Hormone-dependent phosphorylation of the avian progesterone receptor

Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains.     

The ubiquitous nature of the progesterone receptor binding factor-1 (RBF-1) in avian tissues

The avian oviduct receptor binding factor-1 (RBF-1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF-1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF-1. The heart and pancreas had the highest and lowest RBF-1 levels, respectively; the concentration ranging by ～ 50-fold in these tissues. The 10 kDa size of the RBF-1 detected in all tissues suggests no significant tissue-specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF-1 have identical amino-terminal sequence. Using a recently isolated cDNA to RBF-1, the levels of RBF-1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue-specific differences in concentration. The presence of RBF-1 in non-progesterone responsive tissues suggests the possibility that RBF-1 may not be specifically involved in PR-DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor. © 1994 Wiley-Liss, Inc.     

Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.     

Changes in progesterone levels and distribution of progesterone receptor during vitellogenesis in the female mud crab (Scylla paramamosain)

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.     

Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.     

Characterization of a new class of selective nonsteroidal progesterone receptor agonists

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.     

Chromosome 11 aneuploidy detected by fluorescent in situ hybridization (FISH) in breast cancer: relation with progesterone receptor expression

Progesterone receptor (PR) expression is known to be impaired in breast cancer. As the PR gene is located on chromosome 11 which is also often affected, we studied their relationship in 15 patients with breast carcinoma. Tumoural imprints were used for PR immunocytochemistry and for FISH with chromosome 11 centromeric probes. Distribution profiles of chromosome 11 number in PR+ and PR- cell populations were examined. No difference in the number of chromosome 11 was found between PR+ and PR- breast tumours. Thus, loss of PR expression in breast cancer cannot be explained only by loss of chromosome 11; other genetic or non-genetic mechanisms should be advanced.     

Iodination of the progesterone receptor from hen oviduct spares the DNA-binding domain

The progesterone receptor from hen oviduct is isolated as a complex of two subunits, A and B. The A protein binds one molecule of progesterone and also binds to DNA with high affinity. The native A protein can be labeled with iodine with no loss of DNA binding activity. Limited Staphylococcus aureus V8 protease digestion of the labeled preparation results in a number of DNA-binding and non-DNA-binding fragments of the receptor. The progesterone-binding domain contains iodine label. However, two low-molecular-weight DNA-binding fragments do not contain iodine label, indicating a lack of susceptible tyrosine residues near the DNA-binding site of the native receptor. The labeled receptor and its fragments will facilitate studies of the isolated DNA-binding and progesterone-binding domains of the hen A protein as well as of the activity of the native receptor in the presence and absence of hormone.     

Effects of injury and progesterone treatment on progesterone receptor and progesterone binding protein 25-Dx expression in the rat spinal cord

Progesterone provides neuroprotection after spinal cord injury, but the molecular mechanisms involved in this effect are not completely understood. In this work, expression of two binding proteins for progesterone was studied in intact and injured rat spinal cord: the classical intracellular progesterone receptor (PR) and 25-Dx, a recently discovered progesterone membrane binding site. RT-PCR was employed to determine their relative mRNA levels, whereas cellular localization and relative protein levels were investigated by immunocytochemistry. We observed that spinal cord PR mRNA was not up-regulated by estrogen in contrast to what is observed in many brain areas and in the uterus, but was abundant as it amounted to a third of that measured in the estradiol-stimulated uterus. In male rats with complete spinal cord transection, levels of PR mRNA were significantly decreased, while those of 25-Dx mRNA remained unchanged with respect to control animals. When spinal cord-injured animals received progesterone treatment during 72 h, PR mRNA levels were not affected and remained low, whereas 25-Dx mRNA levels were significantly increased. Immunostaining of PR showed its intracellular localization in both neurons and glial cells, whereas 25-Dx immunoreactivity was localized to cell membranes of dorsal horn and central canal neurons. As the two binding proteins for progesterone differ with respect to their response to lesion, their regulation by progesterone, their cellular and subcellular localizations, their functions may differ under normal and pathological conditions. These observations point to a novel and potentially important role of the progesterone binding protein 25-Dx after injury of the nervous system and suggest that the neuroprotective effects of progesterone may not necessarily be mediated by the classical progesterone receptor but may involve distinct membrane binding sites.     

Sensorimotor development in neonatal progesterone receptor knockout mice

Early exposure to steroid hormones can permanently and dramatically alter neural development. This is best understood in the organizational effects of hormones during development of brain regions involved in reproductive behaviors or neuroendocrine function. However, recent evidence strongly suggests that steroid hormones play a vital role in shaping brain regions involved in cognitive behavior such as the cerebral cortex. The most abundantly expressed steroid hormone receptor in the developing rodent cortex is the progesterone receptor (PR). In the rat, PR is initially expressed in the developmentally‐critical subplate at E18, and subsequently in laminas V and II/III through the first three postnatal weeks (Quadros et al. [2007] J Comp Neurol 504:42–56; Lopez & Wagner [2009]: J Comp Neurol 512:124–139), coinciding with significant periods of dendritic maturation, the arrival of afferents and synaptogenesis. In the present study, we investigated PR expression in the neonatal mouse somatosensory cortex. Additionally, to investigate the potential role of PR in developing cortex, we examined sensorimotor function in the first two postnatal weeks in PR knockout mice and their wildtype (WT) and heterozygous (HZ) counterparts. While the three genotypes were similar in most regards, PRKO and HZ mice lost the rooting reflex 2–3 days earlier than WT mice. These studies represent the first developmental behavioral assessment of PRKO mice and suggest PR expression may play an important role in the maturation of cortical connectivity and sensorimotor integration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 16–24, 2014     

Cre-mediated recombination in cell lineages that express the progesterone receptor

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.     

Occurrence of a 6S intermediate form of the progesterone receptor that is sensitive to ribonuclease

Steroid receptors exist in cytosol as 9S, non-DNA-binding species and as 4S (transformed) species that bind to DNA or nuclei. Labeling the progesterone receptor from rabbit uterine cytosol with [³H]progesterone in the presence of 10 mM sodium molybdate revealed a 9S species on sucrose gradient centrifugation. Without molybdate, the receptor sedimented as an intermediate species of 6S, which converted to 4S in 0.3 M NaCl. The 6S species could also be generated from the 4S species by dialysis. Dilution of the same 4S species gave only partial re-aggregation with 50% of the receptor remaining as 4S. Dialysis appeared to retain the association of a macromolecular aggregation factor present in cytosol. Serum did not seem to be the source of the aggregation factor, as perfusion of the uterine vasculature before excision did not affect the S value of the receptor. We tested whether RNA was involved by treating receptor with RNase A (100 µg/400 µl cytosol). While the molybdate-stabilized cytosol receptor (9S) was unaffected, RNase A partially (50%) converted the 6S form of receptor to 4S. RNase A also partially converted the re-aggregated form back to 4S. Protease inhibitors had no effect on this action of RNase. Formation of receptor-ribonucleotide protein particles may play a role in steroid action in the cell.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone). In the immature or ovariectomized quail oviduct, PR was principally localized in the undifferentiated epithelial cells; some mesothelial cells and a very few stromal cells expressed the PR. Only 40-45% of the epithelial cells were immunoreactive. These positive cells were mainly localized in the furrows of the villi where further evagination of the epithelium will occur to form the tubular glands. The onset of sexual maturation was accompanied by an increase of the proportion of positive epithelial cells and stromal cells. In estradiol-treated animals, more than 90% of the tubular gland cells were strongly stained while only 40% of the luminal epithelial cells were immunoreactive. Our results show that there are two subpopulations of epithelial cells: those expressing the PR before the onset of sexual maturation even in ovariectomized quails (constitutive expression) and those expressing the PR during sexual maturation or after estrogen injection (inductive expression). These results, associated with previously published studies dealing with the cytodifferentiation of epithelial cells during natural development or after various hormonal treatments in ovariectomized animals, suggest that the first are the progenitors of tubular gland cells, and the second the progenitors of ciliated and goblet cells. In stromal cells, PR expression is also inducuible.     

ER和PRmRNAs在内异症子宫内膜表达的变化

目的 :探讨雌激素受体 (ER)和孕激素受体 (PR)在子宫内膜异位症 (内异症 )子宫内膜的表达。方法 :利用大鼠内异症动物模型 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测子宫内膜ER和PRmRNAs的表达情况。结果 :内异症模型组大鼠异位内膜ER、PRmRNAs的表达低于在位内膜和对照组正常子宫内膜 ,与后两者比较差异有显著性意义 (P <0 .0 1) ;而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著性意义 (P >0 .0 5 )。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜和正常子宫内膜ER/PRmRNA比值 (P <0 .0 1)。结论 :内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。     

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.     

Estrogen-alpha and progesterone receptor expression in cystic endometrial hyperplasia and pyometra in the bitch

Estrogen-alpha receptor (ER) and progesterone receptor (PR) were examined immunohistochemically in uteri of normal bitches, in uteri of bitches with cystic endometrial hyperplasia-mucometra (CEH-M) and in uteri of bitches with endometritis-pyometra (E-P), under exogenous progesterone treatment.In the CEH-M group, the ER- and PR-scores of all uterine cell types were higher than the ER- and PR-scores of normal uteri, although these differences were not always statistically significant. The ER-scores of E-P group were significantly lower than the ER-scores of the normal uteri and CEH-M group. The PR-scores of the E-P group tended to be higher than the PR-scores of the normal uteri, except for the surface epithelium, although these differences were not statistically significant. Exogenous progesterone treated bitches with CEH-M or E-P showed reduced ER- and PR-scores in the different uterine cell types, compared with the corresponding nontreated CEH-M or E-P group.The differences in ER and PR expression between CEH-M and E-P suggest different factors in the pathogenesis of both entities. Although, these changes in ER and PR expression do not seem to be directly involved in the pathogenesis of CEH-M and E-P. It is suggested that for CEH-M and progestin induced CEH-M a hormone dependent pathway is responsible. For P, the trigger may be bacterial infection.     

Synthesis and biological effect of halogen substituted phenyl acetic acid derivatives of progesterone as potent progesterone receptor antagonists

In this paper, we report the relative binding affinities to the progesterone receptor (PR) of several progesterone derivatives containing an acetoxyphenyl substituent at C-17 and their structure-bioactivity relationship. The inhibitory effect to ovulation as well as their function as interrupters of endometrial maturation is also described. The biological activity of the novel steroids was determined in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. The cytosol used for the determination of the PR, was obtained from the uteri of adult estrogen-primed rabbits and the androgen (AR), mineralocorticoid (MR) and glucocorticoid (GR) receptors were determined in the cytosolic fractions from the prostate of castrated rats and from the kidneys and livers of adenalectomized male rats. We evaluated six related steroidal compounds 8a-8f differing in the nature of the 17alpha ester side chain for the inhibition of [(3)H] R5020 binding to the PR. The IC(50) values for the displacement of [(3)H] R5020 binding to the PR and its relative binding affinities (RBAs) were determined. Progesterone and R5020 had similar IC(50) values; steroids 8a, 8f and 8c bind to the progesterone receptor with RBAs of 100%, whereas 8e, 8b and 8d have RBA values <100%. These data indicate that there is a relationship between the structure of these steroids and their binding activity to the progesterone receptors. Having demonstrated in this study that steroids 8a-8f bind to the PR, we also evaluated the receptor's selectivity, since some progesterone derivatives bind to AR, MR, GR receptors. We demonstrated that the tested steroids did not bind to the AR, MR, GR, since none of the steroids inhibited the labeled mibolerone, aldosterone or dexamethasone binding to the AR, MR or GR, respectively. These results show that the novel compounds have certain selectivity for the PR. After LHRH treatment, the mice of the control group showed the presence of ova in the oviduct, whereas the animals treated with steroids 8a, 8f, 8e and 8c with RBAs of 92-100%, did not exhibit any ovum in the oviducts. As a result of this study, it is evident that the novel steroids 8a, 8f, 8e and 8c inhibited the ovulation in these animals at dose of 0.22mg/kg. After the treatment with LHRH, the uterus of the control group showed the typical progestational activity with an enlarged endometrial thickness with secretory activity. However, the endometrium of the mice treated with steroids 8a, 8f, 8e and 8c (with RBAs of 92-100%) neither did show any enlargement of the endometrium, nor a secretory activity could be detected. The diameter of the uterus was also significantly reduced compared to those of the control group, thus indicating that compounds 8a, 8f, 8e and 8c had antagonistic activity in this tissue. The overall data showed that steroids 8a, 8f, 8e and 8c have a high and selective binding activity to the PR. Furthermore there is a relationship between the structure of these steroids and their binding activity, since the presence of fluorine atom in meta position in the acetoxyphenyl substituent at C-17, improved the binding activity as compared to that for the ortho and para positions. These data also demonstrated that 8a-8f have an anti-progestational activity in vivo, and therefore they have better characteristics than the compounds previously reported.