NADPH oxidase-derived reactive oxygen species mediate decidualization of human endometrial stromal cells in response to cyclic AMP signaling

Differentiation of human endometrial stromal cells into specialized decidual cells is critical for embryo implantation and survival of the conceptus. Initiation of this differentiation process is strictly dependent on elevated cAMP levels, but the signal intermediates that control the expression of decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly characterized. Here we show that cAMP-dependent decidualization can be attenuated or enhanced upon treatment of primary cultures with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) or activator (apocynin), respectively. Time-course analysis demonstrated that cAMP enhances endogenous reactive oxygen species production, apparent after 12 h of stimulation, which coincides with a dramatic increase in decidual PRL and IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of CCAAT/enhancer binding protein-β, a key regulator of human endometrial stromal cell differentiation. Thus, NOX-4 activation and reactive oxygen species signaling play an integral role in initiating the endometrial decidual response in preparation of pregnancy.     

Transdominant suppression of estrogen receptor signaling by progesterone receptor ligands in uterine leiomyoma cells

Uterine leiomyomas develop in reproductive-age women with high frequency and are dependent on the production of ovarian hormones. While it is generally accepted that these tumors are estrogen (E(2))-responsive, the role of progesterone (P(4)) in modulating tumor growth is less clear. In the present study, an in vivo/in vitro rat model was used to characterize progesterone receptor (PR) isoform expression in uterine leiomyoma and investigate PR signaling using progestins and antiprogestins in the leiomyoma-derived cell line ELT-3. PR-A was the predominant isoform expressed in normal myometrium, leiomyomas and ELT3 cells. In the normal myometrium, PR-A and PR-B levels varied during the estrous cycle with low ratios of PR-A relative to PR-B (PR-A/PR-B) coinciding with times of cell proliferation. Although PR ligands had no effect on basal levels of uterine leiomyoma cell proliferation in vitro, both progestins and antiprogestins inhibited E(2)-stimulated cell proliferation. In addition, E(2)-stimulated transactivation of an estrogen-response-element reporter gene as well as E(2)-induced upregulation of the PR were also inhibited by PR ligands. These data indicate that PR ligands can transdominantly suppress estrogen receptor signaling and stimulation of uterine leiomyoma cell growth.     

Activin A promotes human endometrial stromal cell decidualization in vitro

Decidualization of the endometrium is critical for implantation and placental development. Stromal cells differentiate causing widespread tissue remodeling. The mechanisms involved are unknown, although a number of known paracrine factors play contributory roles. From its known actions on cell differentiation and remodeling, we hypothesized that activin A regulates decidualization. Stromal cells produce activin beta A subunits with the onset of decidualization, and these cells coexpress activin receptors. Utilizing an in vitro model of stromal cell decidualization, we demonstrate that high concentrations of dimeric activin A are produced by decidual cells. Addition of activin A to cells undergoing decidualization resulted in a dose-dependent increase in PRL production, an established marker of decidualization. This response was inhibited by co-treatment with follistatin. Neutralization of endogenous activins significantly reduced the decidual response. This is the first report of dimeric activin A production by decidualized cells, and is evidence for a autocrine/paracrine action of activin A within the endometrium. The demonstration that activin A enhances the decidual reaction in vitro suggests that it plays a key role in promoting stromal cell decidualization. This data provides insights into the processes underlying decidualization, which is important for understanding implantation and placentation and has potential clinical applications for the regulation of fertility.     

FOXO1A differentially regulates genes of decidualization

The forkhead box O1A (FOXO1A) has been identified as one gene that is up-regulated early in the decidualization process. To further investigate the role of FOXO1A during this process, six genes, IGFBP1, PRL, TIMP3, LAMB1, CNR1, and DCN, shown to be up-regulated during decidualization, were chosen as potential targets of FOXO1A action. Treatment of human endometrial stromal cells with hormones (estradiol and medroxyprogesterone acetate) plus dibutyryl cAMP (H+dbcAMP) for 48 h increased expression of IGFBP1, PRL, TIMP3, CNR1, and DCN but not LAMB1, as measured by real-time PCR. Silencing of FOXO1A using small interfering RNA oligonucleotides decreased IGFBP1 and DCN levels and increased CNR1, TIMP3, and PRL levels. LAMB1 was not affected. When FOXO1A was overexpressed in human endometrial stromal cells, expression of IGFBP1, DCN, and PRL increased, whereas levels of TIMP3 and CNR1 decreased. Addition of H+dbcAMP caused an increased expression of IGFBP1, PRL, and DCN beyond that of FOXO1A alone. TIMP3 and CNR1 levels decreased even further in response to H+dbcAMP compared with FOXO1A alone. LAMB1, which was unresponsive to FOXO1A, decreased when H+dbcAMP was added. Overexpressing FOXO1A also caused a change in cell shape, in that the stromal fibroblasts acquired a rounded, epithelioid appearance. Finally, reporter studies showed that cotransfection of FOXO1A significantly increased PRL promoter activity but not TIMP3 promoter activity. Addition of H+dbcAMP resulted in a significant increase in PRL promoter activity and a significant decrease in TIMP3 promoter activity. In summary, this study demonstrates the versatile nature of FOXO1A in the regulation of a number of decidualization-specific genes.     

Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor alpha knockout mouse uterus

Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor alpha knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor alpha modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.     

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.     

Cyclic AMP enhances progesterone action in human myometrial cells

Cyclic AMP (cAMP) has been shown to promote progesterone and glucocorticoid action in a variety of cellular settings. In this study, we have used human myometrial cells to investigate whether cAMP potentiates the ability of progesterone to repress IL-1β-driven COX-2 expression. We found that forskolin enhanced progesterone-repression of IL-1β-driven COX-2 expression in association with delayed IL-1β-induced nuclear phospho-p65 entry and reduced NF-κB binding to the COX-2 promoter. Further, forskolin enhanced the progesterone-induced expression of FKBP5 and 11βHSD1, progesterone-driven activity of a progesterone response element (PRE) and progesterone receptor (PR)-B binding to a transfected PRE. In addition, forskolin treatment increased PR-B levels and reduced the PR-A:PR-B ratio while acutely decreasing the association between PR and nuclear receptor co-repressor (NCoR) and reducing NCoR levels after 6 h. These findings are of importance in situations where enhancing progesterone activity is desirable, for example in the management of endometrial cancer, the promotion of endometrial receptivity or the maintenance of myometrial quiescence during pregnancy.     

Estrone sulfate sulfatase activity is increased during in vitro decidualization of stromal cells from human endometrium

Arylsulfatase (EC 3.1.6.1) activity in human stromal cells isolated from specimens of histologically normal proliferative endometrium was increased several-fold during culture for 8-15 days in RPMI-1640 medium plus 10% charcoal-treated fetal bovine serum in the presence of a mixture of ovarian hormones (36 nM estradiol, 1 microM medroxyprogesterone acetate, and 100 micrograms/mL relaxin). The changes in sulfatase activity, determined by measuring the rate of formation of estrone from tritiated estrone sulfate, were associated with in vitro decidualization of the stromal cells, as determined by changes in secretion of PRL into daily renewed culture medium. PRL output by the cells during the last 24 h in culture and sulfatase activity in the cells collected at the end of the culture period were related to their DNA and protein contents. Sulfatase activity in the cells cultured in the presence of the ovarian hormones was comparable to the activity found in decidual cells at term pregnancy. PRL added for 1 day to cultures of stromal cells in the absence of exogenous hormones increased sulfatase activity in the cells, probably by acting in an autocrine manner, as previously demonstrated with human decidual cells during pregnancy. These experiments also revealed a hormonal regulation of stromal cell proliferation in vitro, as estimated from measurements of both DNA and protein levels per dish. Augmentation of sulfatase activity can serve as another marker of in vitro decidualization. Physiologically, an increase in this enzymatic activity may result in a preferential estrogenic stimulation of the decidualized cells by utilization of a circulating substrate, estrone sulfate. This hypothesis could explain the preferential retention of progesterone receptors in decidual cells observed immunohistochemically during the late luteal phase of the menstrual cycle, suggestive of a shift in progestogenic actions from the epithelium to the stroma.     

Differential expression and regulation of progesterone receptor isoforms in rat and mouse pituitary cells and LbetaT2 gonadotropes

Manipulation of endogenous progesterone receptor (PR) does not produce equivalent physiological effects in mouse and rat pituitary cells. To test whether this may be due in part to difference in PR isoform expression, we examined hormonally regulated pituitary PR-A and PR-B mRNA levels using quantitative real-time PCR. The LbetaT2 mouse gonadotrope line or pituitary cells from adult, ovariectomized rats or mice were cultured with or without 0.2 nM 17beta-estradiol (E(2)) for 3 days. PR-A was the predominant form expressed for all groups. For mouse cells, E(2) led to an increase in both isoforms without a change in the A:B ratio; for rat cells, the PR-B response to E(2) was more robust resulting in a decrease in the A:B ratio. Exposure of E(2)-treated pituitary cells to 200 nM progesterone for 6 h decreased both PR-A and PR-B levels in rat cells, but had no effect on PR isoform expression in mouse cells even when exposure was extended to 12 h. The low level of PR expression found in LbetaT2 gonadotropes was unaffected by E(2), alone or with progesterone. The weak PR expression and lack of responsiveness of LbetaT2 cells cannot be explained by a male phenotype as was shown by the more than tenfold higher PR mRNA level in primary cultures of male mouse pituitary cells, which responded to E(2) stimulation with a proportional increase in PR isoforms similar to female cells. Functionally, E(2)-stimulated changes in PR mRNA isoform ratios in rat, mouse or LbetaT2 cells correlated with the degree of progesterone augmentation of GnRH-stimulated LH secretion in these models. These results are consistent with the hypothesis that robust GnRH priming and progesterone augmentation of LH secretion in the rat compared to these events in the mouse are a consequence, in part, of differences in the E(2)-modulated ratio of PR isoforms.     

Phosphodiesterase 4 inhibition synergizes with relaxin signaling to promote decidualization of human endometrial stromal cells

The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.