Progesterone withdrawal and estrogen activation in human parturition are coordinated by progesterone receptor A expression in the myometrium

In human parturition, progesterone withdrawal and estrogen activation are not mediated by changes in progesterone and estrogen levels. Instead, these events could be facilitated by changes in the responsiveness of the myometrium to progesterone and estrogens via changes in PR and ER expression. We hypothesized that functional progesterone withdrawal occurs by increased expression of the type A PR (PR-A), which suppresses progesterone responsiveness, and that functional estrogen activation occurs by increased myometrial expression of ERalpha and/or ERbeta. To test this hypothesis we compared the abundance of mRNAs (assessed by quantitative RT-PCR) encoding PR-A, PR-B, ERalpha, and ERbeta in nonlaboring (n = 12) and laboring (n = 12) term human myometrium. PR-A, PR-B, the PR-A/PR-B mRNA ratio, and ERalpha mRNA were significantly increased in laboring myometrium, whereas ERbeta mRNA was low and unchanged. The PR-A/PR-B mRNA ratio correlated positively with ERalpha mRNA levels in nonlaboring myometrium and with HOXA10 mRNA levels in laboring myometrium. Because progesterone inhibits ERalpha and HOXA10 expression, these findings indicate that myometrial progesterone responsiveness is inversely related to the extent of expression of PR-A relative to PR-B. ERalpha mRNA levels correlated positively with cyclooxygenase type 2 and oxytocin receptor mRNA levels in nonlaboring myometrium, indicating that the increase in ERalpha expression is directly associated with the activation of contraction-associated genes and estrogen responsiveness. These data indicate that in the term human myometrium, responsiveness to progesterone is controlled by the expression of PR-A relative to PR-B and that a significant increase in this ratio underlies functional progesterone withdrawal. Our data also indicate that functional estrogen activation occurs by increased expression of ERalpha and is linked to functional progesterone withdrawal. Interaction between the PR and ER systems in the human myometrium may be critical for the control of human parturition and the coordination of progesterone withdrawal and estrogen activation required for parturition.     

Prostaglandins differentially modulate progesterone receptor-A and -B expression in human myometrial cells: evidence for prostaglandin-induced functional progesterone withdrawal

We tested the hypothesis that prostaglandin (PGs), PGE2, and PGF2 alpha, stimulate labor and delivery in women, in part, by inducing functional progesterone withdrawal in myometrial cells by increasing the progesterone receptor (PR)-A/PR-B expression ration. PHM1-31 cells (an immortal pregnant human myometrial cell line) were exposed to PGE2, PGF2 alpha, cyclic-8-bromoadenosine monophosphate (8-Br-cAMP) and phorbol 12-myristate 13-acetate (PMA) at various concentrations for 24h. Effects on PR-A and PR-B expression were then assessed by quantitative RT-PCR. PGF2 alpha dose dependently increased PR-A mRNA and the PR-A/PR-B expression ration but did not effect PR-B mRNA. PGE2 dose-dependently increased mRNAs encoding PR-A and PR-B. The PGE2 dose-threshold for PR-A (0.01 nM) was lower than that for PR-B (0.1 nM), which resulted in an initial rise then a gradual fall in PR-A/PR-B expression ration to basal levels in response to PGE2. Activation of the protein kinase (PK)-A signaling pathway with 8-Br-cAMP coordinately increased expression of PR-A and PR-B and therefore did not alter the PR-A/PR-B expression ration. In contrast, activation of the PKC signaling pathway with PMA increased expression of PR-A without affecting PR-B and therefore significantly (P<0.05) increased the PR-A/PR-B expression ration. These data demonstrate differential control of myometrial PR-A and PR-B expression by PGE2 and PGF2 alpha and by specific intracellular signaling pathways. We conclude that PGs acting via the PKC pathway facilitate functional progesterone withdrawal by increasing the myometrial PR-A/PR-B expression ratio.     

The progesterone receptor in human term amniochorion and placenta is isoform C

The mechanism that initiates human parturition has been proposed to be functional progesterone withdrawal whereby the 116-kDa B isoform of the progesterone receptor (PR-B) switches in favor of the 94-kDa A isoform (PR-A) in reproductive tissues. Recently other PR isoforms, PR-S, PR-C, and PR-M generated from the same gene have been identified and partially characterized. Using immunohistochemical, Western blotting, and RT-PCR techniques, evidence is provided that the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N terminally truncated 60-kDa PR-C isoform. Evidence is also provided that the PR-C isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A, and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C, and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic PR-C isoform but not PR-A, PR-B, or PR-S. The major PR isoform in the amnion, chorion, and placenta is PR-C, suggesting that the cytoplasmic PR-C isoform has a specific role in extraembryonic tissues and may be involved in the regulation of human parturition.     

A novel selective progesterone receptor modulator asoprisnil (J867) inhibits proliferation and induces apoptosis in cultured human uterine leiomyoma cells in the absence of comparable effects on myometrial cells

CONTEXT: Asoprisnil, a selective progesterone (P4) receptor (PR) modulator (SPRM) with mixed P4 agonist/antagonist activities, reduces uterine leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. The evidence from clinical studies suggests that asoprisnil may directly target the uterine leiomyomata. OBJECTIVE AND METHODS: The present study evaluated the effects of asoprisnil on cell proliferation, the expression of apoptosis-related proteins, and apoptosis in cultured human uterine leiomyoma cells and matched normal myometrial cells. PR-A and PR-B expression in the two types of cells was comparatively evaluated. Cell proliferation, proliferating cell nuclear antigen (PCNA)-positive rate, and TUNEL-positive rate were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, respectively. The expression of apoptosis-related proteins and PR was assessed by Western blot analysis. RESULTS: Compared with untreated cultures, asoprisnil decreased the number of viable cultured cells, the PCNA-positive rate, and PCNA protein expression in cultured leiomyoma cells. Asoprisnil increased the TUNEL-positive rate, cleaved caspase-3, and cleaved poly(adenosine 5'-diphosphate-ribose) polymerase expression and decreased Bcl-2 protein expression in cultured leiomyoma cells. These effects were dose and time dependent. In cultured myometrial cells, however, asoprisnil did not affect cell proliferation and apoptosis. PR-B expression was elevated in cultured leiomyoma cells compared with cultured myometrial cells, whereas no differences in PR-A expression were noted between the two cell types. CONCLUSIONS: These results show that asoprisnil inhibits proliferation and induces apoptosis in cultured uterine leiomyoma cells in the absence of comparable effects on cultured normal myometrial cells, suggesting a cell type-specific effect.     

Estrogen and progesterone metabolism in the cervix during pregnancy and parturition

CONTEXT: Experimental and clinical studies in a variety of nonprimate species demonstrate that progesterone withdrawal leads to changes in gene expression that initiate parturition at term. Mice deficient in 5alpha-reductase type I fail to undergo cervical ripening at term despite the timely onset of luteolysis and progesterone withdrawal in blood. OBJECTIVE: Our objective was to test the hypothesis that estrogen and progesterone metabolism is regulated in cervical tissues during pregnancy, even in species in which parturition is not characterized by progesterone withdrawal in blood. DESIGN: Estradiol and progesterone metabolism was quantified in intact cervical tissues from nonpregnant and pregnant women at term before or after labor. SETTING: The study was conducted at a university hospital. PATIENTS: Tissues were obtained from five nonpregnant and 21 pregnant women (nine before labor and 12 in labor). MAIN OUTCOME MEASURES: Enzyme activity measurements, Northern blot analysis, quantitative real-time RT-PCR, and immunohistochemistry were used to quantify steroid hormone metabolizing enzymes in cervical and myometrial tissues. RESULTS: During pregnancy, 17beta-hydroxysteroid dehydrogenase type 2 was induced in glandular epithelial cells to catalyze the conversion of estradiol to estrone and stroma-derived 20alpha-hydroxyprogesterone to progesterone. During parturition, 17beta-hydroxysteroid dehydrogenase type 2 was down-regulated in endocervical cells, thereby creating a microenvironment favorable for cervical ripening. CONCLUSIONS: Together, the data indicate that cervical ripening during parturition involves localized regulation of estrogen and progesterone metabolism through a complex relationship between cervical epithelium and stroma, and that steroid hormone metabolism in cervical tissues from pregnant women is unique from that in mice.     

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.     

Uterine estrogen receptors are increased by RU486 in late pregnant rhesus macaques but not after spontaneous labor

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.     

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.     

Spatial and topological distribution of progesterone receptor A and B isoforms during human development

Progesterone receptor (PR) is a member of the nuclear receptor superfamily. To date, two isoforms of PR have been identified, PR-A and PR-B. In progesterone responsive tissues, the relative ratio of PR-A and PR-B is considered to contribute to the tissue-specific actions of progesterone. In this study, we examined the distribution of PR-A and PR-B in human fetal tissues ranging from 11 to 40 gestational weeks using immunohistochemistry and RT-PCR analysis. PR immunoreactivity was detected in a wide range of fetal tissues until 20 weeks of gestation, but gradually decreased towards the late gestational period. However, PR continued to remain positive throughout the gestational period in the interstitial cells of Cajal and endocrine tissues. PR-B was demonstrated as the predominant isoform in comparison to PR-A in all fetal tissues examined. These findings suggest that progesterone may be involved in the development of fetal organs throughout the gestational period.     

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.     

A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition

The molecular events that lead to the onset of labor in humans and in other mammalian species remain unclear. We propose that a decline in coactivators containing histone acetylase activity in myometrium may contribute to the onset of labor by impairing the function of the progesterone-progesterone receptor (PR) complex. As assessed by semiquantitative and real-time RT-PCR, immunohistochemistry, and immunoblotting, expression of the PR coactivators cAMP-response element-binding protein (CREB)-binding protein and steroid receptor coactivators 2 and 3 was decreased in fundal uterine tissue of women in labor. Using the mouse as an animal model, we also found decreased coactivator levels in uterine tissues at term. In both human and mouse, the levels of acetylated histone H3 were also decreased in uterine tissues at term. Administration of trichostatin A, a specific and potent histone deacetylase inhibitor, to pregnant mice late in gestation increased histone acetylation and delayed the initiation of parturition by 24-48 h, suggesting the functional importance of the decline in histone acetylation in the initiation of labor. These findings suggest that the decline in PR coactivator expression and in histone acetylation in the uterus near term may impair PR function by causing a functional progesterone withdrawal. The resulting decrease in expression of PR-responsive genes should increase sensitivity of the uterus to contractile stimuli.     

Systematic expression analysis and antibody screening do not support the existence of naturally occurring progesterone receptor (PR)-C, PR-M, or other truncated PR isoforms

Functional progesterone withdrawal associated with human parturition has been ascribed to various mechanisms modulating the function of the classical progesterone receptors (PRs), B and A, in utero. These include up-regulation of the inhibitory PR-C isoform, described as a 60-kDa protein occurring from translation initiation at codon 595. Our initial attempts to detect PR-C yielded uninterpretable results. To systematically validate antibodies for immunodetection of PR isoforms, we generated expression vectors for PR variants originating from putative start codons AUG-289, -301, -595, -632, and -692 in addition to those for PR-B and PR-A, and for alternative splice variants PR-T, PR-S, and PR-M. All constructs were subjected to in vitro and in vivo translation and immunoblotting with a panel of 13 PR antibodies. Antibodies raised against full-length PR were generally not capable of detecting N-terminally truncated forms, whereas C-terminal antibodies did not or only weakly reacted with PR-B and PR-A but produced prominent nonspecific signals. Thus, immunodetection of N-terminally truncated PR isoforms is prone to artifacts. Proteins of about 64 kDa were expressed from PR-289 and -301, but no corresponding endogenous forms were observed. PR-T, PR-S, and PR-M cDNAs yielded no detectable translation products. No protein was translated from AUG-595 in our PR-C expression vector unless a Kozak sequence was introduced, and the product was not 60 but 38 kDa in size. Thus, the 60-kDa protein called PR-C does not originate from AUG-595 and is not a naturally occurring PR isoform.     

Activity and expression of soluble and particulate guanylate cyclases in myometrium from nonpregnant and pregnant women: down-regulation of soluble guanylate cyclase at term.

The role of cGMP in the regulation of human myometrial smooth muscle contractility is at present unclear. cGMP can be synthesized by a cytoplasmic, soluble guanylate cyclase (sGC), which is stimulated by nitric oxide and carbon monoxide, and by particulate membrane-bound GC, which are activated by natriuretic peptides. The aim of this study was to determine whether sGC or pGC are present in nonpregnant and pregnant human myometrium, and whether the activity and expression of these enzymes and the cGMP content change during pregnancy and with labor. Myometrium was obtained from nonpregnant women (n = 12) and pregnant women who were preterm (25-34 wk gestation; n = 12), term (>38 wk) not in labor (n = 14), or term in active labor (n = 12). The cGMP content in myometrium obtained from preterm deliveries was significantly higher than that in tissue obtained from nonpregnant women and decreased at term, especially in laboring groups. Protein and mRNA for sGC, particulate GC-A, GC-B, and the clearance receptor were detected in human myometrium. cGMP in pregnant human myometrium, however, appears to be produced predominantly by sGC and possibly by GC-B, as GC-A was only weakly expressed. sGC activity was greater in myometrium from preterm (nonlabor) deliveries compared those taken at term (in labor), but was down-regulated compared with activity in nonpregnant myometrium. Neither atrial natriuretic peptide nor C-type natriuretic peptide (agonists for GC-A and GC-B, respectively) altered contractility in vitro of myometrium from women at term (not in labor). We conclude that the cGMP/guanylate cyclase system in human myometrium is gestationally regulated and potentially plays an important role in mediating quiescence during early pregnancy. A reduction in cGMP availability may contribute to the switch to contractile activity at term.     

Frequent loss of estrogen and progesterone receptors in human prostatic tumors determined by quantitative real-time PCR

Relative gene expression of the estrogen receptors (ER)-alpha (NR3A1) and ER-beta (NR3A2) along with progesterone receptors PR-A and PR-B (NR3C3) was determined by quantitative real-time PCR in a previously characterized panel of paired human prostate tumor and surrounding unaffected tissue (Prostate 54:275). In approximately half of these cases, a 10-fold or greater reduction in the relative mRNA levels of ER-beta but not ER-alpha was found in the cancer as compared to normal tissue, which was also observed with unpaired samples. Immunohistochemical staining for ER-beta and ER-alpha closely paralleled mRNA expression patterns for both receptors in paired samples. Reduced relative expressions of PR-B and total PR-A and PR-B isoforms were also observed in prostate tumor as compared to unaffected tissue, implying a potential role of PR in prostate tissue. The relative decrease in ER-beta is greater than that observed in prior studies, suggesting that paired samples more accurately reflect differences within individual cases. These findings favor the concept that ER-beta mediates anti-proliferative signals and its loss in prostatic tumor promotes proliferation of these cells.