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.     

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.     

Inflammatory status influences aromatase and steroid receptor expression in endometriosis

Aberrant up-regulation of aromatase in eutopic endometrium and implants from women with endometriosis has been reported. Aromatase induction may be mediated by increased cyclooxygenase-2 (COX-2). Recently, we demonstrated that progesterone receptor (PR)-A and PR-B serve an antiinflammatory role in the uterus by antagonizing nuclear factor kappaB activation and COX-2 expression. PR-C, which antagonizes PR-B, is up-regulated by inflammation. Although estrogen receptor alpha (ERalpha) is implicated in endometriosis, an antiinflammatory role of ERbeta has been suggested. We examined stage-specific expression of aromatase, COX-2, ER, and PR isoform expression in eutopic endometrium, implants, peritoneum, and endometrioma samples from endometriosis patients. Endometrial and peritoneal biopsies were obtained from unaffected women and those with fibroids. Aromatase expression in eutopic endometrium from endometriosis patients was significantly increased compared with controls. Aromatase expression in endometriosis implants was markedly increased compared with eutopic endometrium. Aromatase mRNA levels were increased significantly in red implants relative to black implants and endometrioma cyst capsule. Moreover, COX-2 expression was increased in implants and in eutopic endometrium of women with endometriosis as compared with control endometrium. As observed for aromatase mRNA, the highest levels of COX-2 mRNA were found in red implants. The ratio of ERbeta/ERalpha mRNA was significantly elevated in endometriomas compared with endometriosis implants and eutopic endometrium. Expression of PR-C mRNA relative to PR-A and PR-B mRNA was significantly increased in endometriomas compared with eutopic and control endometrium. PR-A protein was barely detectable in endometriomas. Thus, whereas PR-C may enhance disease progression, up-regulation of ERbeta may play an antiinflammatory and opposing role.     

子宫内膜癌组织中PR及其亚型PRA、PRB的表达变化及意义

目的观察子宫内膜癌组织中孕激素受体（PR）及其亚型PRA、PRB表达变化,并探讨其临床意义。方法采用免疫组化SP法检测正常子宫内膜（正常组）、不典型增生内膜（不典型增生组）及子宫内膜样腺癌内膜（内膜癌组）组织中的PR及其亚型PRA、PRB表达情况。结果不典型增生组和内膜癌组PR阳性表达率明显低于正常组（P均〈0.05）,内膜癌组PR表达强度低于正常组和典型增生组（P均〈0.05）。正常组、不典型增生组、内膜癌组PRA阳性表达率分别为100%、86.7%（13/15）、74.5%（41/55）,PRB阳性表达率分别为100%、80%（12/15）和69.1%（38/55）,内膜癌组PRA、PRB阳性表达率与正常组和不典型增生组相比均明显降低（P均〈0.05）。正常组25例（82.5%）PRA、PRB表达强度相等,不典型增生组和内膜癌组分别为7例（46.7%）、12例（21.8%）,内膜癌组明显低于正常组和不典型增生组,P均〈0.01。内膜癌组只表达PRA或PRA占优势者24例（43.6%）,明显多于正常组的5例（12.5%）和不典型增生组的4例（26.7%）,P均〈0.05。PRA表达与子宫内膜癌分化程度有关,PRB表达与子宫内膜癌分化程度、淋巴结转移和FIGO分期有关（P均〈0.05）。结论子宫内膜癌患者子宫内膜组织中PR、PRA、PRB表达均明显降低。子宫内膜组织PR亚型表达缺失、比例失衡,尤其是PRB表达缺失可能与子宫内膜癌的发生有关。     

Mig-6 modulates uterine steroid hormone responsiveness and exhibits altered expression in endometrial disease

Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) effects. An imbalance caused by increased E2 action and/or decreased P4 action can result in abnormal endometrial proliferation and, ultimately, endometrial adenocarcinoma, the fourth most common cancer in women. We have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of progesterone receptor (PR) and steroid receptor coactivator (SRC-1) action in the uterus. Here, we demonstrate that absence of Mig-6 in mice results in the inability of P4 to inhibit E2-induced uterine weight gain and E2-responsive target genes expression. At 5 months of age, the absence of Mig-6 results in endometrial hyperplasia. Ovariectomized Mig-6^d/d mice exhibit this hyperplastic phenotype in the presence of E2 and P4 but not without ovarian hormone. Ovariectomized Mig-6^d/d mice treated with E2 developed invasive endometrioid-type endometrial adenocarcinoma. Importantly, the observation that endometrial carcinomas from women have a significant reduction in MIG-6 expression provides compelling support for an important growth regulatory role for Mig-6 in the uterus of both humans and mice. This demonstrates the Mig-6 is a critical regulator of the response of the endometrium to E2 in regulating tissue homeostasis. Since Mig-6 is regulated by both PR and SRC-1, this identifies a PR, SRC-1, Mig-6 regulatory pathway that is critical in the suppression of endometrial cancer.     

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.     

Estrogen and progesterone receptor isoforms expression in the stomach of Mongolian gerbils

AIM： We studied the estrogen receptor （ER） and progesterone receptor （PR） isoforms expression in gastric antrum and corpus of female gerbils and their regulation by estradiol （E2） and progesterone （P4）.
METHODS： Ovariectomized adult female gerbils were subcutaneously treated with E2, and E2 ＋ P4. Uteri and stomachs were removed, the latter were cut along the greater curvature, and antrum and corpus were excised. Proteins were immunoblotted using antibodies that recognize ER-alpha, ER-beta, and PR-A and PR-B receptor isoforms. Tissues from rats treated in the same way were used as controls.
RESULTS： Specific bands were detected for ERalpha （68 KDa）, and PR isoforms （85 and 120 KDa for PR-A and PR-B isoforms, respectively） in uteri, gastric antrum and corpus. We could not detect ER-beta isoform. PR isoforms were not regulated by E2 or P4 in uterus and gastric tissues of gerbils. ER-alpha isoform content was significantly down-regulated by E2 in the corpus, but not affected by hormones in uterus and gastric antrum.
CONCLUSION： The presence of ER-alpha and PR isoforms in gerbils stomach suggests that E2 and P4 actions in this organ are in part mediated by their nuclear receptors.     

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.     

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.     

Progesterone receptor regulates Bcl-2 gene expression through direct binding to its promoter region in uterine leiomyoma cells

CONTEXT: Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. OBJECTIVE: Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. RESULTS: The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. CONCLUSION: Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.