Prolactin activation of the long form of its cognate receptor causes increased visceral fat and obesity in males as shown in transgenic mice expressing only this receptor subtype.

To date the best defined function of prolactin (PRL) is its action on the ovary and mammary gland, although it has also been shown to have an effect on lipid metabolism. Using mice engineered to express only the long form of the prolactin receptor (PRL-RL), we demonstrate that PRL acting through PRL-RL alone causes severe adipose accumulation in visceral fat of males at 6 months of age. The increase in visceral fat accumulation is attributed to loss of adipose-derived leptin, which results in diminished lipolysis. The reduction in leptin also corresponds to decreased activation of AMP-activated protein kinase (AMPK), which further results in diminished fatty acid oxidation and increased fatty acid synthesis. Interestingly, the blunted AMPK response was only observed in adipose tissue and not in liver suggesting that this PRL mediated effect is tissue specific. A glucose tolerance study inferred that PRL-RL mice may suffer from insulin resistance or a reduction in insulin production that is not due to aberrant expression of glucose transporter 4 (Glut4). Collectively, our findings demonstrate that PRL signaling through the long form receptor causes reduced fatty acid oxidation, increased lipid storage, glucose intolerance, and obesity. These findings are of great importance towards understanding the etiology of obesity associated with hyperprolactinemia in humans as well as the role of PRL as a metabolic regulator in adipose tissue.

Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20alpha-hydroxysteroid dehydrogenase.

Nuclear factor kappa B (NFkappaB) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NFkappaB in regulating ovarian gene expression, we examined in the rat: (i) whether NFkappaB is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NFkappaB complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NFkappaB is maintained in the cytoplasm bound to IkappaB, whose phosphorylation allows NFkappaB translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of IkappaBalpha. Western analysis revealed that IkappaBalpha was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NFkappaB consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NFkappaB binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NFkappaB DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NFkappaB/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NFkappaB/p65 in luteal cells resulted in inhibition of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD) promoter activity as well as endogenous 20alphaHSD mRNA expression. In summary, we have shown that: (i) NFkappaB is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NFkappaB activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NFkappaB/p65 binding to an NFkappaB DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NFkappaB in luteal cells inhibits 20alphaHSD gene expression. The results further support a role for NFkappaB as a survival factor in the CL.

Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase.

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERalpha expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERalpha expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERalpha mRNA levels, suggesting that PRL signaling to the ERalpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERalpha promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERalpha promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERalpha expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.

PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity.

During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominant-negative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua.

Opposite effect of prolactin and prostaglandin F(2 alpha) on the expression of luteal genes as revealed by rat cDNA expression array.

It is well established that prolactin (PRL) sustains, while prostaglandin F(2 alpha) (PGF(2 alpha)) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20 alpha-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF(2 alpha). In addition to 20 alpha-HSD, PGF(2 alpha) stimulated and PRL inhibited PGF(2 alpha)-receptor, phospholipase C delta(1) and TGF beta(1) expression. In contrast PRL stimulated and PGF(2 alpha) inhibited the LH receptor, 11 beta-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GST mu(2), inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF(2 alpha). PGF(2 alpha) stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-beta, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF(2 alpha) also up-regulated the expression of the sodium channel beta(1), Na/K ATPase, annexin IV, GST7pi, and P450 reductase. In contrast PGF(2 alpha) inhibited the expression of two genes involved in cell cycle: cyclin D2 and retinoblastoma related protein (Rb2/p130). It also inhibited genes involved in estradiol (P-450(AROM)) and cholesterol biosynthesis (HMG-CoA synthase), as well as genes involved in tissue remodeling: VEGF and TIMP3. PRL had a profound inhibitory effect on the expression of genes encoding the ADP-ribosylation factor 3, annexin V and c-jun, yet increased the expression of P450scc, 3beta-HSD, and SR-B1 (HDL-receptor), all genes involved in steroidogenesis. PRL also stimulated the expression of beta(2)-microglobulin, TIMP2, cytochrome c oxidase IV, cathepsin H and L, and copper-zinc superoxide dismutase as well as elongation factor SIII, heat shock protein-60 and mitochondrial ATP synthase-D. In conclusion, this investigation has revealed a "yin-yang" relationship between PRL and PGF(2 alpha) in regulating certain critical genes in the rodent CL, and has demonstrated novel regulation by these factors of other important genes involved in luteal function.

Characterization of a rat uterine cell line, U(III) cells: prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor beta, alpha(2)-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway.

Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (U(III)) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, U(III) cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor beta (ER beta) and alpha(2)-macroglobulin (alpha(2)-MG), which are known to be PRL-regulated genes. However, when U(III) cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 and the transcription factor Stat5 were endogenously phosphorylated and their phosphorylation states were not enhanced in the presence of exogenous PRL. To examine whether the endogenously secreted PRL affects the expression of PRL-regulated genes, U(III) cells were treated with either an anti-PRL receptor antibody or a Jak2 inhibitor, AG490. The anti-PRL receptor antibody decreased alpha(2)-MG expression. AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding to its DNA consensus sequence, and also caused a dose-dependent down-regulation of alpha(2)-MG and ER beta expression. In contrast, AG490 enhanced PRL mRNA levels. In summary, we have established that the U(III) stromal cells of uterine origin produce PRL. Furthermore, we have shown for the first time that decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-regulate important genes involved in decidual growth and placentation.

Janus kinase 2 (JAK2) regulates prolactin-mediated chloride transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na+-K+-2Cl- cotransporter.

Epithelial chloride (Cl-) transport is achieved by the coordinated action of symporters such as the Na+-K+-2Cl- cotransporter (NKCC1) and chloride channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). As a secretory tissue, mammary epithelial cells are obvious candidates for such mechanisms, but Cl- transport and its hormonal regulation have been poorly delineated in mammary epithelial cells. We determined whether the mammary epithelial cell line, HC11, transports chloride and whether this was regulated by PRL, a hormone known to stimulate ion transport. HC11 cells express both CFTR and NKCC1. Exposure to PRL or PGE1 increased Cl- transport in HC11 cells. This was inhibited by the NKCC1 blocker, furosemide, and by the Cl- channel inhibitor, diphenylamine 2-carboxylate. Dose and time course of PRL action indicate that PRL had maximal effect on Cl- transport at 1 microg/ml and at 10 min of stimulation. Examination of the signaling pathways suggests that the PRL effect on Cl- transport does not involve an increase in [Ca2+]i or MAP kinase activity. RT-PCR analyses indicate that HC11 cells express mRNA for Janus kinase 1 (JAK1), JAK2, and signal transducer and activator of transcription 5 (STAT5) but not for JAK3. PRL treatment of HC11 cells increased phosphorylation of STAT5. The JAK2 inhibitor AG490 blocked phosphorylation of STAT5 and PRL-induced, but not PGE1-induced, Cl- transport. NKCC1, but not CFTR, is tyrosine phosphorylated in HC11 cells. PRL enhanced tyrosine phosphorylation of NKCC1, and this effect was attenuated by the JAK2 inhibitor AG490. These results are the first demonstrations of a role for tyrosine phosphorylation of NKCC1 and of the PRL-JAK2 cascade in the regulation of Cl- transport.

Estrogen receptors alpha and beta in rat decidua cells: cell-specific expression and differential regulation by steroid hormones and prolactin.

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), alpha and beta. In this investigation, we analyzed the expression of decidual ERalpha and ERbeta, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERp to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERalpha and ERbeta by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERbeta messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERalpha expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERalpha and had no synergistic effect on ERbeta expression. The use of GG-AD cells, which express only ERbeta, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERbeta is expressed in addition to ERalpha in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERalpha, but concomitantly increases the expression of a functional ERbeta that mediates estradiol up-regulation of the decidual progesterone receptor.

Prostaglandin F2alpha-induced expression of 20alpha-hydroxysteroid dehydrogenase involves the transcription factor NUR77.

Prostaglandin F(2)alpha (PGF(2)alpha) binding to its receptor on the rat corpus luteum triggers various signal transduction pathways that lead to the activation of a steroidogenic enzyme, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which in turn catabolizes progesterone. The molecular mechanism underlying PGF(2)alpha-induced 20alpha-HSD enzyme activity has not yet been explored. In this report we show, using mice lacking PGF(2)alpha receptor and pregnant rats, that PGF(2)alpha is responsible for the rapid and massive expression of the 20alpha-HSD gene at the end of pregnancy leading to a decrease in progesterone secretion. We also present evidence that PGF(2)alpha enhances 20alpha-HSD promoter activity. We have determined a region upstream of the -1590 position in the 20alpha-HSD promoter that confers regulation by PGF(2)alpha in ovarian primary cells. This region encompasses a unique transcription factor-binding site with a sequence of a NUR77 response element. Deletion of this motif or overexpression of a NUR77 dominant negative protein caused a complete loss of 20alpha-HSD promoter activation by PGF(2)alpha. NUR77 also transactivated the 20alpha-HSD promoter in transient transfection experiments in corpus luteum-derived cells (GG-CL). This induction required the NUR77-transactivating domain. We also show that PGF(2)alpha induces a very rapid expression of NUR77 that binds to a distal response element located at -1599/-1606 but does not interact with another proximal putative NUR77 response element located downstream in the promoter. A rapid increase in NUR77 mRNA was observed in mice corpora lutea just before parturition at a time when 20alpha-HSD becomes expressed. This increase in the expression of both genes was not seen in PGF(2)alpha receptor knockout mice. By using cyclosporin A and PGF(2)alpha treatment, we established that inhibition of NUR77 DNA binding in vivo prevents PGF(2)alpha induction of the 20alpha-HSD gene in the corpus luteum. Taken together, our results demonstrate, for the first time, that PGF(2)alpha induces in the corpus luteum the expression of the nuclear orphan receptor and transcription factor, NUR77, which in turn leads to the transcriptional stimulation of 20alpha-HSD, triggering the decrease in serum progesterone essential for parturition.

Involvement of SOCS-1, the suppressor of cytokine signaling, in the prevention of prolactin-responsive gene expression in decidual cells.

The cells forming the rat decidua produce PRL and PRL-related proteins and express both the long and short forms of the PRL receptor. Yet, only a defined subpopulation, the mesometrial cells, express the PRL-dependent alpha2-macroglobulin gene. This gene is silenced in vivo in the antimesometrial cells and in the GG-AD cell line, derived from antimesometrial cells. To examine whether the lack of alpha2-macroglobulin expression is due to defective components in the PRL signaling pathway, we compared the relative expression of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 a and b (Stat5 a and b), suppressor of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2 mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of pseudopregnancy, the time of maximal alpha2-macroglobulin expression. We found no significant differences in the relative expression of either Jak2, Stat5 (a and b), or SHP-2 in the two cell populations. However, we discovered a profound difference in the expression of SOCS-1, an inhibitor of the Jak/Stat pathway. This gene was highly expressed in the antimesometrial cells and in the GG-AD cells, which do not produce alpha2-macroglobulin. Immunoprecipitation experiments with GG-AD cells revealed that although Jak2 and Stat5 coprecipitate in response to PRL stimulation, no phosphorylation of Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a role in silencing the alpha2-macroglobulin gene, we cultured GG-AD cells in the presence of either a SOCS-1 antisense oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28 h. Cells were also treated with PRL. Within 4 h of SOCS-1 antisense treatment, alpha2-macroglobulin mRNA expression was initiated. After 28 h, only cells treated with PRL and SOCS-1 antisense oligonucleotide retained the ability to express the alpha2-macroglobulin gene. In summary, results of this study reveal that constitutive expression of SOCS-1 can prevent PRL signaling and that the lack of PRL-induced expression of alpha2-macroglobulin in a defined decidual cell population is largely due to SOCS-1 expression in these cells.

Rat decidual prolactin. Identification, molecular cloning, and characterization.

Establishment and maintenance of pregnancy require the activity of a highly specialized maternal tissue, the decidua. It is well established that the human decidua synthesizes and releases prolactin. However, in the rat, no study has been able to demonstrate the production of prolactin by the decidua. In this report, we established for the first time using Northern blot analysis and reverse transcription-polymerase chain reaction, Western blot analysis, immunocytochemistry, and enzyme-linked immunosorbent assay, that a defined cell population located in the rat antimesometrial decidua expresses prolactin mRNA, as well as synthesizes and secretes this hormone. By reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, we cloned a full-length cDNA for rat decidua prolactin, whose sequence was identical to that of pituitary prolactin. Our results also showed that pituitary prolactin appeared to down-regulate decidual prolactin levels. Under these circumstances, inhibition of pituitary prolactin secretion led to a rise in both decidual prolactin mRNA and protein expression. Moreover, addition of exogenous prolactin to primary decidual cells in culture also caused a marked decrease in decidual prolactin mRNA expression. Finally, treatment of primary decidual cells with steroid hormones or 8-bromo-cAMP revealed a differential regulation of decidual prolactin expression from that of pituitary suggesting a tissue-specific regulation of prolactin gene expression, possibly through the use of an alternative promoter in rat decidua.

Expression of prolactin and its receptor in the baboon uterus during the menstrual cycle and pregnancy.

PRL is known to be a major secretory product of the human decidua. However, the physiological role of decidual PRL during the menstrual cycle and pregnancy has not been fully defined, primarily due to the lack of an appropriate nonhuman primate model for in vivo studies. Therefore, this initial study examined the expression of PRL and its receptor in the baboon uterus during the cycle and pregnancy. PRL and PRL receptor messenger ribonucleic acid expression were detected by semiquantitative RT-PCR, and protein was localized by immunocytochemistry. PRL was shown to be expressed in myometrial smooth muscle during the follicular phase by both RT-PCR and immunocytochemistry. Expression of PRL messenger ribonucleic acid and protein was first observed in the epithelial cells of the deep basal glands during the late luteal phase. With the onset of pregnancy, PRL expression increased steadily and was evident primarily in the decidual tissue. In contrast to PRL, its receptor was expressed at constant levels in both the myometrium and endometrium during the cycle. An increase in receptor expression was evident in both the decidua and placenta throughout pregnancy. In summary, these results demonstrate that the baboon uterus is a site of both PRL production and action during the cycle and pregnancy. These studies establish the baboon as a nonhuman primate model to investigate the potential roles of PRL in implantation and maintenance of pregnancy.

The expression of interleukin-6 (IL-6), IL-6 receptor, and gp130-kilodalton glycoprotein in the rat decidua and a decidual cell line: regulation by 17beta-estradiol and prolactin.

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-beta (ERbeta). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0-100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-beta.

Regulation and role of the insulin-like growth factor I system in rat luteal cells.

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor beta mRNA contents were determined by semiquantitative RT-PCR. The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor beta. Furthermore, whereas IGF-I upregulated oestrogen receptor beta expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor beta. Luteal cells with increased oestrogen receptor beta can respond fully to oestradiol, leading to cell hypertrophy.

Developmental expression and regulation of basic fibroblast growth factor and vascular endothelial growth factor in rat decidua and in a decidual cell line.

During pregnancy, the decidua is comprised of two separate tissues located either mesometrially or antimesometrially in the uterus. Trophoblast invasion takes place only in the mesometrial decidua, where extensive angiogenesis, essential for successful implantation, occurs. Both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been implicated in this phenomenon. The aim of this study was to determine whether the expression of both growth factors is intrinsic to decidua and occurs in the absence of conceptuses, whether their genes are expressed specifically in the mesometrial decidua, the site of angiogenesis, and whether both growth factors are developmentally and hormonally regulated. Decidual tissue was dissected from pseudopregnant rats and levels of both bFGF and VEGF mRNA were examined in mesometrial and antimesometrial decidua by semi-quantitative RT-PCR at different stages of pseudopregnancy. Although induction of decidualization triggered the mRNA expression of bFGF, VEGF mRNA expression remained unchanged. VEGF mRNA level was similar in both antimesometrial and mesometrial decidua, and remained constant throughout pseudopregnancy. In sharp contrast, bFGF mRNA was highly expressed in the mesometrial decidua at a time when extensive angiogenesis takes place in this tissue. Very little signal was observed in the antimesometrial decidua. To examine the regulation of these growth factors, we used a temperature-sensitive decidual cell line developed by transforming antimesometrial decidual cells with SV-40 tsA 209 mutant virus. These cells express both bFGF and VEGF mRNA. Because progesterone is necessary for decidualization and decidua secretes prolactin (PRL)-related hormones, we examined the role of these hormones on VEGF and bFGF mRNA expressions. Neither progesterone nor PRL had any effect on VEGF mRNA levels. However, bFGF mRNA expression was greatly stimulated by PRL. In conclusion, results of this investigation have revealed that bFGF, but not VEGF, mRNA becomes highly expressed in the mesometrial decidua, where angiogenesis occurs, and where trophoblasts, by invading decidual cells, may promote the release of bFGF. In addition, these results indicate that the locally secreted PRL-like hormone up-regulates the mRNA expression of bFGF.

Molecular cloning and characterization of the rat ovarian 20 alpha-hydroxysteroid dehydrogenase gene.

The rat 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is an enzyme responsible for the catabolism of progesterone to the inactive 20 alpha-hydroxprogesterone. We have previously shown that the expression of this enzyme is not regulated by post-translational modification, but at the level of transcription. In this study we have established that the 20 alpha-HSD gene contains nine exons and have isolated a 2.5 kb promoter region. The transcription start site was identified and a TATA box was found. 5' deletions of this promoter significantly decreased basal promoter activity. Treatment with forskolin led to a dose dependent inhibition of the 2.5kg-20 alpha-HSD-luciferase construct. Computer analysis identified one CRE, two Nur77 response elements, two putative AP1 sites and one progesterone response element half-site. In summary, we have identified and partially characterized the promoter region of the rat ovarian 20 alpha-HSD and demonstrated that the regulatory elements for 20 alpha-HSD are present within a 2.5 kb 5' flanking region of the gene.

Hormonal regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase messenger ribonucleic acid in the rat corpus luteum: induction by prolactin and placental lactogens.

The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,Zn-SOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

The expression of interleukin-6 in the pregnant rat corpus luteum and its regulation by progesterone and glucocorticoid.

Interleukin (IL)-6, a multifunctional cytokine originally described as a T cell-derived factor, is also produced by different cell types, and it influences a wide variety of physiological and pathophysiological processes. Recent studies further suggest that IL-6 has a role in down-regulating hormone production by endocrine organs and can negatively affect the steroidogenic capacity of both ovaries and testes. Thus, the aims of this investigation were to examine whether IL-6 plays a role in luteolysis and, more specifically, to determine whether luteal cells express the IL-6 gene, whether this expression is developmentally and hormonally regulated in pregnancy, and whether the corpus luteum could be a target for IL-6 action. Using semiquantitative RT-PCR, messenger RNA (mRNA) encoding both components of the IL-6 receptor [the ligand-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130)] were found to be highly expressed in corpora lutea throughout pregnancy. In contrast, IL-6 mRNA expression was barely detectable from day 4 through the end of pregnancy, whereas a sharp and abrupt expression of IL-6 mRNA occurred immediately after parturition. Although the corpus luteum does not express IL-6 mRNA during most of pregnancy, it could be induced to express this gene with an in vivo injection of the bacterial endotoxin, lipopolysaccharide. In addition, when corpora lutea from day-15 pregnant rats were isolated and maintained in culture, IL-6 mRNA that was undetectable at 0 h increased in a time-related manner and reached significant levels after 4 h of incubation, followed by a similar increase in IL-6 protein secreted in the culture media. Isolation of the small and large luteal cells by elutriation indicated that both cell populations can secrete IL-6 in culture. The apparent ability of luteal cells to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression during most of pregnancy, led us to examine whether the IL-6 gene is silenced throughout pregnancy by luteotropic hormones. Corpora lutea from day-15 pregnant rats were cultured in the presence of different doses of progesterone; the synthetic glucocorticoid, dexamethasone; 17beta-estradiol; and PRL. Progesterone and dexamethasone markedly inhibited IL-6 mRNA expression, whereas 17beta-estradiol had a minimal inhibitory effect, and PRL did not affect IL-6 mRNA expression. In summary, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and that luteal cells are able to secrete IL-6. However, IL-6 gene expression is silenced during most of pregnancy, probably by the high levels of progesterone locally produced in the corpus luteum. The salient finding that progesterone and glucocorticoid strongly inhibit the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might have a deleterious effect on luteal function.

Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase in the rat corpus luteum: induction of manganese superoxide dismutase messenger ribonucleic acid by inflammatory cytokines.

This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1alpha (IL-1alpha), IL-beta, IL-6, and tumor necrosis factor alpha. LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,Zn-SOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.