Inhibition of IL-6 signaling pathway by curcumin in uterine decidual cells.

IL-6 is a multifunctional pro-inflammatory cytokine and has been implicated in many gestational disorders including preterm birth. Currently, there are no appropriate therapeutic interventions available to circumvent inflammatory-mediated gestational disorders. Therefore, the goal of this study was to identify a safe and effective pharmacological compound to counterbalance inflammatory responses in the uterus. Curcumin, a naturally-occurring polyphenolic compound, has been widely used in alternative medicine to treat inflammatory diseases. However, the anti-inflammatory effect of curcumin has not been explored in uterine decidual cells, a major source of IL-6. Therefore, we examined the effect of curcumin on IL-6 expression using two types of uterine decidual cells 1) HuF cells, primary human fibroblast cells obtained from the decidua parietalis; 2) UIII cells, a rodent non-transformed decidual cell line. Curcumin treatment completely abrogated the expression of IL-1ß-induced IL-6 in these cells. Curcumin also strongly inhibited the expression of gp130, a critical molecule in IL-6 signaling, whereas expression of IL-6R and sIL-6R was not affected. Curcumin also inhibited phosphorylation and nuclear localization of STAT3, a well-known downstream mediator of IL-6 signaling. Furthermore, curcumin attenuated IL-1ß-induced IL-6 promoter reporter activity suggesting transcriptional regulation. To further understand whether NF-Ò¡B is involved in this inhibition, we examined the effect of curcumin on the expression of p50 and p65 subunits of NF-Ò¡B in decidual cells. Expression of IL-1ß-induced p50 mRNA was repressed by curcumin while p65 mRNA was not affected. However, curcumin treatment dramatically inhibited both p50 and p65 protein levels and prevented its nuclear localization. This effect is at least partly mediated through the deactivation of IKK, since IL-1ß-induced IKKα/ß phosphorylation is decreased upon curcumin treatment. Our results not only revealed molecular mechanisms underlying curcumin action in uterine decidual cells but also suggest that this compound may have therapeutic potential for the prevention of inflammation-mediated preterm birth and other gestational disorders.

Prolactin and dexamethasone regulate second messenger-stimulated cl(-) secretion in mammary epithelia.

Mammary gland ion transport is essential for lactation and is regulated by prolactin and glucocorticoids. This study delineates the roles of prolactin receptors (PRLR) and long-term prolactin and dexamethasone (P-D)-mediation of [Ca(2+)](i) and Cl(-) transport in HC-11 cells. P-D (24 h) suppressed ATP-induced [Ca(2+)](i). This may be due to decreased Ca(2+) entry since P-D decreased transient receptor potential channel 3 (TRPC3) but not secretory pathway Ca(2+)-ATPase 2 (SPCA2) mRNA. ATP increased Cl(-) transport, measured by iodide (I(-)) efflux, in control and P-D-treated cells. P-D enhanced I(-) efflux response to cAMP secretagogues without altering Cl(-) channels or NKCC cotransporter expression. HC-11 cells contain only the long form of PRLR (PRLR-L). Since the short isoform, PRLR-S, is mammopoietic, we determined if transfecting PRLR-S (rs) altered PRLR-L-mediated Ca(2+) and Cl(-) transport. Untreated rs cells showed an attenuated [Ca(2+)](i) response to ATP with no further response to P-D, in contrast to vector-transfected (vtc) controls. P-D inhibited TRPC3 in rs and vtc cells but increased SPCA2 only in rs cells. As in wild-type, cAMP-stimulated Cl(-) transport, in P-D-treated vtc and rs cells. In summary, 24 h P-D acts via PRLR-L to attenuate ATP-induced [Ca(2+)](i) and increase cAMP-activated Cl(-) transport. PRLR-S fine-tunes these responses underscoring its mammopoietic action.

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 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.

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.

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.

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.

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.

Involvement of cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) in interleukin 11 stimulation of decidualization in mice.

Interleukin 11 receptor alpha (Il11ra) null mice are infertile due to defective decidualization and abnormal trophoblast invasion. We have previously shown in these mice that downregulation of decidual proteinase inhibitors plays a role in uncontrolled trophoblast invasion. However, the decidua is abnormally smaller in pseudopregnant Il11ra null mice, where trophoblast invasion is not a factor. Here, we examined whether defective decidualization is due to dysregulation of key molecules involved in decidual cell growth and differentiation. We found a dramatic downregulation of cyclin D3 in Il11ra null mice. We also found that IL11 robustly stimulates the expression of cyclin D3 in cell culture. CDK4 and CDK6, known partners of cyclin D3, are not affected. Immunolocalization studies show absence of cyclin D3 in the mesometrial site and absence of differentiated polyploid cells in the antimesometrial site of Il11ra null mice. We also examined the expression of cell differentiation factors CDKN1A (p21) and CDKN1B (p27), and found that in both in vivo and cell culture the expression of CDKN1A (p21) but not CDKN1B (p27) is under the control of IL11. Another clear target of IL11 in the decidua is BIRC5 (Survivin), whose expression is repressed in the decidua of Il11ra null mice and stimulated by IL11 in cell culture. Taken together, these results provide, at least in part, an explanation for the defective small decidua of mice lacking the Il11ra gene, and reveal for the first time that cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) are targets of IL11 in the decidua.

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.

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.