Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.     

Agonist and antagonist-induced qualitative and quantitative alterations of progesterone receptor from breast cancer cells

T47D cells, cultured in medium containing serum stripped of endogenous steroids, proliferate in response to treatment with the progesterone receptor (PR) agonist, R5020 or the PR agonist/antagonist, RU486, whereas the full PR antagonist, ZK98299 has no proliferative effects. Under estrogenized conditions, all of the PR ligands tested inhibit cell growth [23]. In order to determine whether the levels or phosphorylation state of PR are reflected in the growth patterns of T47D cells, we monitored the effects of these PR ligands on the immunoblotted PR band intensities, the relative intensities, of PR-A and PR-B, and their phosphorylation states that are reflected in their altered mobility during SDS-PAGE. Under conditions where the PR ligands inhibit cell proliferation, each ligand had distinctively different qualitative and quantitative effects on PR. Short term treatment of the cells with R5020 or RU486 induced a characteristic phosphorylation-dependent upshift of both PR-A and PR-B. The phosphorylated PR was stable for up to 4 days after treatment of the cells with RU486, but was down regulated between 6-24 h after treatment with R5020. No replenishment of PR in cells treated with R5020 was detected. ZK98299, at concentrations tested, had no qualitative or quantitative effects on PR. Culturing cells for 8 days in medium containing steroid-depleted serum caused a significant reduction in the PR band intensity without causing a change in the ratio of PR-A and PR-B or their phosphorylation states. This decrease in the PR band intensity was reversed by maintaining the cells in 1 nM estrogen, but was potentiated by RU486 or ZK98299. These observations support the view that decreased PR levels may play a role in the stimulatory effects of R5020 and RU486 when cells are cultured under non-estrogenized conditions.     

Progesterone-independent effects of human progesterone receptors (PRs) in estrogen receptor-positive breast cancer: PR isoform-specific gene regulation and tumor biology

Progesterone receptors (PRs) are prognostic markers in breast cancers irrespective of the patient's progestational status. However, there are two PR isoforms, PR-A and PR-B, that are equimolar in the normal breast but dysregulated in advanced disease. Postmenopausal, tamoxifen-treated patients with estrogen receptor (ER)-positive, PR-A-rich tumors have much faster disease recurrence than patients with PR-B-rich tumors. To study the mechanisms we engineered ER+ breast cancer cells that express each PR isoform under control of an inducible promoter. We identified 79 genes regulated by progesterone (P), mainly by PR-B, and 51 genes regulated without progesterone, mainly by PR-A. Only nine genes were regulated with and without ligand, leading to definition of three classes: I) genes regulated only by liganded PR; II) genes regulated only by unliganded PR; III) genes regulated by both. Unliganded PR-A and PR-B differentially regulate genes that coordinate extracellular signaling pathways and influence tumor cell biology. Indeed, in the absence of P, compared with ER+/PR-B+ or PR- cells, ER+, PR-A+ cells exhibit an aggressive phenotype, are more adhesive to an extracellular matrix, and are more migratory. Additionally, unliganded PR-A and PR-B both inhibit cell growth and provoke resistance to Taxol-induced apoptosis. We propose that PR-A:PR-B ratios, even in the absence of P, influence the biology and treatment response of ER+ tumors, that PR-A isoforms are functionally dominant in P-deficient states, and that PR-A rich tumors are especially aggressive.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Regulation of progesterone receptor isoforms content in human astrocytoma cell lines

Progesterone regulates several functions through the interaction with its intracellular receptor (PR) which expresses two isoforms with different functions and regulation: PR-A and PR-B. Both PR isoforms have been detected in human astrocytomas, the most common and aggressive primary brain tumours, but their regulation and function are unknown. We studied the effects of estradiol, progesterone and their receptor antagonists (ICI 182,780 and RU 486) on PR isoforms content in U373 and D54 human astrocytoma cell lines, respectively derived from grades III and IV astrocytomas, by Western blot analysis. In U373 cells we also evaluated the effects of PR-A overexpression on cell growth. We observed that in U373 cells estradiol increased the content of both PR isoforms whereas in D54 cells it had no effects. Estradiol effects were blocked by ICI 182,780. In both cell lines, PR isoforms content was down-regulated by progesterone after estradiol treatment. This effect was blocked by RU 486. We observed that overexpression of PR-A significantly diminished the increase in U373 cells number produced after progesterone treatment. Our results suggest a differential PR isoforms regulation depending on the evolution grade of human astrocytoma cells, and an inhibitory role of PR-A on progesterone effects on astrocytomas cell growth.     

Human umbilical vascular endothelial cells express estrogen receptor beta (ERβ) and progesterone receptor A (PR-A), but not ERα and PR-B

Several reports deal with possible effects of female sex hormones on human umbilical vein endothelial cells (HUVEC) including elasticity, activation of plasma membrane Na(+)/H(+) exchange, VEGF receptor Flk-1/KDR and many others. In contrast to those findings, some publications pointed out that HUVEC lack expression of both the estrogen receptor (ER) and/or the progesterone receptor (PR). Because the majority of these investigations were carried out at a time period, when only one ER and one PR was known, the aim of this study was the systematic analysis of ERalpha and ERbeta as well as PR-A and PR-B expression in HUVEC with specific monoclonal antibodies by immunocytochemistry and quantitative RT-PCR (TaqMan). As a result, we could show that HUVEC lack ERalpha but express ERbeta. The expression of ERbeta could be significantly upregulated with 17beta-estradiol on mRNA and protein level. In addition, HUVEC express PR-A but not PR-B. PR-A expression could be significantly upregulated with progesterone, again on mRNA and protein level. We conclude that estrogenic effects on HUVEC are mediated via the ERbeta and gestagens act via the PR-A pathway.     

Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function

Progesterone and its interaction with nuclear progesterone receptors (PR) PR-A and PR-B play a critical role in the regulation of female reproductive function in all mammals. However, our knowledge of the regulation and possible cellular function of PR protein isoforms in the fallopian tube and uterus in vivo is still very limited. In the present study, we revealed that equine chorionic gonadotropin (eCG) treatment resulted in a time-dependent increase in expression of both isoforms, reaching a maximal level at 48 h in the fallopian tube. Regulation of PR-A protein expression paralleled that of PR-B protein expression. However, in the uterus PR-B protein levels increased and peaked earlier than PR-A protein levels after eCG treatment. With prolonged exposure to eCG, PR-B protein levels decreased, whereas PR-A protein levels continued to increase. Furthermore, subsequent treatment with human (h)CG decreased the levels of PR protein isoforms in both tissues in parallel with increased endogenous serum progesterone levels. To further elucidate whether progesterone regulates PR protein isoforms, we demonstrated that a time-dependent treatment with progesterone (P(4)) decreased the expression of PR protein isoforms in both tissues, whereas decreases in p27, cyclin D(2), and proliferating cell nuclear antigen protein levels were observed only in the uterus. To define the potential PR-mediated effects on apoptosis, we demonstrated that the PR antagonist treatment increased the levels of PR protein isoforms, induced mitochondrial-associated apoptosis, and decreased in epidermal growth factor (EGF) and EGF receptor protein expression in both tissues. Interestingly, immunohistochemistry indicated that the induction of apoptosis by PR antagonists was predominant in the epithelium, whereas increase in PR protein expression was observed in stromal cells of both tissues. Taken together, these observations suggest that 1) the tissue-specific and hormonal regulation of PR isoform expression in mouse fallopian tube and uterus, where they are potentially involved in regulation of mitochondrial-mediated apoptosis depending on the cellular compartment; and 2) a possible interaction between functional PR protein and growth factor signaling may have a coordinated role for regulating apoptotic process in both tissues in vivo.     

Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus

Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A + PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24 h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A + B antisense (PR A + B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A + B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A + B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the regulation of the content of TPH, TH and GAD in the rat hypothalamus.     

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.