Form Of Progesterone Receptor Research Articles

Drastic decrease of progesterone receptor form B but not A mRNA reflects poor patient prognosis in endometrial cancers

Objectives. Progesterone receptor (PR) has been recognized as an important factor that correlates with success of endocrine treatment and patients' prognoses in endometrial cancers (EC). This study was designed to determine the clinical implications of expression of PR form A (PR-A) and B (PR-B) in EC. Methods. We have quantified the mRNA levels of total PR (PR-AB) and PR-B by real-time RT-PCR in 120 EC and 40 normal endometria (NE). We then analyzed the correlation between expression levels of each receptor and clinical characteristics including clinical stages, histological grades, depth of myometrial invasion and patients' prognoses. Results. Although the expression level of PR-B is correlated with that of PR-A, the expression pattern of PR-B classified according to clinical characteristics was different from that of PR-A. We found that high PR-B mRNA levels but not PR-A mRNA levels correlated significantly with survival, independent of clinical characteristics including clinical stages, histological grades and depth of myometrial invasion. Percentage of PR-B to total PR (B/AB) in EC is widely distributed and the ratios of middle B/AB group (0.15 < B/AB < 0.35) are 75% in NE, 47% in surviving cases of EC and 18% in dead cases of EC at 5 years, respectively. Conclusions. These results taken together indicate that PR-B may have a different function from PR-A and the intact coordinate expression of PR isoforms was disrupted in EC, especially in poor prognostic case. The measurement of PR isoforms has prognostic value in EC.

Overexpressed progesterone receptor form B inhibit invasive activity suppressing matrix metalloproteinases in endometrial carcinoma cells

In this study, we focused on the influence of progesterone and its receptor in invasion and MMPs on endometrial carcinoma cells. The growth of Ishikawa cells, to which an progesterone receptor form B (PR-B) expressing vector was transfected, was inhibited by progesterone as was the inhibition of the expression of cyclin D1. By invasion assay, in conditions with progesterone, the invasiveness of Ishikawa cells was inhibited as well as the expression of (metalloproteinase) MMP-1, -2, -7 and -9 and Ets-1 decreased. These results suggest that activation of PR-B by progesterone results in tumor suppression by inhibiting cell growth and invasiveness via suppression of the expression of MMPs.

Mammary epithelial cells of PR-A transgenic mice exhibit distinct alterations in gene expression and growth potential associated with transformation.

Expression of the 'A' and 'B' forms of progesterone receptor (PR), in an appropriate ratio is critical for normal mammary development. As such, mammary glands of PR-A transgenic mice, carrying additional 'A' form of PR as transgene, exhibit morphological and histological characteristics associated with transformation. Accordingly, in the present studies, we analyzed these mammary glands for the presence of transformed epithelial cells by examining for alterations in gene expressions and growth potential, known to be associated with different stages of transformation. These studies reveal that, in the aberrant mammary epithelial structures, there is a decrease in p21 expression, an increase in cyclin D1 expression accompanied by an increase in cell proliferation, and a decrease in estrogen receptor alpha (ER alpha). In mammary ducts with normal histology, there is a decrease in p21 expression without an elevation in cyclin D1 expression or cell proliferation or a decrease in ER alpha expression. Treatment of PR-A transgenics with anti-progestin, mifepristone, has no effect on cell proliferation, cyclin D1 or ER alpha expression in the aberrant epithelial structures. In contrast, mifepristone restored the loss of p21 expression in the epithelial cells of both the ducts with normal histology and aberrant structures. Parallel studies reveal no apparent differences between the mammary glands of wild-type and PR-B transgenic mice, which carry additional PR 'B' form. Accordingly, we conclude that (i) mammary glands of PR-A transgenics contain at least two distinct populations of transformed epithelial cells, (ii) the epithelial cell population in the ducts with normal histology contain presumptive immortalized cells, indicative of early stages of transformation, (iii) the aberrant epithelial structures contain later stages of transformation associated with hyperplasias/pre-neoplasias and (iv) the transformation of mammary epithelial cells in PR-A transgenics might be due to a misregulation in progesterone action resulting from overexpression of PR 'A' form.

Stimulatory effect of progesterone on the expression of steroidogenic acute regulatory protein in MA-10 Leydig cells.

The steroidogenic acute regulatory protein (StAR), by virtue of its ability to facilitate the intramitochondrial transport of cholesterol, plays an important role in regulating steroid hormone biosynthesis in steroidogenic cells. In agreement with published data, both StAR expression and progesterone production in MA-10 mouse Leydig tumor cells could be stimulated with hCG and 8Br-cAMP. Addition of aminoglutethimide, an inhibitor of cholesterol side chain cleavage (P450(scc)) enzyme, not only resulted in a drastic inhibition of progesterone production but also in an attenuation of StAR expression in response to either hCG or 8-Br-cAMP. Therefore, we addressed the question of whether progesterone, the end product of the steroidogenic cascade in these cells, could be in a position to regulate the StAR gene expression. In MA-10 cells, we report here that progesterone in microgram amounts can induce StAR gene expression in a time- and dose-dependent manner. StAR expression in response to a maximally effective concentration of progesterone of 10 microg/ml was highest at 6 h and started decreasing thereafter. The effect of progesterone on StAR protein and StAR mRNA induction was mimicked by its synthetic analog, progestin R5020, but not by other steroids, including dexamethasone, estradiol, testosterone, and dihydrotestosterone. Dexamethasone, in contrast, was able to inhibit StAR expression in MA-10 cells. Surprisingly, RU486, a potent antagonist of progesterone and glucocorticoid action, had a stimulatory effect on StAR mRNA levels. Reverse transcription-polymerase chain reaction analysis demonstrated the absence of the classical form of progesterone receptor in MA-10 cells. Thus, for the first time, a direct stimulatory effect of a steroid on StAR gene expression has been demonstrated. Furthermore, these results provide a new insight, indicating that progesterone mediates the activation of StAR expression exerted presumably through a novel, nonclassical progesterone receptor in mouse Leydig cells.

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.

Detection of progesterone receptor forms A and B by immunohistochemical analysis

Aim—The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of...

Impact of progesterone receptor on cell-fate decisions during mammary gland development.

Mammary epithelium contains lineage-limited progenitors that give rise to cells that form distinct morphological structures, ducts vs. lobules, depending on the endocrine status of the female. Progesterone signaling through progesterone receptor (PR) is essential for lobulo-alveolar development that accompanies pregnancy, but not for ductal growth accompanying puberty. PR exists in two molecular forms, A and B, and an imbalance in the native ratio of the two isoforms can lead to alterations in PR signaling. Indeed, as we reported previously, in transgenic mice carrying additional A form of PR, mammary development is abnormal, characterized by excessive lateral ductal branching. This suggests that alterations in PR signaling may have important consequences to mammary development, particularly with regard to ductal vs. alveolar growth. To test this further, we created transgenic mice carrying additional B form of PR and report that mammary development in these mice is also abnormal, characterized by inappropriate alveolar growth. More importantly, these mammary glands, on serial transplantation, undergo a premature arrest in ductal growth without any alteration in the potential for lobulo-alveolar growth. Such an arrest in ductal growth does not occur with transgenics carrying additional A form of PR. These studies, therefore, provide strong evidence to indicate that PR signaling may be of paramount importance for appropriate cell-fate decisions during normal mammary development, and also that this requires a regulated expression of the two isoforms.

Is ICI 182,780 an antiprogestin in addition to being an antiestrogen?

The pure antiestrogen ICI 182,780 has been shown to have antiprogestin activity in reporter gene constructs. Cell lines, naturally devoid of progesterone receptors (PR) were transfected with either the A or B forms of the human PR and a luciferase construct driven by a progesterone-response element (PRE). Because this system is an artificial one, our purpose was to determine whether these observations could be made in a human breast cancer cell line, naturally containing PR. We further evaluated the dose-response of ICI 182,780 and RU-486 (mifepristone) on PR and estrogen receptors (ER) in the presence of either progesterone, norgestrel or estradiol. These effects were measured using immunoassays for prostate-specific antigen (PSA) and human glandular kallikrein (hK2) and pS2. We found that ICI 182,780 blocked progesterone-stimulated PSA and hK2 production 100% at 10(-5) M, which decreased significantly by 10-6 M. This inhibition did not occur when norgestrel was the progestin used. RU-486 showed 100% blockade for both progestins at all concentrations used. We concluded that the antiprogestin activity of ICI 182,780 exists for progesterone only. This weak antiprogestin activity may be unlikely to have significant clinical implications.

Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody.

Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.

Dominant Expression of Progesterone Receptor Form B mRNA in Ovarian Endometriosis

This study was designed to examine the biological implications of progesterone receptor form A (PR-A) and B (PR-B) mRNA expressions in human ovarian endometriosis (ectopic endometrium). A high ratio of PR-B to PR-AB (PR-A+PR-B) mRNA expression was found in 8 of 14 cases of endometriosis, compared with the ratio in eutopic endometrium. The mean ratio in ectopic endometria was significantly (p < 0.01) higher than in eutopic endometria. The ratio in eutopic and ectopic endometria showed no significant change during the menstrual cycle. The mean ratio in ectopic endometria in the proliferative and secretory phases of the endometrium was significantly (p < 0.01) higher than in eutopic endometria. In conclusion, PR-B mRNA was relatively highly expressed in some endometriomas, which might lead to aberrations in the control of progestational effects involving responsiveness to sex steroidal growth regulation.

Expression of progesterone receptor isoforms in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentrations.

To understand the biological significance of progesterone receptor forms A (PR-A) and B (PR-B) in human corpus luteum, the expression of mRNA and serum steroid hormone concentrations were determined simultaneously in the luteal stages. The expression of PR-A mRNA predominated over PR-B mRNA in all samples analysed. Total PR (PR-AB) and PR-B mRNA concentrations at the late secretory phase were significantly (P < 0.01) lower than those at the early and mid secretory phases of the menstrual cycle. The ratio of PR-B to PR-AB mRNA concentration showed no significant change during the secretory phase. In the early and mid secretory phases, there was a negative correlation between PR-B mRNA concentration and serum progesterone concentration, and between the ratio of PR-B to PR-AB mRNA concentrations and serum progesterone concentration (P < 0.01). These findings suggest that human corpus luteum might intracellularly synthesize PR-A and PR-B, and thus be involved in the steroid functional regulation of the corpus luteum, especially at the early and mid secretory phases, and that progesterone might regulate the synthesis of PR-A and PR-B.

A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor.

Synthetic steroid hormone antagonists are clinically important compounds that regulate physiological responses to steroid hormones. The antagonists bind to the hormone receptors, which are ligand-inducible transcription factors, and modulate their gene-regulatory activities. In most instances, a steroid receptor, such as progesterone receptor (PR) or estrogen receptor (ER), is transcriptionally inactive when complexed with an antagonist and competitively inhibits transactivation of a target steroid-responsive gene by the cognate hormone-occupied receptor. In certain cellular and promoter contexts, however, antagonist-occupied PR or ER acquires paradoxical agonist-like activity. The cellular mechanisms that determine the switch from the negative to the positive mode of transcriptional regulation by an antagonist-bound steroid receptor are unknown. We now provide strong evidence supporting the existence of a cellular inhibitory cofactor that interacts with the B form of human PR (PR-B) complexed with the antiprogestin RU486 to maintain it in a transcriptionally inactive state. In the presence of unliganded thyroid hormone receptor (TR) or ER complexed with the antiestrogen 4-hydroxytamoxifen, which presumably sequesters a limiting pool of the inhibitory cofactor, RU486-PR-B functions as a transcriptional activator of a progesterone-responsive gene even in the absence of hormone agonist. In contrast, hormone-occupied TR or ER fails to induce transactivation by RU486-PR-B. Recent studies revealed that a transcriptional corepressor, NCoR (nuclear receptor corepressor), interacts with unliganded TR but not with liganded TR. Interestingly, coexpression of NCoR efficiently suppresses the partial agonistic activity of antagonist-occupied PR-B but fails to affect transactivation by agonist-bound PR-B. We further demonstrate that RU486-PR-B interacts physically with NCoR in vitro. These novel observations suggest that the inhibitory cofactor that associates with RU486-PR-B and represses its transcriptional activity is either identical or structurally related to the corepressor NCoR. We propose that cellular mechanisms that determine the switch from the antagonistic to the agonistic activity of RU486-PR-B involve removal of the corepressor from the antagonist-bound receptor so that it can effect partial but significant gene activation.

Expression of Progesterone Receptor Form A and B mRNAs in Uterine Leiomyoma

This study was designed to determine the expression pattern of progesterone receptor form A (PR-A) and B (PR-B) mRNAs in uterine leiomyoma. Approximately equal expression of PR-A and PR-B mRNAs was designated as type AB and dominant expression of PR-B mRNA as type B. In all cases of normal uterine myometrium, PR mRNA expression was type AB. Alteration to type B in the superficial part of uterine leiomyoma was observed in approximately 40% of cases. Therefore, the relative overexpression of PR-B mRNA in the surface of uterine leiomyoma might reveal an activated phenotype of progestational proliferation, plausibly related to the growth of uterine leiomyoma.

Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands.

In this report we document the creation of transgenic mice in which the native ratio of A and B forms of progesterone receptor (PR) has been altered by the introduction of additional A form as transgene. We also show that in these mice there is an aberration in mammary development. In ovariectomized prepubertal PR-A transgenic mice, end buds with unusual morphology persist after ovariectomy, and in young adult nonovariectomized mice, mammary glands have extensive lateral branching. The glands of adult mice also exhibit ductal hyperplasia with a disorganized basement membrane and decreased cell-cell adhesion, features commonly associated with neoplasia. Because progesterone is a mitogenic hormone in mammary glands and PR is required for mammary development, these data provide direct evidence that in vivo a regulated expression of the two isoforms of PR is critical for appropriate cellular response to progesterone and that for mammary glands this may have major implications to carcinogenesis.

Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle

Recent studies suggest that the progesterone receptor isoforms (PR-A and PR-B) activate genes differentially and that PR-A may act as a repressor of PR-B function. Hence, the absolute and relative expression of the two isoforms will determine the response to progesterone. We have measured their relative expression in the uterus of cycling women who underwent endometrial biopsy. PR isoforms were identified on blots of SDS-PAGE gels by reaction with the AB-52 antibody after immunoprecipitation from endometrial extract. Both isoforms were highest in the peri-ovulatory phase, but levels of PR-A were always higher than those of PR-B. The ratio of PR-A to PR-B changed during the menstrual cycle. Between days 2 and 8, PR-B is almost undetectable and the A:B ratio is >10:1. From days 9 to 13, the ratio is about 5:1, and it is about 2:1 between days 14 and 16. Thereafter, PR-B dwindles rapidly and is virtually undetectable at the end of the cycle. In various hypoestrogenic environments, PR-B expression was reduced. However, exogenous estrogens in the follicular phase in the form of oral contraceptives, enhanced PR-B expression. These data support the possibility that progesterone acts through cycle-specific PR isoforms.

Clinical implication of expression of progesterone receptor form A and B mRNAs in secondary spreading of gynecologic cancers

This study was designed to determine the clinical implication of expression of progesterone receptor form A (PR-A) and B (PR-B) mRNAs in secondary spreading of gynecologic cancers. Approximately equal expression of PR-A and PR-B mRNAs was designated as type AB and dominant expression of PR-B mRNA as type B. Alteration from type AB to type B in the metastatic cancers occurred in 3 8 cases of uterine endometrial cancers, 2 8 cases of uterine cervical cancers, and 2 8 cases of ovarian cancers. Other cancers revealed type B regardless of primary or metastatic status. thus, all metastatic cancers studied revealed type B. These results suggest that transcription of PR-A mRNA may be damaged, which might lead to uncontrolled overexpression of PR-B mRNA in metastatic lesion, and that the type B status could reveal a highly malignant phenotype in these three gynecologic cancers.

Progesterone Binding to Plasma Membrane and Cytosol Receptors in the Amphibian Oocyte

We have found a single class of progesterone binding sites at the amphibian oocyte plasma membrane, whereas two progesterone receptor forms, similar to those in chick and human, are present in the cytosol. In this study both plasma membranes and 105,000 × g cytosol fromRana pipiensoocytes were photoaffinity labeled with the synthetic progestin [3H]R5020. SDS-polyacrylamide gel electrophoresis of the photolabeled proteins in the oocyte cytosol indicate that the two forms have molecular weights essentially identical to that found for human breast tissue and chick oviduct, i.e., 80 and 110 kDa, and that the forms were present in approximately equimolar ratios. In contrast, the plasma membrane form is present as a single 110 kDa species and accounts for at least 50% of the total 110 kDa species. The presence of large amounts of the 110 kDa protein in both membrane and cytosol suggests that the plasma membrane receptor may not be unique, and that the 110 kDa form may function both in membrane and cytosol and/or that part of the cytosolic 110 kDa form represents progesterone receptor in the process of being transported to or from the plasma membrane.

An amino-terminal truncated progesterone receptor isoform, PRc, enhances progestin-induced transcriptional activity.

Previously we reported the identification of two unique progesterone receptor (PR) messenger RNA transcripts that encode a smaller PR isoform, termed the C-receptor (PRc). These two PR transcripts encode a protein that is N-terminally truncated, so that it lacks the first zinc finger of the DNA binding domain, but still contains a complete hormone binding region with sequences for dimerization and nuclear localization. We also have demonstrated the existence of a 60-kDa progestin-specific binding protein in progestin target cells using a monoclonal antibody directed to the C-terminus of PRs, suggesting that these two novel transcripts generate a truncated form of PR. In this paper, we address the hypothesis that the C-receptor arises from the initiation of translation of a methionine C-terminal to the methionine start sites that generate the larger 94-kDa A and 116-kDa B human PR isoforms. The studies shown here support the postulate that another downstream in-frame methionine within the PR-coding region can serve as a translation initiation site for the generation of a third PR protein. A partial PR complementary DNA, lacking the translation start sites for B- and A-receptors was translated in vitro. The synthetic protein product bound [3H]progestins and unlabeled progestins. The antiprogestin RU486 also competed for this binding. Transfection of this partial PR complementary DNA into PR-negative HeLa cells resulted in progestin-specific binding activity. Because the third PR isoform lacks the first zinc finger of the DNA binding domain, but contains sequences for dimerization, we reasoned that the C-receptor isoform would be transcriptionally in-active and not bind DNA directly. Surprisingly, however, in the presence of A- and/or B-receptors, we found that C-receptors can modulate the transcriptional activity of A- and/or B-receptors using a reporter gene. These studies emphasize that multiple receptor isoforms may have distinct biological properties, and that the truncated C-receptor may play a role in explaining some of the pleiotropic effects of progestins.

Hormone-dependent changes in A and B forms of progesterone receptor.

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied. These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.

Progesterone receptor-induced bending of its target DNA: distinct effects of the A and B receptor forms.

We have used circular permutation and phasing electrophoretic mobility shift assays to determine the ability of the A and B forms of human progesterone receptor (PR) to bend target DNA. Studies were done with baculovirus-expressed full-length receptors purified to apparent homogeneity. By circular permutation analysis, both forms of PR induced substantial distortions in the structure of target DNA with calculated distortion angles (alpha D) of 57 degrees for PR-A and 84 degrees for PR-B. The apparent bend centers for both forms of PR were similarly located a few base pairs (-4 to -2 bp) from the middle of the progesterone response element. No differences were detected in the magnitude of distortion or apparent bend centers when PR was bound to hormone agonist (R5020) or the antagonist RU486. Phasing analysis, which can determine the orientation of a DNA bend, revealed that both forms of PR mediated directional bends toward the major groove of the DNA helix. Calculated directed bend angles (alpha B) were 40 degrees for PR-B and 31 degrees for PR-A. The chromatin high mobility group protein HMG-1, which acts as an accessory factor to enhance the binding affinity of purified PR for progesterone response elements, had minimal influence on PR-mediated DNA bending. This result, taken together with the fact that HMG-1 can form a ternary complex with PR and DNA, is consistent with the conclusion that HMG-1 facilitates PR binding by stabilizing a receptor-induced DNA conformation that is required for assembly of a high affinity PR-DNA complex. The results of this study also suggest that DNA bending may be coupled to transcriptional regulation since PR-B is generally a stronger transcriptional activator than PR-A and also mediates a larger bend in target DNA than PR-A.