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.     

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.     

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.     

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.     

Ontogenic variations in the content and distribution of progesterone receptor isoforms in the reproductive tract and brain of chicks

Progesterone participates in the regulation of several functions in chicks such as ovulation, gonadal differentiation, and sexual and nesting behaviors. Many progesterone actions are mediated by specific intracellular receptors (PR) which are ligand-induced transactivators. Two PR isoforms that are functionally distinct in their ability to activate genes and regulate distinct physiological processes have been described in chicks: a full length form PR-B and the N-terminally truncated one PR-A which lacks the amino-terminal 128 amino acids of PR-B. PR isoforms have been detected in several tissues of both the adult and the embryo chick such as brain, ovary and oviduct. PR isoforms expression ratio varies among progesterone target tissues and under different hormonal and environmental conditions such as those presented during avian sexual maturity and the seasons of the year. These data let us to conclude that progesterone actions in brain, ovary, and oviduct highly depend on PR isoforms expression pattern and regulation.     

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.     

Estradiol increases cell growth in human astrocytoma cell lines through ERα activation and its interaction with SRC-1 and SRC-3 coactivators

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.     

Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappaB activation of cyclooxygenase 2 expression

Spontaneous labor in women and in other mammals is likely mediated by a concerted series of biochemical events that negatively impact the ability of the progesterone receptor (PR) to regulate target genes that maintain myometrial quiescence. In the present study, we tested the hypothesis that progesterone/PR inhibits uterine contractility by blocking nuclear factor kappaB (NF-kappaB) activation and induction of cyclooxygenase-2 (COX-2), a contractile gene that is up-regulated in labor. To uncover mechanisms for regulation of uterine COX-2, immortalized human fundal myometrial cells were treated with IL-1beta +/- progesterone. IL-1beta alone caused a marked up-regulation of COX-2 mRNA, whereas treatment with progesterone suppressed this induction. This was also observed in human breast cancer (T47D) cells. In both cell lines, this inhibitory effect of progesterone was blocked by RU486. Using chromatin immunoprecipitation, we observed that IL-1beta stimulated recruitment of NF-kappaB p65 to both proximal and distal NF-kappaB elements of the COX-2 promoter; these effects were diminished by coincubation with progesterone. The ability of progesterone to inhibit COX-2 expression in myometrial cells was associated with rapid induction of mRNA and protein levels of inhibitor of kappaBalpha, a protein that blocks NF-kappaB transactivation. Furthermore, small interfering RNA-mediated ablation of both PR-A and PR-B isoforms in T47D cells greatly enhanced NF-kappaB activation and COX-2 expression. These effects were observed in the absence of exogenous progesterone, suggesting a ligand-independent action of PR. Based on these findings, we propose that PR may inhibit NF-kappaB activation of COX-2 gene expression and uterine contractility via ligand-dependent and ligand-independent mechanisms.