Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.

Human endometrial stromal (ES) cells in culture express PRL, a marker of decidualization, in response to sustained activation of protein kinase A (PKA). Cotreatment with the progestin medroxyprogesterone acetate (MPA) enhanced decidual PRL gene activation in the presence of elevated intracellular cAMP levels. This synergy became apparent, at protein and promoter level, after a lag period of 2 days and increased in a time-dependent manner thereafter. Pretreatment with cAMP advanced the time at which synergy between cAMP and MPA was apparent, suggesting that PKA activation sensitized ES cells to the effects of progestins. Analysis of the progesterone receptor (PR) indicated that PR-A was the predominant form in differentiating ES cells, but its abundance decreased markedly during the course of the decidualization response. The decline in PR levels was of functional relevance, as expression of PR-B or PR-A, by transient transfection, dramatically inhibited the activity of a decidual PRL promoter-reporter construct in response to cAMP. Furthermore, the expression of endogenous PRL protein in response to cAMP or cAMP plus MPA was substantially decreased by constitutive expression of green fluorescence protein-tagged PR, which was localized in the nucleus even in the absence of added ligand. Ligand-independent PR inhibition of the decidual PRL promoter was receptor specific, independent of known PR phosphorylation sites, and required minimally a functional DNA-binding domain. Transient expression of steroid receptor coactivator-1e (SRC-1e), but not SRC-1a, allowed synergy between cAMP and MPA without the requirement of sensitization by pretreatment with cAMP. This raised the possibility that SRC-1e was a component of cAMP-dependent sensitization of ES cells, but there was no evidence of altered messenger RNA expression of either SRC-1 isoform during decidualization. In conclusion, cellular PR levels determine the onset of the decidualization response. Initiation of this process requires elevated intracellular cAMP levels that sensitize ES cells to the actions of progestins through down-regulation of cellular PR levels and possibly via modulation of function of an intermediate factor(s) such as SRC-1e.     

Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells

Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).     

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.     

Premenstrual disappearance of aminopeptidase A in endometrial stromal cells around endometrial spiral arteries/arterioles during the decidual change

Aminopeptidase A (APA, BP-1) is a membrane-bound zinc metallopeptidase that converts angiotensin II (AngII) into AngIII by selectively hydrolyzing the N-terminal aspartyl residue. AngII has been proposed as a candidate for the initial vasoconstrictor of endometrial spiral arteries/arterioles in the preliminary step of menstruation. In the late secretory phase, endometrial stromal cells (ESC) around the blood vessels begin to differentiate into decidual cells, and AngII has been reported to accumulate around such vessels. However, whether there is a concurrent increase in renin or angiotensin-converting enzyme in this area has not been determined. We hypothesized that APA may be involved in the metabolism of AngII in the cycling endometrium. Western blot analysis in the present study demonstrated that a considerable amount of APA was present in the secretory phase endometrium. ESC in the secretory phase showed strong expression of APA by immunohistochemical analysis and of APA mRNA by in situ hybridization. In contrast, both APA mRNA and protein were absent in decidual cells. The enzyme activity and the biosynthesis of [(35)S]methionine-labeled APA significantly decreased during the in vitro decidualization of cultured ESC. These results suggest that the perivascular disappearance of APA is a differentiation-specific change that occurs along with the decidualization, and that the disappearance of APA might accelerate the accumulation of AngII around the vessels.     

The role of human chorionic gonadotropin on decidualization of endometrial stromal cells in vitro

Although decidualization of endometrial stromal cells (ESC) is crucial for blastocyst implantation and maintenance of pregnancy, its complex mechanism still remains largely unknown. It has long been believed that hCG can directly induce in vitro decidualization of ESC via cAMP signaling. Recently, however, it has been reported that the LH/CG receptor is not present in human endometrium, and the direct effect of hCG on decidualization has become controversial. To reevaluate the exact effect of hCG on decidualization, human ESC were isolated and cultured with hCG and/or ovarian steroids. ESC treated with 17beta-estradiol plus progesterone (E(2)/P) transformed morphologically and produced significant PRL, whereas ESC treated with hCG alone showed no significant increase in PRL in culture medium and exhibited no morphological changes. Moreover, hCG did not promote E(2)/P-induced PRL production or intracellular cAMP accumulation, and protein kinase A inhibitor failed to block E(2)/P-induced PRL production. These results suggest that hCG does not directly affect in vitro decidualization of human ESC and that the process of E(2)/P-induced in vitro decidualization might consist of several pathways, including the intracellular cAMP signaling cascade.     

Differential effects of progestin and relaxin on the synthesis and secretion of immunoreactive prolactin in long term culture of human endometrial stromal cells

PRL secretion from human endometrium is a continuous process extending from the luteal phase of the menstrual cycle throughout the entire gestational stage. We have developed a long term primary cell culture system to elucidate the hormonal requirements for this sustained production of PRL. The effects of medroxyprogesterone acetate (MPA), progesterone, and relaxin (RLX) on the production of immunoreactive PRL were investigated. MPA stimulated cell growth and PRL production rate during days 5-20 of culture. Progesterone was 20-40% less effective in stimulating PRL than MPA. Stimulation of PRL was continued 1-2 weeks after MPA withdrawal. Relaxin did not promote cell growth. However, it induced the PRL production which fluctuated during the long term culture. The maximal response to RLX was 2- to 3-fold higher or similar to that of MPA. Only five of nine endometrial specimens examined responded to RLX alone. The effect of MPA plus RLX was significantly greater than that of MPA or RLX alone. The highest production rate was shown in cells treated with MPA and then RLX in sequence. After a month of culture, the production rates (micrograms of PRL per 0.1 mg cell DNA/day) under various culture conditions (A, control; B, MPA; C, MPA for 10-15 days and no hormone afterward; D, both MPA and RLX; and E, MPA and RLX in sequence) were: A, about 0-0.01 (n = 12); B, 2.5 +/- 0.9 (n = 8); C, 4.8 +/- 2.5 (n = 8); D, 5.7 +/- 3.0 (n = 5); and E, 11 +/- 3.7 (n = 7); mean +/- SD; n, number of specimens). Endometrial stromal cells were incubated with [35S]methionine, and [35S]immunoreactive PRL and other secretory proteins were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to characterize the size and isoforms of immunoreactive PRL. PRL was one of the five major secretory proteins (23-25K, 32K, 42K, 78K, and 150K daltons, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition) induced by MPA and RLX in endometrial stromal cells. More than 90% of immunoreactive PRL was secreted into the medium. The apparent mol wt of immunoreactive PRL were 21K, 23K (the predominant size), and 25K daltons. Results obtained from the incorporation of [14C]glucosamine into immunoreactive PRL indicated that both 23K and 25K PRL contained glycosylated PRL. A 45K-dalton glycosylated immunoreactive PRL was also present in the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells.

The decidualized endometrium during the first trimester of pregnancy synthesizes and secretes a 32-kDa insulin-like growth factor-binding protein (termed hIGFBP-1) at high levels. IGFBP-1 is the major soluble protein product of this tissue and is principally localized to the differentiated endometrial stromal cell, the decidual cell. In the present study long term culture of stromal cells from the nonpregnant endometrium have been employed to elucidate the hormonal requirements for IGFBP-1 production. Immunoreactive IGFBP-1 was undetectable in control cultures. However, inclusion of medroxyprogesterone acetate (MPA) induced rates of 0.35 +/- 0.09 microgram/0.1 mg cell DNA.day (mean +/- SEM; n = 5) after 20-30 days. In these cultures cells exhibited morphological changes consistent with decidual cell differentiation. In all cultures removal of MPA after exposure for 10-16 days, with or without subsequent inclusion of relaxin (RLX), increased production of IGFBP-1 450- to 4600-fold to rates of 150-710 micrograms/0.1 mg cell DNA.day or 26-131 micrograms/10(6) cells.day on days 24-26. The rates tended to be higher with the inclusion of RLX and were sustained in contrast to cultures without RLX, where rates fell by day 30. Individual cultures responded differently to RLX when added from the initiation of culture, with either a response similar to MPA alone or a cyclical change in production, achieving maximal rates of 190-290 micrograms/0.1 mg cell DNA.day. Cultures in which RLX alone induced high IGFBP-1 high production were obtained from endometrium during the progesterone-dominated luteal phase. In cultures exhibiting high rates of immunoreactive IGFBP-1 production, the protein represented their major secretory protein product. This was confirmed by [35S]methionine incorporation and the presence of IGFBP-1 as the predominant protein in serum-free culture medium. The immunoreactive IGFBP-1 isolated from culture medium was found to be identical, by a number of criteria, with IGFBP-1 derived from decidual tissue. These results were consistent with a primary role of progestin exposure, whether in vivo or in vitro, in converting endometrial stromal cells to cells potentially able to exhibit the high rates of IGFBP-1 production typical of the decidualized endometrium of pregnancy.     

Effect of relaxin on aromatase activity in human endometrial stromal cells

Previous studies have shown that the aromatase activity in human endometrial stromal cells was stimulated by progestin and enhanced by estrogen and forskolin (Fk), an agent that stimulates the accumulation of intracellular cAMP. Present study was undertaken to investigate whether any peptide hormone would affect endometrial aromatase activity. Stromal cells were isolated from normal proliferative and secretory endometria and cultured in nutrient medium. Porcine relaxin (RLX) was added to culture medium individually or in combination with medroxyprogesterone acetate (MPA) and estradiol (E2). Cells treated with RLX alone did not affect the aromatase activity. RLX, however, exerted a synergistic effect on aromatase activity in the presence of MPA or MPA plus E2. On the other hand, human CG, epidermal growth factor, human PRL, and insulin did not increase the aromatase activity in the presence or absence of MPA and E2 studied in a limited number of specimens. The progestin-dependent effect of RLX on aromatase activity was dose dependent indicating that the biological effect of RLX is mediated through a saturable mechanism. When RLX was added to MPA-pretreated cells, additional increase of aromatase activity was seen after 24 h incubation indicating that the action of RLX on stromal cells is not an acute effect. Antiprogestin, RU486, inhibited the stimulation of aromatase activity in both MPA and MPA plus RLX treated cells. RLX has either no effect or a moderate increase (up to 2-fold over the control) on intracellular cAMP content. On the other hand, Fk increased the intracellular cAMP level and enhanced the aromatase activity in the presence of progestin. Also RLX did not replace the effect of Fk since additional increase of aromatase activity was noted when stromal cells were incubated with MPA plus RLX plus Fk in comparison to MPA plus RLX or MPA plus Fk. These results suggest that the action of RLX on stromal cells may be mediated through an intracellular messenger independent of cAMP. Present studies provide evidence that RLX exerts a synergistic effect on aromatase activity in the presence of progestin in human endometrial stromal cells. It is evident that human endometrium is a target organ of RLX.     

Changes in endometrial PTEN expression throughout the human menstrual cycle

Frequent mutation of the PTEN tumor suppressor gene in endometrial adenocarcinoma has led to the prediction that its product, a phosphatase that regulates the cell cycle, apoptosis, and possibly cell adhesion, is functionally active within normal endometrial tissues. We examined PTEN expression in normal human endometrium during response to changing physiological levels of steroid hormones. PTEN ribonucleic acid levels, assessed by RT-PCR, increase severalfold in secretory compared to proliferative endometrium. This suggested that progesterone, a known antineoplastic factor for endometrial adenocarcinoma, increases PTEN levels. Immunohistochemistry with an anti-PTEN monoclonal antibody displayed a complex pattern of coordinate stromal and epithelial expression. Early in the menstrual cycle under the dominant influence of estrogens, the proliferative endometrium shows ubiquitous cytoplasmic and nuclear PTEN expression. After 3-4 days of progesterone exposure, glandular epithelium of early secretory endometrium maintains cytoplasmic PTEN protein in an apical distribution offset by expanding PTEN-free basal secretory vacuoles. By the midsecretory phase, epithelial PTEN is exhausted, but increases dramatically in the cytoplasm of stromal cells undergoing decidual change. We conclude that stromal and epithelial compartments contribute to the hormone-driven changes in endometrial PTEN expression and infer that abnormal hormonal conditions may, in turn, disrupt normal patterns of PTEN expression in this tissue.     

Interleukin-2 inhibits the synthesis and release of prolactin from human decidual cells

PRL is synthesized and released by several extrapituitary tissues, including decidualized endometrial stromal cells. As interleukin-2 (IL-2) stimulates the synthesis and release of pituitary PRL, and decidual stromal cells have receptors for IL-2, we examined whether IL-2 also regulates the release of decidual PRL. Exposure of primary cultures of human decidual cells (10(6) cells/well) from term pregnancies to IL-2 (50 ng/mL) inhibited PRL release beginning 48 h after exposure. The inhibition by IL-2 was dose dependent, and the maximal inhibition of PRL release after 5 days of exposure to IL-2 was 71.0 +/- 0.9% (mean +/- SE). IL-2, however, had no effect on decidual cell viability. The inhibitory effect of IL-2 on PRL release was secondary to inhibition of PRL synthesis. Decidualized human endometrial stromal cells transfected with 3 kb of the extrapituitary PRL (exon 1a) promoter coupled to a luciferase expression vector responded to IL-2 (10 ng/mL) with a significant decrease in luciferase activity. These findings strongly suggest that IL-2 inhibits the synthesis and release of decidual PRL and provide further support for a critical role of cytokines in the regulation of decidual PRL gene expression.     

Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells

Interleukin-1 (IL-1), a critical cytokine for the initiation of the immune response to infection or antigenic challenge, is known to also possess a variety of biological functions outside the immune system. We examined whether IL-1 could affect the decidualization of human endometrial stromal cells (ESC), a conspicuous part in the process of implantation, by assessing PRL production and morphological transformation in an in vitro system. Purified human ESC were cultured in the presence of progesterone (P) with or without the addition of IL-1. IL-1 markedly suppressed the induction of PRL production by P in a dose-dependent manner. The morphological decidualization of ESC in response to P was also inhibited by IL-1. This report demonstrates for the first time the possibility that IL-1 blocks decidualization, the functional differentiation of human endometrial stromal cells in response to ovarian steroids.     

Differences in the cell surface antigens of prolactin-producing cells of human decidual and pituitary tissues

Anterior pituitary cells and other endocrine cells which synthesize and release protein hormones share the cell surface ganglioside antigens A2B5 and 3G5. In addition, these peptide hormone-producing cells contain chromogranin, a major protein component of secretory vesicles. Since human decidual cells synthesize and release a PRL that has chemical and biological properties indistinguishable from those of pituitary PRL, we examined by immunochemical techniques whether decidual cells also contain cell surface antigens A2B5, 3G5, and chromogranin. In these studies, human decidual tissue from three pregnancies and human and rat pituitary tissues were processed and stained with mouse monoclonal antibodies to A2B5, 3G5, and chromogranin by indirect immunofluorescence using fluorescein-conjugated sheep antimouse gamma-globulin. In all instances, the pituitary and pancreatic tissue stained strongly with the monoclonal antibodies to the cell surface antigens, but the decidual tissues from the three pregnancies did not. The pituitary and pancreatic cells also stained strongly with the monoclonal antibody (LK 2H10) to chromogranin, but the decidual tissues had no reactivity. In control experiments, the decidual tissues stained strongly with a monoclonal antibody to HLA antigens, and none of the tissues stained with nonimmune gamma-globulin. These results strongly suggest that the PRL-producing cells of human decidua and pituitary have different cell surface antigens. The observation that decidual cells do not contain chromogranin is consistent with previous biochemical studies suggesting that decidual PRL is not stored in secretory granules. The differences in the subcellular localization of PRL in decidual and pituitary tissues may explain, in part, the differences in the regulation of PRL release from these tissues.