WW-domain binding protein-2 (WBP-2) was cloned as an E6-associated protein (E6-AP) interacting protein and its role in steroid hormone receptor (SHR) function was investigated. We show that WBP-2 differs from other SHR coactivators, as it specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER alpha), whereas it had no significant effect on the androgen receptor, glucocorticoid receptor or the activation functions of p53 or VP-16. We also demonstrated that, like other well characterized coactivators, WBP-2 contains an intrinsic activation domain. Depletion of endogenous WBP-2 with small interfering RNAs indicated that normal physiological protein level of WBP-2 was required for the proper functioning of ER alpha and PR. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. As we initially identified WBP-2 as an E6-AP interacting protein, we investigated whether WBP-2 and E6-AP function in concert. Our data shows that WBP-2 and E6-AP each enhance PR function and when co-expressed they additively enhance the transactivation functions of PR. However, WBP-2 was also able to enhance the transactivation functions of ER alpha and PR in mouse embryonic fibroblast cells generated from E6-AP knockout mice lines, suggesting that the coactivation functions of WBP-2 was not dependent on E6-AP. The further elucidate the molecular mechanism of action of WBP-2; we dissected the functional importance of the polyproline (PY) motifs contained within the WBP-2 protein. Mutational analysis suggests that one of three PY motifs, PY3 of WBP-2 was essential for its coactivation and intrinsic activation functions. In this study, we also demonstrate that the WBP-2 binding protein, Yes-kinase associated protein 1 (YAP1) acts as a secondary coactivator of ER alpha and PR. However, the coactivation function of YAP1 is revealed only in the presence of wild-type WBP-2 and not with the PY motif 3 mutant WBP-2. This is consistent with our observations that, unlike the wild-type WBP-2, the PY motif 3 mutant WBP-2 does not interact with YAP1. Our quantitative reChIP assays demonstrates an estrogen-dependent recruitment and association of ER alpha with both WBP-2 and YAP1. The hormone-dependent recruitment of YAP1 to ER alpha responsive promoter is dependent on the physiological expression levels of WBP-2. This is consistent with, our observation that the coactivation functions of YAP1 is dependent on WBP-2, and is also in agreement with other known secondary coactivators that get recruited to SHR responsive promoter via their interaction with primary coactivators. Surprisingly, the association of WBP-2 with ER alpha and its recruitment to the ER alpha target promoter was abrogated by YAP1 knock-down, suggesting that WBP-2 and YAP1 may stabilize each other at the promoter, and consequently, are functionally interdependent. Taken together our data establish the role of WBP-2 and YAP1 as selective coactivators for ER alpha and PR transactivation pathways.

ABSTRACT Selective progesterone receptor modulators (SPRM) represent a new class of synthetic steroids which can interact with the progesterone receptor (PR) and can exert agonist, antagonist or mixed effects on various progesterone target tissues in vivo. VA-2914 is a selective progesterone receptor modulator with potential contraceptive activity. We evaluated VA 2914 for ovulation inhibition in women, using three experimental doses ( 2,5 mg/d, 5 mg/d, 10 mg/d ) given continuously for three months. We examined the endometrial impact in each group. Anovulation (defined by absence of progesterone above 3 ng/ml ) was obtained in nearly 80% women in the 5 and 10 mg/d groups with a great rate of amenorrhea ( 81.2 and 90% cases in the 5 and 10 mg/d groups ). Estradiol levels remained in the physiological follicular phase range. Endometrial histological analysis show predominantly usual patterns of secretory phase. Some cystic glandular dilatations are observed in rare cases ; any hyperplasia was detected. VA-2914 induces amenorrhea while progestins cause endometrial spotting and bleeding. This abnormal bleeding due to progestins is a consequence of focal stromal proteolysis by increase in naked vessel size and density. We quantified the effects of VA 2914 on endometrial vascularisation, fibrillar matrix and VEGF-A expression in endometrial biopsies from 41 women before and after 12 weeks daily treatment (2,5 , 5 or 10 mg/d VA 2914 versus placebo). We did not observed changes in endometrial vessels, collagen network and VEGF-A distribution between the luteal phase at baseline and under VA 2914. From these observations, we can assess that VA 2914 does not behave like a progestin since it does not alter the endometrial matrix nor the pattern of endometrial vessels. Résumé : Les modulateurs sélectifs du récepteur de la progestérone ( SPRMs) constituent une nouvelle famille de stéroïdes présentant des propriétés mixtes agonistes ou antagonistes en fonction des gènes cibles, du contexte cellulaire, de la présence simultanée dautres ligands du récepteur de la progestérone (Spitz 2003). Le chef de file de cette famille, la mifépristone, est utilisé depuis plusieurs décennies pour ses propriétés abortives. Son intérêt dans la contraception post coïtale a été démontré. Des molécules aux propriétés abortives réduites sont à létude ou en cours de développement dans les domaines de la contraception, du cancer du sein, du traitement médical de lendométriose et des fibromes utérins (Pintiaux et al. 2009). Le VA2914 que nous étudions, fait partie de ces SPRMs en cours de développement. Nous avons démontré sa capacité à inhiber lovulation à partir de 5 mg administrés par voie orale quotidiennement. Il exerce une action endométriale participant vraisemblablement à leffet contraceptif . Il ninhibe pas le développement folliculaire et permet déviter la carence estrogénique observée sous progestatif antigonadotrope. A partir de la dose de 5 mg par jour, son utilisation saccompagne dun haut taux daménorrhée (Chabbert-Buffet et al. 2007). Lutilisation dun SPRM au long cours pose le problème dun endomètre soumis aux estrogènes endogènes sans opposition progestative. Limpact dun SPRM sur lendomètre varie en fonction de la molécule, de son dosage, de la présence concomitante dautres stéroïdes et de lespèce à laquelle ce SPRM est administré (Chwalisz et al. 2000). Des aspects histologiques particuliers sont décrits dans les endomètres soumis aux SPRMs. Il sagit de la coexistence daspects histologiques non présents de façon simultanée physiologiquement ( aspects sécrétoires et prolifératifs coexistant au sein du même endomètre, signes dapoptose et dactivité mitotique au sein dune même glande) (Mutter et al. 2008). La persistance dune activité mitotique est observée dans les glandes endométriales des patientes anovulatoires soumises au VA2914 (Chabbert-Buffet et al. 2007). Lutilisation de cette molécule quotidiennement et à long terme nest donc pas dactualité contrairement à son utilisation ponctuelle dans le cadre de la contraception postcoïtale (Creinin 2006). La place des SPRMs en contraception classique doit être définie. Différents schémas dadministration et différents dosages devront être évalués en termes defficacité et de sécurité. Lutilisation des SPRMs pourrait être utile pour contrer les saignements indésirables observés lors de la prise de progestatif seul. Contrairement aux patientes soumises aux progestatifs seuls, les patientes sous VA2914 présentent un haut taux d'aménorrhée. Lors du saignement physiologique menstruel comme lors de l'administration de progestatif seul, la dégradation locale du stroma endométrial et une lyse du réseau fibrillaire riche en collagène sont classiquement observées (Galant et al. 2000). Sous VA2914, nous nobservons pas de dégradation de la matrice extracellulaire (Ravet et al. 2009). Le rôle des métalloprotéases matricielles dans les saignements normaux et pathologiques de l'endomètre humain est bien connu . Le profil d'expression des métalloprotéases matricielles dans l'endomètre des patientes traitées par VA2914 à différents dosages peut contribuer à lintégrité endométriale observée. Une angiogenèse aberrante est observée sous progestatif. Une modification de la densité vasculaire endométriale et une altération de la maturation de la paroi des vaisseaux, déficitaire en péricytes et en cellules musculaires lisses ont été décrites (Hickey et al. 1999; Hickey et al. 2000; Jondet et al. 2005; Rogers et al. 1993; Stephanie et al. 2007). L'observation de la vascularisation de lendomètre exposé au dispositif intrautérin au lévonorgestrel durant 1 à 3 mois montre une augmentation très importante (11,5 fois) des petits vaisseaux non matures constitués exclusivement d'un endothélium. Le nombre de vaisseaux partiellement matures est augmenté de 6 fois. Au plus long cours, ces vaisseaux immatures ou partiellement matures restent néanmoins 4 fois plus fréquents que dans les endomètres non soumis à cette thérapeutique. La surface vasculaire et la densité augmentent au cours du temps sous ce dispositif hormonal (Stephanie et al. 2007). De telles modifications ne sont pas observées sous VA2914 et peuvent contribuer à l'absence de saignement. Au cours du cycle témoin, nous avons observé la présence de vaisseaux matures, représentant 80 % de la surface vasculaire totale. Après 3 mois de traitement sous VA2914 aux différentes doses, aucun changement vasculaire n'est observé. Sous VA2914, nous ne constatons pas de modification de l'expression du VEGFA ni de modification de sa distribution qui apparaît prédominante au niveau de la portion apicale des cellules épithéliales de surface et glandulaires. L'absence de modification de la distribution et de l'intensité du marquage du VEGFA (Vascular endothelial growth factor) et la stabilité du rapport Ang-1/Ang-2 avant et sous traitement par VA2914 n'est pas en faveur d'un remodelage vasculaire important (Ravet et al. 2009). Au niveau du réseau vasculaire endométrial, le VA2914 ne paraît donc pas se comporter comme un agoniste du récepteur de la progestérone.

<p>The progesterone receptor (PR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. The steroid hormone progesterone binds to PR and induces a conformational change that enables the receptor to bind DNA, recruit cofactors, and directly regulate the transcription of target genes. In addition, extra-nuclear PR can indirectly regulate gene expression by rapidly activating other signaling pathways such as Src/MAPK. Although the direct and indirect functions of PR have been well studied in isolation, it is important to understand the molecular mechanisms by which these pathways can cross talk and integrate to ultimately impact gene expression.</p><p>Towards this end, we initiated studies to assess the overall impact of MAPK inhibition on PR transcriptional activity in T47D breast cancer cells treated with the synthetic progestin R5020. During the course of microarray and biochemical analyses that were undertaken to address this issue, we discovered a subset of PR target genes that are enriched for E2F binding sites. Subsequently, we determined that PR-B is a component of several distinct pathways that function both directly and indirectly to positively up-regulate E2F1 expression in T47D breast cancer cells. Firstly, PR directly regulates E2F1 transcription by binding to proximal and distal enhancer sites located near E2F1. Secondly, progestin induces the hyperphosphorylation of Rb, which results in increased recruitment of E2F1 to its own promoter, thereby activating a positive feedback loop that further amplifies its transcription. Finally, PR induces expression of Krüppel-like factor 15 (KLF15) and potentially other Sp/KLF family members, which can bind to GC-rich DNA within the E2F1 promoter and further activate transcription. Together, these results suggest a paradigm for multimodal PR gene regulation that entails cooperation between direct and indirect pathways of PR signaling to achieve the desired downstream transcriptional cascade.</p><p>In the breast and other tissues of the female reproductive system, progesterone plays an important role in normal development and function. Therefore, synthetic PR modulators (PRMs) are widely used to manipulate the downstream biology of PR for purposes including contraception and hormone replacement therapy (HRT). However, progestins and PR have also been implicated in disease pathologies such as breast cancer. While the molecular mechanisms by which PR regulates breast tumor growth have not been fully elucidated, recent studies highlight the fact that progestins may have a dose-dependent role in breast cancer progression. Consequently, we undertook studies to identify and characterize any differential effects of low-dose versus high-dose progestins on the downstream activities of PR. Specifically, we found that treatment of breast cancer cells with low-dose progestins can induce maximal transcriptional activation of a subset of PR target genes, including the cell cycle regulators cyclin D1 and E2F1. Furthermore, low-dose and high-dose progestins have differential effects on the phosphorylation of PR and subsequent receptor turnover. Cumulatively, these findings underscore the importance of establishing the effects of a wide range of progestin concentrations on target gene expression and other PR actions, so that we are able to accurately predict the potential consequences of PRMs on downstream PR signaling pathways and biology.</p> / Dissertation

Endometrial cancer is the fourth most common cancer in women and the most common gynecological malignancy. While patient outcome has improved for the majority of cancers, the outlook for endometrial cancer has steadily decreased. In order to address this problem, we must better understand the different mechanisms involved in endometrial cancer development and progression. To this end, we quantified expression of 667 miRNAs in four endometrioid adenocarcinoma and four serous adenocarcinoma using Taqman Low Density Arrays (TLDAs). miR-888 was one of the most highly overexpressed miRNAs in both endometrial cancer subtypes. Analysis of miR-888 expression across multiple cancer types using the The Cancer Genome Atlas database revealed that miR-888 was selectively expressed in endometrial cancer, with a significant association to invasive and high grade tumors. In addition, miR-888 was most predominantly expressed in endometrial carcinosarcoma, a rare but deadly form of endometrial cancer. Therefore, we conclude that miR-888 expression marks an aggressive endometrial tumor phenotype. One of the top predicted targets of miR-888 by TargetScan is the progesterone receptor (PR). PR is a potent tumor suppressor of the endometrium whose expression is often lost in advanced endometrial cancers. We quantified PR mRNA expression in a panel of endometrial tumors and found a statistically significant, negative correlation between miR-888 and PR mRNA expression. Furthermore, overexpression of miR-888 in endometrial cancer cell lines was capable of decreasing PR at the protein level. To determine if miR-888 directly targets PR, we cloned each of the four miR-888 binding sites downstream of Renilla luciferase into the psiCHECK2 reporter vector. miR-888 overexpression was capable of decreasing luciferase activity for all four binding sites, with the second and third binding sites producing the most prominent results. Here we describe a novel mechanism by which miR-888 inhibits PR mRNA translation to negatively regulate PR expression in endometrial tumors. To determine the endogenous function of miR-888 in human cells, we quantified miR-888 in a panel of 21 normal human tissues. Interestingly, miR-888 was highly expressed in testes, with minimal or absence of expression in all other tissues investigated. The restricted expression pattern of miR-888 in testes and cancer suggested that miR-888 may qualify as a novel cancer-testis (CT) antigen. CT-antigens are a large class of genes that demonstrate selective expression normally in testes germ cells and abnormally in various types of cancer. Furthermore, CT-antigen genes are predominantly located on the X chromosome and are part of evolutionarily novel multicopy gene families. Indeed, miR-888 is part of a multicopy, primate-specific miRNA gene family located on the X-chromosome. Furthermore, miRNA in situ hybridization localized miR-888 expression to the early stages of spermatogenesis, as is often observed for CT antigens. Together, these data identify miR-888 as the first miRNA CT antigen and expand the CT antigen field to noncoding RNAs.

<p dir="ltr">The vaginal microbiome and female sex hormones are both involved in the development and progression of gynecological pathologies. The individual mechanisms by which the vaginal microbiome leads to disease progression and how female sex hormones are known. However, the mechanisms by which the vaginal microbiome regulates female sex hormones, such as progesterone, are not well understood. This study seeks to understand how the vaginal microbiome regulates progesterone receptor (PGR) expression via secondary analysis of host and vaginal microbiome multi-omics data from the Partners PrEP cohort. This dataset consists of cervicovaginal samples of women enrolled in the Partners PrEP study. Partial Least Squares Regression (PLSR) models were created for each biological data type (microbial composition, metabolomics, metaproteomics) to assess how these factors regulate PGR expression. Significant factors were identified through variable importance of projection (VIP) and correlation analysis. Partial correlation analysis and follow-up PLSR models incorporating clinical and demographic variables were performed to assess the robustness of the vaginal microbiome-PGR associations. The PLSR models indicated lower PGR expression was associated with <i>G. vaginalis,</i> and higher PGR expression was associated with <i>Lactobacillus </i>species. Cytosine, guanine, and tyrosine were among metabolites significantly associated with higher PGR expression and experimentally determined to be produced by <i>Lactobacillus</i> species. Conversely, citrulline and succinate were associated with lower PGR expression and experimentally determined to be produced by <i>G. vaginalis</i>. The models indicated that bacterial metabolic pathways involved in glucose metabolism, such as glucagon signaling and starch and sugar metabolism, may regulate PGR expression. Demographic phenotypes were also considered from the dataset and did not significantly alter the association between the biological explanatory variables and PGR expression. The results indicate that guanine, cytosine, succinate, starch and sucrose metabolism, and glycolysis gluconeogenesis may be regulators of PGR abundance and function. The models suggest vaginal microbiome factors could play a role in gynecological conditions where progesterone signaling is suppressed. Future experimental work is needed to validate the results of these models and support their use as predictive tools to understand the role of the vaginal microbiome.</p>

INTRODUÇÃO: Apesar de a endometriose profunda (EPF) ser a forma da doença de maior repercussão clínica, os estudos sobre a doença costumam ser baseados em lesões de endometriose ovariana (EOV) e peritoneal (EPT). A patogênese da EPF ainda é objeto de amplo debate, pois há poucos estudos feitos exclusivamente com lesões de EPF. O fator de transcrição codificado pelo gene homeobox A10 (HOXA10) regula a conferência de identidade tecidual de útero ao ducto paramesonéfrico indiferenciado durante o período embrionário. O gene mantém um padrão de expressão temporal e espacial bem definido e, durante a fase adulta, continua expresso no miométrio e endométrio. Sugere-se que HOXA10 esteja implicado na patogênese da endometriose, pois é expresso em EOV, EPT, endometriose pulmonar e endometriose retovaginal, um tipo de EPF. Possivelmente, o gene HOXA10 seja necessário para conferir identidade de endometriose a um tecido indiferenciado. O estradiol e a progesterona ativam a transcrição do gene HOXA10 e regulam diretamente sua ação. Esses hormônios estão envolvidos na patogênese da EPF, e suas atividades podem ser inferidas pelo estudo da expressão tecidual de seus receptores. A endometriose de reto-sigmoide (ERS) é um modelo representativo para o estudo da EPF. Neste estudo, avaliamos a expressão proteica do fator de transcrição HOXA10, das isoformas ? (ER-alfa) e beta (ER-beta) dos receptores de estrogênio, e do receptor de progesterona AB (PR-AB) e sua isoforma B (PR-B) na lesão (LES) e no tecido muscular liso perilesional (TMLP) de ERS de pacientes inférteis, durante as fases proliferativa e secretora do ciclo menstrual. MÉTODOS: amostras de LES e TMLP de ERS de 18 pacientes (9 operadas em cada fase do ciclo menstrual) foram agrupadas em blocos de microarranjos de tecidos (tissue microarray). As amostras foram coradas com anticorpos específicos para análise imunoistoquímica de cada uma das proteínas. Foram então avaliadas por microscopia ótica (MO) e pela análise das imagens digitalizadas das lâminas com por um software específico, a análise morfométrica (AM). RESULTADOS: HOXA10 foi expresso no estroma de LES de ERS durante a fase secretora, de acordo com a MO. ER-alfa e ER-betaforam expressos em glândulas e estroma de LES e TMLP de ERS durante ambas as fases do ciclo, de acordo com a MO e a AM. PR-AB e PR-B foram expressos em glândulas e estroma de LES de ERS durante ambas as fases do ciclo, de acordo com a MO. PR-B foi mais expresso durante a fase secretora, independentemente do local de expressão, segundo a AM. A expressão de HOXA10 correlacionou-se diretamente com PR-AB e PR-B na ERS, segundo a AM. Não houve correlação entre ER-alfa e ER-beta com HOXA10, PR-AB ou PR-B em nenhuma fase do ciclo ou local de expressão de ERS. CONCLUSÕES: HOXA10 é expresso em ERS, um local fora do seu eixo espacial de expressão. A presença de HOXA10 pode ser necessária para conferir a identidade \"de novo\" na EPF, incluindo ERS. A progesterona pode ativar o gene HOXA10 e regular esta ação, possivelmente mediada por PR-B. O estradiol exerce sua ação mitógena na ERS através ER-alfa e ER-beta / INTRODUCTION: Although deep endometriosis (DE) is the major clinical form of endometriosis, studies regarding the disease are typically based on ovarian (OE) and peritoneal (PE) lesions. DE pathogenesis is still a matter of great discussion because there are few studies exclusively involving DE lesions. The transcription factor encoded by the homeobox gene A10 (HOXA10) regulates the identity imparted to the undifferentiated paramesonephric duct during embryogenesis. The gene is expressed in the myometrium and endometrium during adult life in a well-defined spatial and temporal mode. It has been suggested that HOXA10 plays a role in endometriosis pathogenesis because it is expressed in OE, PE, pulmonary endometriosis, and rectovaginal endometriosis, which is a clinical form of DE. Thus, HOXA10 may be necessary for \"de novo\" endometrial development from undifferentiated tissues. Both estradiol and progesterone activate HOXA10 transcription and directly regulate its action. These hormones are involved in DE pathogenesis, and therefore their activities could be assessed by studying the tissue expression of their receptors. Rectosigmoid endometriosis (RE) is a representative model for studying DE. In this study, we evaluated the protein expression of HOXA10, the estrogen receptor (ER) isoforms alfa (ER-alfa) and beta (ER-beta), the progesterone receptor AB (PR), and the PR isoform B (PR-B) in lesions (LES) and adjacent smooth muscle (SM) of RE from infertile patients during the proliferative and secretory phases of the menstrual cycle. METHODS: LES and SM samples from RE patients were grouped in tissue microarray blocks. Each of the proteins was analyzed by immunohistochemistry using regular optical microscopy (OM) and a software-assisted analysis of digitalized images as well as morphometric analysis (MA). RESULTS: HOXA10 was expressed in the stroma of the LES during the secretory phase based on OM. ER-alfa and ER-beta were expressed in the glands and stroma of LES and SM during both phases based on OM and MA. PR and PR-B were expressed in the glands and stroma of LES during both phases; however, PR-B had higher expression during the secretory phase, independent of its expression in the LES or SM. HOXA10 expression was directly correlated with PR and PR-B expression in RE. In addition, there was no correlation between the expression of ER-alfa and ER-beta with HOXA10, PR, or PR-B during any phase of the menstrual cycle or site of expression. CONCLUSIONS: HOXA10 is expressed in RE outside of its spatial domain of expression, and may be necessary for \"de novo\" development of DE, including RE. Progesterone might stimulate HOXA10 expression and regulate this action, which is most likely mediated by PR-B. Moreover, estradiol exerts its mitogenic effect in RE though ER-alfa and ER-beta

INTRODUÇÃO: Apesar de a endometriose profunda (EPF) ser a forma da doença de maior repercussão clínica, os estudos sobre a doença costumam ser baseados em lesões de endometriose ovariana (EOV) e peritoneal (EPT). A patogênese da EPF ainda é objeto de amplo debate, pois há poucos estudos feitos exclusivamente com lesões de EPF. O fator de transcrição codificado pelo gene homeobox A10 (HOXA10) regula a conferência de identidade tecidual de útero ao ducto paramesonéfrico indiferenciado durante o período embrionário. O gene mantém um padrão de expressão temporal e espacial bem definido e, durante a fase adulta, continua expresso no miométrio e endométrio. Sugere-se que HOXA10 esteja implicado na patogênese da endometriose, pois é expresso em EOV, EPT, endometriose pulmonar e endometriose retovaginal, um tipo de EPF. Possivelmente, o gene HOXA10 seja necessário para conferir identidade de endometriose a um tecido indiferenciado. O estradiol e a progesterona ativam a transcrição do gene HOXA10 e regulam diretamente sua ação. Esses hormônios estão envolvidos na patogênese da EPF, e suas atividades podem ser inferidas pelo estudo da expressão tecidual de seus receptores. A endometriose de reto-sigmoide (ERS) é um modelo representativo para o estudo da EPF. Neste estudo, avaliamos a expressão proteica do fator de transcrição HOXA10, das isoformas ? (ER-alfa) e beta (ER-beta) dos receptores de estrogênio, e do receptor de progesterona AB (PR-AB) e sua isoforma B (PR-B) na lesão (LES) e no tecido muscular liso perilesional (TMLP) de ERS de pacientes inférteis, durante as fases proliferativa e secretora do ciclo menstrual. MÉTODOS: amostras de LES e TMLP de ERS de 18 pacientes (9 operadas em cada fase do ciclo menstrual) foram agrupadas em blocos de microarranjos de tecidos (tissue microarray). As amostras foram coradas com anticorpos específicos para análise imunoistoquímica de cada uma das proteínas. Foram então avaliadas por microscopia ótica (MO) e pela análise das imagens digitalizadas das lâminas com por um software específico, a análise morfométrica (AM). RESULTADOS: HOXA10 foi expresso no estroma de LES de ERS durante a fase secretora, de acordo com a MO. ER-alfa e ER-betaforam expressos em glândulas e estroma de LES e TMLP de ERS durante ambas as fases do ciclo, de acordo com a MO e a AM. PR-AB e PR-B foram expressos em glândulas e estroma de LES de ERS durante ambas as fases do ciclo, de acordo com a MO. PR-B foi mais expresso durante a fase secretora, independentemente do local de expressão, segundo a AM. A expressão de HOXA10 correlacionou-se diretamente com PR-AB e PR-B na ERS, segundo a AM. Não houve correlação entre ER-alfa e ER-beta com HOXA10, PR-AB ou PR-B em nenhuma fase do ciclo ou local de expressão de ERS. CONCLUSÕES: HOXA10 é expresso em ERS, um local fora do seu eixo espacial de expressão. A presença de HOXA10 pode ser necessária para conferir a identidade \"de novo\" na EPF, incluindo ERS. A progesterona pode ativar o gene HOXA10 e regular esta ação, possivelmente mediada por PR-B. O estradiol exerce sua ação mitógena na ERS através ER-alfa e ER-beta / INTRODUCTION: Although deep endometriosis (DE) is the major clinical form of endometriosis, studies regarding the disease are typically based on ovarian (OE) and peritoneal (PE) lesions. DE pathogenesis is still a matter of great discussion because there are few studies exclusively involving DE lesions. The transcription factor encoded by the homeobox gene A10 (HOXA10) regulates the identity imparted to the undifferentiated paramesonephric duct during embryogenesis. The gene is expressed in the myometrium and endometrium during adult life in a well-defined spatial and temporal mode. It has been suggested that HOXA10 plays a role in endometriosis pathogenesis because it is expressed in OE, PE, pulmonary endometriosis, and rectovaginal endometriosis, which is a clinical form of DE. Thus, HOXA10 may be necessary for \"de novo\" endometrial development from undifferentiated tissues. Both estradiol and progesterone activate HOXA10 transcription and directly regulate its action. These hormones are involved in DE pathogenesis, and therefore their activities could be assessed by studying the tissue expression of their receptors. Rectosigmoid endometriosis (RE) is a representative model for studying DE. In this study, we evaluated the protein expression of HOXA10, the estrogen receptor (ER) isoforms alfa (ER-alfa) and beta (ER-beta), the progesterone receptor AB (PR), and the PR isoform B (PR-B) in lesions (LES) and adjacent smooth muscle (SM) of RE from infertile patients during the proliferative and secretory phases of the menstrual cycle. METHODS: LES and SM samples from RE patients were grouped in tissue microarray blocks. Each of the proteins was analyzed by immunohistochemistry using regular optical microscopy (OM) and a software-assisted analysis of digitalized images as well as morphometric analysis (MA). RESULTS: HOXA10 was expressed in the stroma of the LES during the secretory phase based on OM. ER-alfa and ER-beta were expressed in the glands and stroma of LES and SM during both phases based on OM and MA. PR and PR-B were expressed in the glands and stroma of LES during both phases; however, PR-B had higher expression during the secretory phase, independent of its expression in the LES or SM. HOXA10 expression was directly correlated with PR and PR-B expression in RE. In addition, there was no correlation between the expression of ER-alfa and ER-beta with HOXA10, PR, or PR-B during any phase of the menstrual cycle or site of expression. CONCLUSIONS: HOXA10 is expressed in RE outside of its spatial domain of expression, and may be necessary for \"de novo\" development of DE, including RE. Progesterone might stimulate HOXA10 expression and regulate this action, which is most likely mediated by PR-B. Moreover, estradiol exerts its mitogenic effect in RE though ER-alfa and ER-beta

Clinical immunohistochemistry (IHC) techniques are not yet fully standardized. In this project, a standardization method was developed and tested for proficiency testing (PT) in external quality assurance (EQA) and quality control (QC) in clinical IHC laboratories. The breast cancer markers estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) were used as a model system. Digital image analysis (IA) was used in conjunction with new calibrated and standardized cell line microarrays (CLMA). CLMAs built from nine formalin-fixed paraffin-embedded (FFPE) breast cancer cell lines were used for both QC controls and PT samples, instead of traditionally used FFPE tissues, in the standardization of breast cancer IHC. IA was used for measurement of IHC results, and compared to evaluation by the traditional expert-assessment method. Laboratory Score: Reference Score Ratio (LSRSR) was derived from Histo-Scores (HScores) determined by IA. HScores and LSRSRs were examined statistically and evaluated as histograms and boxplots to summarize and rank participant laboratory EQA results, in comparison to a reference sample or reference laboratories in two consecutive Canada-wide EQA runs. LSRSR-derived reference ranges were highly sensitive in evaluating laboratory EQA performance in PT as well as for monitoring of controls for QC. Laboratory on-slide tissue and cell-line IHC QA controls were assessed using IA and Levey Jennings QC charts. These charts were determined to be an excellent way to observe trending in laboratory IHC staining over time, particularly when cell line controls were used. This approach also reduced the time and labor costs for PT evaluation. Overall, cell line calibration controls were functionally equivalent or better than tissue-based controls in QC and PT mainly because of cell line biological homogeneity and sample availability. This study identified an optimal design for preparation of IHC cell line controls and PT samples for breast cancer markers. Optimal, intermediate staining cell line IHC controls were identified for all three breast cancer markers. Using IA with LSRSR and cell line samples is recommended for standardization of IHC methodology. This approach advances QA for diagnostic IHC and when implemented will improve patient care

Angiogenesis is thought to play a pivotal role in the cycling endometrium. Coordinated by oestrogen and progesterone, endometrial blood vessel development is primarily mediated by vascular endothelial growth factor-A (VEGF-A), which promotes endothelial cell (EC) proliferation and protects ECs from induced apoptosis. Studying changes at transcript level in human endometrial endothelial cells (HEECs) in response to mitogenic and inhibitory stimuli is one way towards understanding the regulation of physiological endometrial angiogenesis. Endometriosis, the presence of endometrial-like tissue outside the uterine cavity, is a common gynaecological disorder in women of reproductive age, often causing pelvic pain and reduced fertility. Chronic inflammation in the peritoneal environment and defective endometrial protein expression are some of the contributors to the complex pathophysiology of endometriosis. The aim of this work was to study the changes in the transcriptome induced by VEGF-A and partial serum deprivation in primary HEECs, and to investigate biochemical factors associated with subfertility and chronic pelvic pain in endometriosis patients. Exposing primary HEECs to VEGF-A, and serum withdrawal was found to regulate transcripts associated with survival, migration, apoptosis and progression through the cell cycle, when assessed using microarray technology and bioinformatic tools. A subset of 88 transcripts was reciprocally regulated under the two experimental conditions; thus probably important in HEEC biology. Higher endometrial epithelial staining scores of oestrogen receptor-α and reduced staining of progesterone receptors were seen in subfertile endometriosis patients. Lower levels of the receptivity biomarker leukaemia inhibitory factor (LIF) and its receptor, as well as signs of dysregulated αB-crystallin expression and increased peritoneal fluid concentrations of interleukin (IL)-1α and IL-6 were associated with reduced pregnancy rates. Endometriosis patients with chronic pelvic pain had higher levels of vasoactive intestinal peptide (VIP) in eutopic endometria and in endometriotic lesions compared with patients without chronic pain. The presence of chronic pelvic pain was also associated with increased concentrations of VIP and IL-6 in peritoneal fluid. The present results may constitute a basis for further investigation of regulatory pathways in endometrial angiogenesis as well as for studies of endometrial receptivity and pain in women with endometriosis.

Le récepteur de la progestérone (PR) est un cible pharmacologique majeure pour la contraception, et pour le traitement de certaines pertubations endocriniennes ainsi que des cancers hormono-dépendants de l’utérus et du sein. Chez la femme, PR est exprimé sous deux isoformes majeures PRA et PRB qui sont des facteurs de transcription fonctionnellement distincts. L’expression de PRA vs PRB est souvent altérée dans certaines situations pathologiques selon des mécanismes encore mal identifiés. Dans cette thèse, nous démontrons que des phosphorylations clés regulées par des MAPK distincts contrôlent la stabilité de PRB et PRA. PRA est sélectivement stabilisée par la p38 MAPK tandis que PRB est préférentiellement stabilisé par la p42/44 MAPK. Ces mécanismes différentiels régulent donc le rapport d’expression PRA/PRB de façon ligand-dépendente et mettent les fonctions progestatives sous le contrôle de l’activité des facteurs de croissance et des cytokines pro-inflammatoires. Or, dans les cellules cancéreuses, la suractivité de certains stimuli extracellulaires provenant de telles signalisations et activant préférentiellement p42/44 et/ou p38 MAPK, pourrait être à l’origine des pertubations du rapport PRA/PRB observées dans les tumeurs du sein. Afin d’explorer la contribution différentielle des isoformes du PR dans la signalisation cellulaire, nous avons élaboré un modèle cellulaire original permettant de contrôler l’expression de PRA et/ou PRB de façon conditionnelle, réversible et dose-dépendante. Par une approche transcriptomique, nous avons identifiés les gènes régulés de façon différentielle par PRA et/ou PRB en absence ou présence de l’hormone. Nous montrons que plusieurs aspects de la signalisation de PR comme la sélectivité de la régulation transcriptionnelle, la dialogue-croisée avec des facteurs de croissance ainsi que l’efficacité antiproliférative des antiprogestatifs dépendent de l’expression differentielle des isoformes du PR. Une nouvelle approche thérapeutique ou préventive possible dans les cancers hormono-dépendants pourrait consister à administrer des antagonistes du PR. Cependant, la plupart des antiprogestatifs disponibles comme la mifépristone présentent des effets agonistes partiels et ne sont pas sélectifs du PR, produisant ainsi des effets indésirables majeurs. Dans un projet collaboratif, et sur la base d’études cristallographiques de PR, nous avons synthétisé et caractérisé plusieurs dizaines de molécules antagonistes du PR, nommés APRn. L’étude des relations structure-fonctions de ces APRn a permis d’identifier les substitutions introduites dans la structure stéroïdienne qui sont responsables des propriétés agonistes/antagonistes de ces molécules. Plusieurs APRn sélectionnés sont dépourvus d’effets agonistes partiels, sont spécifiques du PR et inhibent son activité transcriptionnelle par un nouveau mécanisme d’action dit « passif », en raison de leur capacité particulière à inhiber le recrutement des corégulateurs transcriptionnels. Ces antagonistes sélectifs de PR offrent des perspectives thérapeutiques intéressantes dans les maladies de la reproduction et des cancers hormono-dépendants de l’utérus et du sein. L’ensemble de nos résultats apportent des informations nouvelles sur les mécanismes impliqués dans la sélectivité fonctionnelle des isoformes du PR en physiopathologie, ainsi que sur la possibilité d’un ciblage pharmacologique spécifique par de nouveaux antagonistes utilisables dans le traitement du cancer du sein. / Progesterone receptor (PR) is an essential pharmacological target for contraception, female reproductive disorders as well as for hormone-dependent breast and uterine cancers. Human PR is expressed as two major isoforms PRA and PRB which behave as distinct transcriptional factors. PRA vs PRB expression is often altered under pathological conditions notably breast cancer through unknown mechanisms. In this thesis we demonstrate that down-regulations of PRB and PRA proteins are negatively controlled by key phosphorylation events involving distinct MAP kinase signaling. PRA is selectively stabilized by p38 MAPK whereas p42/44 MAPK specifically controls PRB stability leading to unbalanced PRA/PRB ratios in a ligand sensitive manner. In cancer cells, elevated extracellular stimuli such as epidermal growth factors or pro-inflammatory cytokines that preferentially activate p42/44 or p38 MAPK respectively may result in opposite variations in PRA/PRB expression ratio. These results may explain altered PRA/PRB ratios often associated with breast tumors. To get a mechanistic understanding of how varied PRA/PRB ratio contributes in cell signaling, we generated an original bi-inducible PR-isoform cell model allowing selective, reversible and dose-dependent expression of PRA and/or PRB, enabling fine-tune adjustment of PRA/PRB ratio in the same cells. Using this cell-based system, we undertook genome-wide transcriptomic studies to investigate transcriptional regulation driven by unliganded and liganded PR isoforms. We report that several aspects of PR signaling such as target gene selection/transcriptional regulation, cross-talk with growth factors and antiproliferative efficacy of antiprogestin are highly dependent upon variation in PRA/PRB ratio. A new potential therapeutic strategy in PR-dependent pathological conditions may rely on the use of PR antagonists. Most of the currently available antiprogestins such as mifepristone present partial agonist activity and are not selective to PR leading to undesirable side effects. Therefore, in a collaborative project we have synthesized and characterized several new PR antagonist compounds named as APRn. Structure-activity relationship studies allowed identification of the key substitutions in steroidal skeleton responsible for agonist/antagonist character of these molecules. Several selected APRn lack partial agonist effect, are PR specific and inhibit PR transcriptional properties through a new passive mechanism of action i.e. impaired recruitment of transcriptional coregulators. Such PR selective antagonists devoid of partial agonist character might provide important therapeutic perspectives for various reproductive tract abnormalities and hormone-dependent uterine and breast cancers. Altogehter, our results provide mechanistic insights into the functional selectivity of PR isoforms and their pharmacological targeting by the use of PR antagonists.