Progesterone Receptor A Research Articles

Ethnic differences in expression of the dysregulated proteins in uterine leiomyomata

Black ethnicity is one of the risk factors for uterine leiomyomata (ULM). Little is known about the ethnic differences in leiomyoma-associated gene products in women with uterine leiomyomata. A total of 120 hysterectomies with ULM were collected from black, Asian, Hispanic and white women (30 cases from each group). Twenty-two gene products were selected for the study. The expressions of the selected dysregulated gene products were measured by the semiquantification and the immunoscores were normalized by matched myometrium. The relative expressions of progesterone receptor A (PR-A) (up-regulation), retinoid acid receptor alpha (down-regulation), and retinoid X receptor alpha (RXRalpha) (no change) in leiomyomata compared to normal myometrium in black women were significantly different compared to other ethnic groups (P < 0.05). About one-third of ULM from black women subclustered together in association with a group of up-regulated gene products. Many other gene products, including local growth factors, insulin-like growth factor (IGF)-signalling proteins, and cell proliferation markers, were dysregulated in ULM but showed non-significant differences between the ethnic groups. There are substantial differences of the sex steroid receptors and other nuclear receptors between black women and other ethnic groups. Based on tissue microarray data, there are at least two broad groups of leiomyomata presented by the dysregulation of different groups of gene products. One is dominated by up-regulation of amplified in breast cancer 1, CD24, hamartin, human mobility group gene 2, IGF2, PR-A and RXR, and the other is characterized by up-regulation of epithelial growth factor receptor, down-regulation of hamartin, PR-A and tuberin.

Role of FOXO1A in the Regulation of Insulin-Like Growth Factor-Binding Protein-1 in Human Endometrial Cells: Interaction with Progesterone Receptor1

Insulin-like growth factor-binding protein-1 (IGFBP1) is a major secretory product of the decidualized endometrium. In the present study, we investigated the role of two transcription factors, progesterone receptor (PGR) and a member of the forkhead box class O family of transcription factors (FOXO1A), in the regulation of the IGFBP1 gene in endometrial cells. Human endometrial fibroblasts (HuF) expressed FOXO1A, progesterone receptor A (PGRA), and progesterone receptor B (PGRB) proteins, whereas the endometrial adenocarcinoma cell line, HEC-1B cells, expressed only FOXO1A and no detectable PGR proteins. When FOXO1A expression was silenced using small interference RNA, IGFBP1 expression decreased in both HuF and HEC-1B cells. Using the chromatin immunoprecipitation technique, we demonstrated that liganded PGR was recruited to the IGFBP1 promoter region (-358 to -49). In addition, immunoprecipitation of HuF nuclear proteins with a PGR antibody followed by immunoblotting with anti-FOXO1A revealed that these two proteins interact in these cells. Reporter studies demonstrated that whereas liganded PGRA or PGRB increased a progesterone response element-linked reporter construct, pPRE/ GRE.E1b.Luc, coexpression of FOXO1A inhibited the PGRB response in HuF and synergistically increased PGRA and PGRB response in HEC-1B cells. Furthermore, in HEC-1B cells, FOXO1A increased IGFBP1 promoter activity, and coexpression of PGRA or PGRB further increased the promoter activity in a cooperative manner. In HuF, the response to FOXO1A and PGR was not additive; in fact, it was lower than the sum of the individual responses. Thus, FOXO1A and PGR associate with one another, and each influences the transactivating potential of the other. The cell type-dependent responses strongly implicate the involvement of other cofactors.

Altered Progesterone Receptor Isoform Expression Remodels Progestin Responsiveness of Breast Cancer Cells

The ovarian hormone progesterone is essential for normal breast development and function. However, it is also implicated in breast cancer development. Progesterone signals through two nuclear receptors [progesterone receptor A (PRA) and progesterone receptor B (PRB)], which display striking differences in transcriptional activity when analyzed separately. The two species are coexpressed equally in normal breast, but expression becomes markedly disrupted in breast cancer, where a predominance of PRA is common. To determine the impact on PR transcriptional activity of the shift from coexpression of PRA and PRB, observed in normal cells, to predominance of PRA, common in cancers, we modeled these changes in PR expression patterns using an inducible model of PRA predominance. At short treatment times progestin regulation was directed toward transcriptional modulators, whereas longer exposure more frequently targeted genes associated with regulation of cell shape, adhesion, and metabolism, and a number of these targets acquired responsiveness only when PRA predominance was achieved. Consistent with this, overexpression of PRA altered progestin effects on cell-substrate attachment and focal adhesion signaling. Our data suggest that disrupted balance of PRA and PRB remodels progestin responsiveness and that altered regulation of morphology and adhesion are important components of altered progestin response in breast cancer.

Significance of progesterone receptor-A and -B expressions in endometrial adenocarcinoma

The progesterone receptor (PR) has two isoforms, A and B, among which PR-B is mainly involved in regulating proliferation of the uterine endometrium. In this study, immunohistochemical analysis was carried out to investigate the correlation of PR-A and -B expressions with cell cycle-regulatory proteins and clinicopathological parameters in endometrial adenocarcinoma. One hundred and forty-one endometrioid adenocarcinomas [76 with well-differentiated (G1), 35 with moderately differentiated (G2) and 30 with poorly differentiated (G3)] were used. Specimens of formalin-fixed and paraffin-embedded tissue were immunohistochemically stained using the high polymer method (HISTOFINE, NICHIREI). The percentage of positive nuclei of tumor cells observed in three high power fields was expressed as a labeling index (LI). PR-B expression significantly occurred more frequently in G1. It was inversely correlated with p53 gene mutation and p53 over expression, and also with clinicopathological variables, including myometrial and lymph-vascular space invasion and the FIGO stage. Patients with negative PR-B had a poorer prognosis than positive cases. PR-A expression was also significantly higher in G1 and was inversely correlated with Ki-67 expression and myometrial invasion, but not with prognosis. PR-A and -B expressions were significantly correlated with biologically malignant potential. Especially, PR-B expression is useful as a prognostic indicator of endometrial adenocarcinoma.

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis.

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.

Retraction: Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis.

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.

Progesterone receptors A and B differentially modulate corticotropin-releasing hormone gene expression through a cAMP regulatory element.

Corticotrophin-releasing hormone (CRH) plays a major role in mechanisms controlling human pregnancy and parturition. Gene regulation by progesterone may be a key point in the control of placental CRH production. Studies in primary placental cells show that antagonism of progesterone activity or production by RU486 or trilostane leads to an increase in CRH promoter activity. This effect can be reversed by the addition of progesterone. Overexpression of progesterone receptor A (PR-A) or glucocorticoid receptor resulted in a decrease in CRH promoter activity following progesterone treatment, whereas an increase in promoter activity was observed with overexpressed PR-B. Studies including mutation of the cAMP regulatory element (CRE) confirm this site to be essential for the progesterone-mediated effects. In summary, our results demonstrate that progesterone regulates CRH gene transcription via a CRE in the CRH promoter and that PR-A and PR-B exhibit different actions in the regulation of CRH gene expression.

Regulation of hepatic progesterone and estrogen receptors in the female turtle, Chrysemys picta: relationship to vitellogenesis

Previous studies using the fresh water turtle Chrysemys picta have demonstrated the differential expression of the two progesterone receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with ER mRNA levels and hepatic vitellogenesis. During the inter-vitellogenic periods the ratio of PRB:PRA favors PRA, suggesting that the PRB:PRA ratio may be important in the regulation of vitellogenesis. Based on these and other studies we hypothesize that progesterone may have differential effects on the estrogen (E)-induced vitellogenin response (inhibitory or stimulatory), depending on the PRB:PRA ratio. In this study, we determined the expression pattern and the hormonal regulation of progesterone receptor (PR) isoforms in the liver, during the estrogen-induced vitellogenin response in female C. picta. Northern blot analysis was used to evaluate the changes in vitellogenin mRNA, estrogen receptor (ER) mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vitellogenin. The expression of PR isoforms in the liver of female turtles at the mRNA and protein levels was differentially regulated by estradiol and progesterone. Estradiol treatment enhanced the transcription of PR mRNA isoforms and possibly translation and/or increased stability of PRB protein in the female turtle liver. In contrast, this hormone decreased PRA protein levels. Progesterone alone down-regulated progesterone receptor isoform A (PRA) and progesterone receptor isoform B (PRB) proteins equally, but did not affect PR mRNA levels. Estradiol markedly increased hepatic ER mRNA, vtg mRNA, and vtg in plasma, but this effect was not impacted significantly by progesterone. The results presented here indicate that in female turtles hepatic PRB:PRA ratios are markedly changed by estradiol treatment due primarily to a decrease in PRA. The change in the PRB:PRA ratio after hormonal treatment confirms that progesterone and estrogen exposure will be a determinant in the regulation of vitellogenesis, and, in turn, that the regulation of vitellogenesis will be determined by the ratio of PR isoforms and the physiological levels of steroid hormones.

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.

Hormone trajectories leading to human birth.

The mechanisms regulating human parturition and labor remain unknown. This ignorance is expensive as preterm birth is responsible for 70% of neonatal mortality and 50% of cerebral palsy. Methods for the prediction of preterm birth and treatments for women in preterm labor have poor efficacy reflecting our limited knowledge of the mechanisms involved. Recent research has supported the view that parturition is a cascade of events that commences early in pregnancy and involves the mother, fetus, placenta, membranes, cervix and myometrium. Although a number of the key hormones and proteins involved have been identified, the relationships between these factors in time and tissues remain unclear. Placental production of Corticotropin-releasing hormone (CRH) is proposed as an early event regulating the cascade of events. Central to the onset of parturition will be a mechanism for progesterone withdrawal and estrogen activation in human. Two forms of progesterone receptor with opposing actions exist in the human myometrium. Progesterone receptor A (PR-A) is a dominant negative repressor of progesterone receptor B (PR-B). Preliminary studies strongly support the hypothesis that the onset of human parturition is initiated by rising concentrations of PR-A in the myometrium.

Progesterone and progesterone receptors in reptiles

The role of progesterone (P) has been most extensively studied in the female reproductive tissues (ovary, reproductive tract, mammary gland) and in the brain, in which it is an important regulator and modulator in conjunction with estradiol (E). In nonmammalian vertebrate species, less work has been done on P metabolites involved in ovulation. In addition, P induces the expression of egg-white proteins, decreases myometrial contractility, and facilitates processing of eggs, formation of eggshell, and deposition of egg-white proteins. Actions of P may be synergistic with, or antagonist to, the actions of E, depending on hormone ratios, timing of exposure, and physiological state. These effects of P are mediated through progesterone receptor isoform A (PRA), a general transcription inhibitor of P target genes, and isoform B (PRB), a specific transcriptional stimulator of some reproductive tract genes. In this review, we focus on P action and PR expression in the liver and reproductive tissues of several reptilian species, especially Chelonia, since most of the data obtained are from this group. We also present novel data showing the expression of PR in nonreproductive tissues (such as kidney, spleen, gastrointestinal tract) in the painted turtle, Chrysemys picta.

Ovarian hormones induce TGF-beta(3) and fibronectin mRNAs but exhibit a disparate action on cardiac fibroblast proliferation.

Prior to menopause, women have a lower risk of cardiovascular disease compared to age-matched men. Despite the well-documented beneficial physiological effects of ovarian hormones on vascular reactivity and growth, very little is known with regard to the direct action on cardiac cells. The following study examined the pattern of ovarian hormone receptor subtype expression in cardiac fibroblasts, the modulator role of 17 beta-estradiol and progesterone on growth and their respective influence on putative molecular events of extracellular matrix remodeling. Neonatal rat cardiac fibroblasts were isolated from 1- to 3-day-old Sprague--Dawley rats. Immunofluorescence and Western blot analysis revealed the presence of estrogen receptor-alpha (ER-alpha), and -beta (ER-beta) subtypes, with the ER-alpha subtype localized on the plasma membrane. Likewise, both progesterone receptor-A (PR-A), and -B (PR-B) subtypes were expressed in cardiac fibroblasts, and the PR-B appeared to be the predominant subtype associated with the plasma membrane. Despite the presence of both ER subtypes, the treatment of cardiac fibroblasts with 1 microM 17 beta-estradiol exerted a modest decrease in DNA synthesis. By contrast, progesterone treatment caused a dose-dependent decrease in [3H]thymidine uptake, without a concomitant induction of apoptosis. The progesterone-mediated decrease in DNA synthesis was associated with the upregulation of the cyclin-dependent kinase inhibitor p27(Kip1), whereas p21(cip) and proliferating cell nuclear antigen protein levels were unchanged. Lastly, despite the modest effect on DNA synthesis, 17 beta-estradiol increased the steady-state mRNA levels of transforming growth factor-beta(3) and fibronectin. Likewise, progesterone increased the expression of both transforming growth factor-beta(3), and fibronectin mRNA. Collectively, these data are the first to highlight the presence of estrogen and progesterone receptor subtypes on the plasma membrane of neonatal rat cardiac fibroblasts, and further underscore the ability of ovarian hormones to directly suppress DNA synthesis, and influence putative molecular events associated with extracellular matrix remodeling.

Split Response to Steroids

In mammals, a single gene encodes the receptor for the steroid reproductive hormone progesterone, but the protein exists in two forms, progesterone receptor-A (PR-A) and PR-B, that result from alternative starting points for transcription and translation. Mulac-Jericevic et al . describe mice in which a mutation selectively prevents expression of PR-A. Acting alone, PR-B regulated only a subset of known progesterone-responsive genes in the uterus. Progesterone normally antagonizes estrogen-induced proliferation in the uterine epithelium, yet interactions with PR-B promoted proliferation. The separation of distinct physiological functions for PR-A and PR-B raises the possibility that selective modulators of the PR isoforms could provide more specific therapeutic effects. Mulac-Jericevic, B., Mullinax, R.A., DeMayo, F.J., Lydon, J.P., and Conneely, O.M. (2000) Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science 289 : 1751-1754. [Abstract] [Full Text]

Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform.

Progesterone regulates reproductive function through two intracellular receptors, progesterone receptor-A (PR-A) and progesterone receptor-B (PR-B), that arise from a single gene and function as transcriptional regulators of progesterone-responsive genes. Although in vitro studies show that PR isoforms can display different transcriptional regulatory activities, their physiological significance is unknown. By selective ablation of PR-A in mice, we show that the PR-B isoform modulates a subset of reproductive functions of progesterone by regulation of a subset of progesterone-responsive target genes. Thus, PR-A and PR-B are functionally distinct mediators of progesterone action in vivo and should provide suitable targets for generation of tissue-selective progestins.

Transcriptional regulation of the murine multidrug resistance gene mdr1b by progesterone occurs via an indirect mechanism.

The murine multidrug resistance gene mdr1b is highly induced in the endometrium during pregnancy. Evidence suggests that induction occurs mainly as a result of progesterone action. To study the molecular mechanisms involved in this induction, 5'-flanking sequences between -540 and +97 of the mdr1b gene were fused to the reporter gene, bacterial chloramphenicol acetyltransferase (p540CAT). Unlike most progesterone-responsive genes, mdr1b is preferentially activated by the A form of the progesterone receptor. We now report that activation is not observed with a DNA-binding domain mutant of progesterone receptor A (PRA) suggesting that induction occurs at the transcriptional level. Time course experiments demonstrated that induction was first observed 12 hr after hormone addition, suggestive of a secondary (or late) response gene. Sequence comparison highlighted the region M1 (-234 to -206), which contains a partially conserved progesterone response element. Its functional significance was evaluated by expression assays and gel shift analysis. Reporter plasmids with modifications of this element were transfected into HeLa cells. Constructs containing the native M1 element, or a mutated element (M1mt) that eliminated any similarity to a progesterone response element, were induced four-fold by progesterone whereas an element containing a consensus progesterone response element (M1PRE) was induced eight-fold. In addition, by gel shift analysis, the M1 element did not bind the progesterone receptor or any other factors. This suggested that the M1 region does not participate in the response to progesterone. 5' Nested deletion analysis, used to identify other regions of the upstream regulatory region that contributed to induction by progesterone, demonstrated that enhancer sequences between -122 and -65, which contain binding sites for C/EBPbeta and NF-Y, were important. Mutations in the binding sites for these factors decreased induction by progesterone. On the basis of our studies using 540 bp of upstream sequence, mdr1b is activated transcriptionally by progesterone, in an indirect manner dependent on basal factors.

Interaction of the chicken progesterone receptor with heat shock protein (HSP) 90.

The chicken progesterone receptor A (PRA) was expressed from cDNA by in vitro transcription and translation and also by transient transfection of receptor-negative COS M6 cells. These receptors synthesized from cDNA exhibited functional properties similar to those of oviduct PRA. The ability of PRA to form an 8S complex and to bind to DNA was studied. PRA, synthesized by either expression system, formed an 8S complex which was dissociated by incubation in vitro with 0.4 M NaCl or 20 nM progesterone to generate a 4S species able to bind to DNA-cellulose. The presence of HSP 90 in the PRA 8S complex was confirmed by use of an HSP 90-specific antibody, AC-7. Expression constructs coding for various receptor deletions were studied in order to identify the site of interaction of PRA with HSP 90. Deletions of 290 amino acids from the C-terminus resulted in the loss of ability to form an 8S complex. Truncated receptor proteins lacking 153 amino acids from the C-terminus or 369 amino acids from the N-terminus were able to interact with HSP 90. These data suggest that the site of interaction between PRA and HSP 90 responsible for 8S complex formation may be in this region (amino acid 369-506). However, small internal amino acid deletions in this region of PRA did not result in the loss of interaction of mutant receptor proteins with HSP 90. Thus, it appears that there may be more than one site of interaction between PRA and HSP 90 in this region.