16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium.

Detection and functional portrayal of a novel class of dihydrotestosterone derived selective progesterone receptor modulators (SPRM).

In early pregnancy, abortion can be induced by blocking the actions of progesterone receptors (PR). However, the PR antagonist, mifepristone (RU38486), is rather unselective in clinical use because it also cross-reacts with other nuclear receptors. Since the ligand-binding domain of human progesterone receptor (hPR) and androgen receptor (hAR) share 54% identity, we hypothesized that derivatives of dihydrotestosterone (DHT), the cognate ligand for hAR, might also regulate the hPR. Compounds designed and synthesized in our laboratory were investigated for their affinities for hPRB, hAR, glucocorticoid receptor (hGRα) and mineralocorticoid receptor (hMR), using whole cell receptor competitive binding assays. Agonistic and antagonistic activities were characterized by reporter assays. Nuclear translocation was monitored using cherry-hPRB and GFP-hAR chimeric receptors. Cytostatic properties and apoptosis were tested on breast cancer cells (MCF7, T-47D). One compound presented a favorable profile with an apparent neutral hPRB antagonistic function, a selective cherry-hPRB nuclear translocation and a cytostatic effect. 3D models of human PR and AR with this ligand were constructed to investigate the molecular basis of selectivity. Our data suggest that these novel DHT-derivatives provide suitable templates for the development of new selective steroidal hPR antagonists.

Proteasome-dependent degradation of the human estrogen receptor

In eukaryotic cells, the ubiquitin–proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin–proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor β proteins.

The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.

Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.

Evaluation of estrogen receptor alpha and beta and progesterone receptor expression and correlation with clinicopathologic factors and proliferative marker Ki-67 in breast cancers

To elucidate the molecular profile of hormonal steroid receptor status, we analyzed ER-alpha, ER-beta, and PGR mRNA and protein expression in 80 breast carcinomas using reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical analysis. Qualitative analysis revealed positive expression of ER-alpha, ER-beta, and PGR mRNA in 48%, 59%, and 48% of the breast carcinomas, respectively. ER-alpha, ER-beta, and PGR transcript overexpression was observed in 51%, 0%, and 12% of the cases, respectively, whereas moderate or strong protein expression was detected in 68%, 78%, and 49% of the cases, respectively. Tumor grade was negatively correlated with transcript and protein levels of ER-alpha (P = .0169 and P = .0006, respectively) and PGR (P = .0034 and P = .0005, respectively). Similarly, proliferative index Ki-67 was negatively associated with transcript and protein levels of ER-alpha (P = .0006 and P < .0001, respectively) and PGR (P = .0258 and P =. 0005, respectively). These findings suggest that ER-alpha and PGR expression are associated with well-differentiated breast tumors and less directly related to cell proliferation. A significant statistical difference was observed between lymph node status and ER-beta protein expression (P = .0208). In ER-alpha-negative tumors, we detected a correlation between ER-beta protein expression and high levels of Ki-67. These data suggest that ER-beta could be a prognostic marker in human breast cancer. (C) 2008 Elsevier B.V. All rights reserved.

Marginal activity of progesterone receptor B (PR-B) in dogs but high incidence of mammary cancer

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

No significant association between progesterone receptor exon 4 Val660Leu G/T polymorphism and risk of ovarian cancer

Epidemiological studies suggest that ovarian cancer is an endocrine-related tumour, and progesterone exposure specifically may decrease the risk of ovarian cancer. To assess whether the progesterone receptor (PR) exon 4 valine to leucine amino acid variant is associated with specific tumour characteristics or with overall risk of ovarian cancer, we examined 551 cases of epithelial ovarian cancer and 298 unaffected controls for the underlying G-->T nucleotide substitution polymorphism. Stratification of the ovarian cancer cases according to tumour behaviour (low malignant potential or invasive), histology, grade or stage failed to reveal any heterogeneity with respect to the genotype defined by the PR exon 4 polymorphism. Furthermore, the genotype distribution did not differ significantly between ovarian cancer cases and unaffected controls. Compared with the GG genotype, the age-adjusted odds ratio (95% confidence interval) for risk of ovarian cancer was 0.78 (0.57-1.08) for the GT genotype, and 1.39 (0.47-4.14) for the TT genotype. In conclusion, the PR exon 4 codon 660 leucine variant encoded by the T allele does not appear to be associated with ovarian tumour behaviour, histology, stage or grade. This variant is also not associated with an increased risk of ovarian cancer, and is unlikely to be associated with a large decrease in ovarian cancer risk, although we cannot rule out a moderate inverse association between the GT genotype and ovarian cancer.

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has similar to 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P-103 in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants ((PY)-Y-103, G(105)D and the double mutant, (PY)-Y-103/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and (PY)-Y-103/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR. (C) 2001 Elsevier Science Inc. All rights reserved.

The progesterone receptor exon 4 Va1660Leu G/T polymorphism and risk of breast cancer in Australian women

An Alu insertion polymorphism of the progesterone receptor (PR) was reported recently to be associated with a reduced risk of breast cancer, with risks of 0.8- and 0.3-fold associated with the heterozygote and homozygote genotypes, respectively. This intronic variant is considered to be in linkage disequilibrium with an exon 4 hinge region G to T Val660Leu polymorphism. We investigated whether the exon 4 PR polymorphism was associated with breast cancer in Australian women, using a population-based study of 1452 cases and 793 controls, half of whom were

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Signatures of selection in the human olfactory receptor OR5I1 gene

The human olfactory receptor repertoire is reduced in comparison to other mammalsand to other non-human primates. Nonetheless, this olfactory decline opens an opportunity forevolutionary innovation and improvement. In the present study, we focus on an olfactoryreceptor gene, OR5I1, which had previously been shown to present an excess of amino acidreplacement substitutions between humans and chimpanzees. We analyze the geneticvariation in OR5I1 in a large worldwide human panel and find an excess of derived allelessegregating at relatively high frequencies in all populations. Additional evidence for selectionincludes departures from neutrality in allele frequency spectra tests but no unusually extendedhaplotype structure. Moreover, molecular structural inference suggests that one of thenonsynonymous polymorphisms defining the presumably adaptive protein form of OR5I1may alter the functional binding properties of the olfactory receptor. These results arecompatible with positive selection having modeled the pattern of variation found in the OR5I1gene and with a relatively ancient, mild selective sweep predating the “Out of Africa”expansion of modern humans.

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5' flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogenin minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells. In contrast to the transfected genes, the endogenous chromosomal vitellogenin genes remain silent, demonstrating that in spite of the presence of the hER and the hormone, the conditions necessary for their activation are not fulfilled.

Immuno-electron microscopic identification of human estrogen receptor-DNA complexes at the estrogen-responsive element and in the first intron of a Xenopus vitellogenin gene.

Using an extract of nuclei from the estrogen-responsive human breast cancer cell line MCF-7, protein-DNA complexes were assembled in vitro at the 5' end of the Xenopus laevis vitellogenin gene B2 that is normally expressed in liver after estrogen induction. The complexes formed were analyzed by electron microscopy after labeling by the indirect colloidal gold immunological method using a monoclonal antibody specific for the human estrogen receptor. As identified by its interaction with protein A-gold, the antibody was found linked to two protein-DNA complexes, the first localized at the estrogen responsive element of the gene and the second in intron I, thus proving a direct participation of the receptor in these two complexes. The procedure used allows the visualization and rapid localization of specific transcription factors bound in vitro to a promoter or any other gene region.