is to sustain progesterone production by the corpus luteum, progesterone treatment of PRL or PRL receptor (PRL-R) null mice is unable to prevent fetal loss. We have previously shown that the rat decidua is a site of PRL production and action. In this report, we examined the hypothesis, using PRL null mice and rat decidual cell culture, that the absence of this hormone leads to the expression in the decidua of genes detrimental to pregnancy. The results show that decidual growth is normal in PRL null mice treated with PRL, progesterone, or their combination. However, the decidua of mice treated with progesterone starts expressing IL-6 and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), two proteins absent from the decidua of wild-type mice and involved, respectively, in inflammation and progesterone catabolism. The expression of both IL-6 and 20alpha-HSD is prevented by PRL treatment. Our results further suggest that PRL inhibition of 20alpha-HSD expression is at the level of transcription and that decidual PRL (dPRL) inhibits 20alpha-HSD promoter activity. Inhibitors of Janus kinase 2 (Jak2) but not other kinases prevent dPRL down-regulation of the 20alpha-HSD promoter. Furthermore, cotransfection of the 20alpha-HSD promoter with expression vectors of constitutively active PRL-R, Jak2, or signal transducer and activator of transcription 5b (Stat5b) leads to substantial inhibition of promoter activity. Taken together, our investigation provides an explanation for the inability of progesterone to sustain pregnancy in PRL null mice and suggests that dPRL plays an important role in pregnancy by repressing the expression of IL-6 and 20alpha-HSD in the decidua. The study also demonstrates that PRL signals through the Jak2/Stat5 pathway to down-regulate 20alpha-HSD expression in the decidua.

The role of interleukin-11 in pregnancy involves up-regulation of alpha2-macroglobulin gene through janus kinase 2-signal transducer and activator of transcription 3 pathway in the decidua.

IL-11 expressed by endometrial stromal cells is crucial for normal pregnancy. IL-11 receptor alpha (IL-11Ralpha) null mice are infertile due to abnormal development of the placenta. In these mice, the mesometrial decidual tissue, which is the site of trophoblast invasion, thins and disappears at mid-pregnancy. Degeneration of the decidua is accompanied by uncontrolled trophoblast invasion. In this report, we show, using IL-11Ralpha null mice, that a defect in IL-11 signaling in the decidua leads to severe down-regulation of alpha(2)-macroglobulin (alpha(2)-MG), a metalloproteinase inhibitor crucial for limiting trophoblast invasion. We also present evidence, using uterine stromal cells that decidualize in culture, that IL-11 robustly stimulates the endogenous alpha(2)-MG expression and enhances alpha(2)-MG promoter activity. Serial 5' deletion and internal deletion of the promoter reveal two important signal transducer and activator of transcription (Stat) binding sites. Mutation of either one of these motifs decreases IL-11 stimulation, whereas double mutation prevents IL-11 action. We also found that IL-11 activates Janus kinase 2 (Jak2) and induces rapid phosphorylation, nuclear translocation, and promoter binding activity of Stat3 in decidual cells, whereas Jak1, Tyk2, and Stat5 activities are not affected. In addition, Jak2 inhibitor totally prevents alpha(2)-MG expression in decidual cells. Taken together, results of this investigation provide, at least in part, an explanation for the overinvasiveness of the trophoblast in IL-11Ralpha null mice and reveal, for the first time, that IL-11 signals through the Jak2/Stat3 pathway in decidual cells to stimulate the expression of alpha(2)-MG, a protease inhibitor essential for normal placentation in pregnancy.

Rat decidual cell cultures.

Pregnancy requires profound reorganization of the different tissues forming the uterus. Growth and differentiation of the uterine endometrial cells give rise to the decidual tissue, a transitory organ, which plays a key role in fetal survival. In this chapter, we describe a technique for the dispersion and the separation of the two different decidual cell subpopulations with high yield and viability. We also detail a cell culture method, which allows the maintenance of the function and life span of these highly purified decidual cells when cultured either separately or in a co-culture system.

Cloning and characterization of a 5' regulatory region of the prolactin receptor-associated protein/17{beta} hydroxysteroid dehydrogenase 7 gene.

Prolactin receptor-associated protein (PRAP) originally cloned in our laboratory was shown to be a novel, luteal isoform of 17beta hydroxysteroid dehydrogenase 7 (17betaHSD7). In this study, we cloned the promoter region of rat PRAP/17betaHSD7 and investigated the mechanisms regulating both basal activity and LH-induced repression of this promoter. Truncated and site-specific mutants of PRAP/17betaHSD7 promoter identified two enhancer regions that contained highly conserved Sp1 binding site and bound Sp1 from nuclear extracts of both corpora lutea and a rat luteal cell line. Repression of PRAP/17betaHSD7 expression and promoter activity by human chorionic gonadotropin/forskolin was localized to a -52-bp proximal segment of the promoter. This region contained a conserved CCAAT site and bound nuclear factor Y; binding of this transcription factor was inhibited by human chorionic gonadotropin in vivo. Furthermore, mutation of the nuclear factor Y site in the -52-bp promoter-reporter construct abolished forskolin-mediated inhibition of the promoter in a rat luteal cell line. In summary, we have identified the promoter elements involved in the basal expression of PRAP/17betaHSD7. We have also found that LH-mediated repression of this gene is at the level of transcription and involves inhibition of nuclear factor YA binding to the CCAAT site within the proximal promoter.

PGF2alpha induced differential expression of genes involved in turnover of extracellular matrix in rat decidual cells.

In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2alpha) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2alpha in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2alpha sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2alpha are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2alpha. The results strongly suggest that the abortificient role of elevated levels of PGF2alpha after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.

Prolactin regulation of estrogen receptor expression.

The ability of the rat corpus luteum to respond to estrogen requires prolactin (PRL), which can stimulate the expression of the estrogen receptor (ER). This review will focus on the signaling mechanisms by which this occurs. Transcription of the genes encoding both ERalpha (Esr1) and ERbeta (Esr2) is stimulated by PRL through the Jak2-Stat5 pathway and Stat5-response elements that are located in each of the Esr promoters. A single nucleotide difference between these two response elements is responsible for the observation that either Stat5a or Stat5b can stimulate Esr1 transcription, whereas only Stat5b can activate transcription of Esr2. The tyrosine kinase Jak2 is required for PRL activation of Esr1 promoter activity; however, additional pathways are involved in PRL-induced Stat5b phosphorylation, nuclear translocation and DNA binding. In addition to the corpus luteum, PRL-induced ER expression might provide a mechanism for fine-tuning the responsiveness of other target tissues, such as the decidua and mammary gland, to these two hormones.

Prostaglandin F(2alpha) (PGF(2alpha)) and prolactin signaling: PGF(2alpha)-mediated inhibition of prolactin receptor expression in the Corpus luteum.

It is well established that prolactin (PRL) sustains, whereas prostaglandin F(2alpha) (PGF(2alpha)) curtails, progesterone production by the rodent corpus luteum (CL). We have previously shown that PGF(2alpha) inhibits the expression of several luteal genes stimulated by PRL, whereas it stimulates other genes inhibited by this hormone. We have also found that PGF(2alpha) stimulation of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD), an enzyme that catabolizes progesterone, at the end of pregnancy is accompanied by a dramatic decrease in PRL receptor (PRL-R) expression. These findings, and the fact that the factors that inhibit PRL-R are not known, led us to examine in vivo whether the decline in PRL-R at the end of pregnancy is due to PGF(2alpha) and to also find out whether PGF(2alpha) opposes PRL action by inhibiting PRL-R expression. Using the PGF(2alpha) receptor (PGF(2alpha)-R) knockout, we examined whether the absence of the PGF(2alpha)-R prevents the decline in the expression of both the short and long forms of the PRL-R in the CL. We found that, in sharp contrast to the wild-type mice, in which both forms of the PRL-R decline to low levels between d 18-20 of pregnancy, expression of these receptors remained elevated in the PGF(2alpha)-R null mice. Furthermore, administration of PGF(2alpha) to pregnant rats inhibited PRL-R expression. Time-course analysis revealed that PGF(2alpha) treatment decreases both isoforms of PRL-R within 1 h of treatment in vivo, whereas its stimulatory effect on 20alphaHSD expression was further delayed. Similar results were obtained with luteinized granulosa cells in culture. To examine whether the decline in PRL-R is involved/necessary for PGF(2alpha) action, cells were transfected with a constitutively active PRL-R. The expression of this receptor did not prevent PGF(2alpha) effect on PRL-R or 20alphaHSD expression. Taken together, these results demonstrate that PGF(2alpha) inhibits the expression of the PRL-R and that the decline in both forms of the PRL-R that occurs at the end of pregnancy in the CL is due to PGF(2alpha). The results further suggest that PGF(2alpha)-mediated stimulation of 20alphaHSD is independent from PGF(2alpha) inhibition of PRL signaling in luteal cell.

Pituitary hypoplasia and lactotroph dysfunction in mice deficient for cyclin-dependent kinase-4.

The lactotroph undergoes dynamic regulation of cell cycle progression during pregnancy, as well as throughout the development of the pituitary. We recently reported that female mice with targeted disruption of Cdk4, one of the G(1)-regulatory cyclin-dependent kinases, are unable to support embryo implantation because of defective progesterone secretion from the corpus luteum. In this study, we demonstrate that this phenotype is not attributable to a primary defect in the corpus luteum but is a consequence of defective prolactin (PRL) production caused by inappropriate development of the pituitary lactotroph population. Specifically, the pituitary of Cdk4-deficient mice is extremely hypoplastic. Lactotrophs and somatotrophs of prepubertal Cdk4-deficient mice were 80% decreased in number, relative to those in wild-type mice, whereas gonadotrophs were unaffected. Lactotrophs of Cdk4-deficient mice did not proliferate in response to estrogen administration, whereas estrogen could induce the expression of galanin, an estrogen-responsive factor required for lactotroph proliferation. The reduction in lactotroph numbers was reflected by markedly diminished serum PRL levels in both prepubertal and postcoital Cdk4-deficient mice. Administration of PRL, after mating, significantly increased serum progesterone levels and restored implantation in Cdk4-deficient female mice. These observations demonstrate that Cdk4 is required for normal proliferation of the lactotroph population.

Intact follicular maturation and defective luteal function in mice deficient for cyclin- dependent kinase-4.

Cell cycle progression of granulosa cells is critical for ovarian function, especially follicular maturation. During follicular maturation, FSH induces cyclin D2, which promotes G1 progression by activating cyclin-dependent kinase-4 (Cdk4). Because cyclin D2-deficient mice exhibit a block in follicular growth, cyclin D2/Cdk4 has been hypothesized to be required for FSH-dependent proliferation of granulosa cells. Here we investigate ovarian function in Cdk4-knockout mice we recently generated. Cdk4(-/-) females were sterile, but the morphology of their ovaries appeared normal before sexual maturation. The number of preovulatory follicles and the ovulation efficiency were modestly reduced in gonadotropin-treated Cdk4(-/-) mice. However, unlike cyclin D2-deficient mice, Cdk4(-/-) mice showed no obvious defect in FSH-induced proliferation of granulosa cells. Cdk4(-/-) ovaries displayed normal preovulatory expression of aromatase, PR, and cyclooxygenase-2. Postovulatory progesterone secretion was markedly impaired in Cdk4(-/-) mice, although granulosa cells initiated luteinization with induction of p450 side-chain cleavage cytochrome and p27(Kip1). Progesterone treatment rescued implantation and restored fertility in Cdk4(-/-) mice. Serum PRL levels after mating were significantly reduced in Cdk4(-/-) mice, suggesting the involvement of perturbed PRL regulation in luteal failure. Thus, Cdk4 is critical for luteal function, and some redundant protein(s) can compensate for the absence of Cdk4 in proliferation of granulosa cells.

The involvement of apoptotic regulators during in vitro decidualization.

OBJECTIVES: The uterus responds to an implanting blastocyst by undergoing extensive tIssue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. DESIGN: In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39 degrees C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33 degrees C. METHODS: We performed Northern blot analysis for bax, bcl-x(L) and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. RESULTS: We found an increase in the expression of bax when GG-AD cells were grown at 39 degrees C. We also showed apoptosis with Annexin V staining at 39 degrees C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. CONCLUSIONS: These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression.

Prolactin signaling mechanisms in ovary.

Prolactin is a hormone that is essential for normal reproduction and signals through two types of receptors. Not only is the classical long form of the prolactin receptor identified, but so are many short form receptors in rodents and human tissues. Mouse mutagenesis studies have offered insight into the biology of prolactin family, providing compelling evidence that the different isoforms have independent biological activity. The possibility that short forms mediate cell proliferation is important for a variety of tissues including mammary gland and ovarian follicles. This review summarizes our current knowledge about prolactin signaling and its role in reproduction through either long or short isoform receptors.

The stimulation of HSD17B7 expression by estradiol provides a powerful feed-forward mechanism for estradiol biosynthesis in breast cancer cells.

Our laboratory has previously cloned and purified an ovarian protein found to be a novel 17ß-hydroxysteroid dehydrogenase type 7 enzyme (HSD17B7) (formerly prolactin receptor-associated protein) that converts the weak estrogen, estrone, to the highly potent estradiol. The regulation of this enzyme has not yet been explored. In this report, we show high expression of HSD17B7 in human ductal carcinoma and breast cancer cell lines and present evidence for a strong up-regulation of this enzyme by estradiol at the level of mRNA, protein expression, and promoter activity in MCF-7 cells. The effect of estradiol is mediated by estrogen receptor (ER)α, whereas ERß prevents this stimulation. ER antagonists, ICI 182,780 and 4-hydroxytamoxifen, prevent estradiol-induced stimulation of the endogenously expressed HSD17B7, suggesting that these inhibitors not only block estradiol action but also its production. We have identified a -185-bp region of the hsd17b7 promoter that is highly conserved among rat, mouse, and human and confers regulation by estradiol in MCF-7 cells. This region is devoid of a classical estradiol-response element but contains a nuclear factor 1 (NF1) site that is essential for estradiol action. We found that estradiol stimulates the recruitment and DNA binding of NF1 to this region of the hsd17b7 promoter. Furthermore, knockdown of NF1 family members, NF1B, NF1A, and NF1X, completely prevents induction of this gene by estradiol. In summary, our findings demonstrate that estradiol stimulates HSD17B7 transcriptional activity in breast cancer cells through a novel mechanism requiring NF1 and strongly suggest a positive feedback mechanism to increase local estradiol synthesis causing growth of estrogen-dependent breast cancers.

Regulation of transcription factors and repression of Sp1 by prolactin signaling through the short isoform of its cognate receptor.

Prolactin (PRL) affects the development and function of the reproductive system by binding to two types of receptors, which differ by the size of their intracellular domain in rodents. Whereas the signaling pathway through the long form of the receptor (PRL-RL) is well characterized, signaling through the short form (PRL-RS) remains obscure. In this investigation, we examined transcription factors regulated by PRL in the ovary and decidua of mice expressing only PRL-RS in a PRL receptor null background. These mice provide a powerful in vivo model to study the selective signaling mechanism of PRL through PRL-RS independent of PRL-RL. We also examined the regulation of transcription factors in ovarian and uterine cell lines stably transfected with PRL-RS or PRL-RL. We focused our investigation on transcription factors similarly regulated in both these tissues and clearly established that signaling through PRL-RS does not activate the JaK/Stat in vivo but leads to severe down-regulation of Sp1 expression, DNA binding activity, and nuclear localization, events that appear to involve the calmodulin-dependent protein kinase pathway. Our in vivo and in culture data demonstrate that the PRL-RS activates a signaling pathway distinct from that of the PRL-RL.