RESUMO
Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.
Assuntos
Endométrio/metabolismo , Redes Reguladoras de Genes/genética , Progesterona/metabolismo , Animais , Sequência de Bases , Sequência Conservada/genética , Implantação do Embrião , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Camundongos , Fosfoproteínas/metabolismo , Gravidez , Progesterona/sangue , Ligação Proteica/genética , Receptores de Progesterona/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.
Assuntos
Receptor alfa de Estrogênio/genética , Ativação Transcricional , Animais , Sequência de Bases , Sequência Consenso , Estradiol/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Elementos de Resposta , Útero/metabolismoRESUMO
Progesterone (P4) signaling through its nuclear transcription factor, the progesterone receptor (PR), is essential for normal uterine function. Although deregulation of PR-mediated signaling is known to underscore uterine dysfunction and a number of endometrial pathologies, the early molecular mechanisms of this deregulation are unclear. To address this issue, we have defined the genome-wide PR cistrome in the murine uterus using chromatin immunoprecipitation (ChIP) followed by massively parallel sequencing (ChIP-seq). In uteri of ovariectomized mice, we identified 6367 PR-binding sites in the absence of P4 ligand; however, this number increased at nearly 3-fold (18,432) after acute P4 exposure. Sequence analysis revealed that approximately 73% of these binding sites contain a progesterone response element or a half-site motif recognized by the PR. Many previously identified P4 target genes known to regulate uterine function were found to contain PR-binding sites, confirming the validity of our methodology. Interestingly, when the ChIP-seq data were coupled with our microarray expression data, we identified a novel regulatory role for uterine P4 in circadian rhythm gene expression, thereby uncovering a hitherto unexpected new circadian biology for P4 in this tissue. Further mining of the ChIP-seq data revealed Sox17 as a direct transcriptional PR target gene in the uterus. As a member of the Sox transcription factor family, Sox17 represents a potentially novel mediator of PR action in the murine uterus. Collectively, our first line of analysis of the uterine PR cistrome provides the first insights into the early molecular mechanisms that underpin normal uterine responsiveness to acute P4 exposure. Future analysis promises to reveal the PR interactome and, in turn, potential therapeutic targets for the diagnosis and/or treatment of endometrial dysfunction.
Assuntos
Proteínas HMGB/metabolismo , Receptores de Progesterona/metabolismo , Fatores de Transcrição SOXF/metabolismo , Útero/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Sequência Consenso , Feminino , Genoma , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Progesterona/fisiologia , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Elementos de Resposta , Análise de Sequência de DNA , Transdução de Sinais , TranscriptomaRESUMO
In mammals, such as mouse and human, timely production of the progesterone receptor (PR) in the proper uterine compartments is critical for preparing the uterus for the initiation and maintenance of pregnancy. Developmentally, the expression of GATA2, a member of the six member zinc-finger family of transcription factors, has been shown to be necessary for multiple non-related tissues, such as the hematopoietic system, adipose maturation and the urogential system. We recently identified Gata2 as a potential progesterone target gene in the mouse uterus; however, the expression of the GATA genes in the mouse uterus during pregnancy has not been demonstrated. In the present study, we examined the expression of GATA2 protein during the phases of pregnancy, including early pregnancy where progesterone (P4) signaling is critical in order to facilitate the window of receptivity for embryo implantation and during the decidualization of the uterine stroma, a process of cellular proliferation and differentiation which is necessary for maintenance of the invading embryo until placentation occurs. Here, we report that GATA2 protein is expressed in the uterine luminal and glandular epithelium pre-implantation, spatio-temporally co-localizing with that of the PR. Additionally, GATA2 continues to be expressed in the decidualized stroma throughout early pregnancy indicating a role in the maintenance of decidual cells. Based on these findings, we conclude that GATA2 is expressed during critical phases of early pregnancy, similar to that of the PR, and that it may play a major role in mediating P4 signaling in the mouse uterus.
Assuntos
Fator de Transcrição GATA2/metabolismo , Gravidez/metabolismo , Útero/metabolismo , Animais , Linhagem Celular , Decídua/metabolismo , Implantação do Embrião , Endométrio/metabolismo , Feminino , Humanos , Camundongos , Promegestona/metabolismo , Pseudogravidez/metabolismo , Receptores de Progesterona/agonistas , Receptores de Progesterona/análiseRESUMO
The ovarian steroid progesterone, acting through the progesterone receptor (PR), coordinates endometrial epithelial-stromal cell communication, which is critical for its development and function. PR expression in these cellular compartments is under tight temporal and endocrine control. Although ex vivo studies demonstrated the importance of stromal PR expression, they failed to show a role for epithelial PR in uterine function. Here, the in vivo role of PR in the uterine epithelium is defined using floxed PR (PR(f/f)) mice crossed to Wnt7a-Cre mice. Progesterone was unable to stimulate the expression of its epithelial target genes, including Ihh, in the Wnt7a-Cre(+)PR(f/-) mice. Analysis was conducted on Ihh to determine whether PR directly regulates epithelial gene transcription. ChIP-on-chip analysis identified PR binding sites in the 5'-flanking region of Ihh. Cotransfection of the proximal Ihh promoter with PR demonstrated that PR directly regulates Ihh transcription. Female Wnt7a-Cre(+)PR(f/-) mice are infertile due to defects in embryo attachment, stromal cell decidualization, and the inability to cease estrogen-induced epithelial cell proliferation. Finally, progesterone was unable to inhibit neonatal endometrial glandular development in Wnt7a-Cre(+)PR(f/-) mice. Thus, epithelial PR is necessary for the regulation of progesterone epithelial target gene expression, as well as uterine function and development.
Assuntos
Células Epiteliais/metabolismo , Receptores de Progesterona/fisiologia , Útero/fisiologia , Proteínas Wnt/fisiologia , Animais , Sítios de Ligação/genética , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina/métodos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Estrogênios/farmacologia , Feminino , Fertilidade/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Progesterona/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Útero/efeitos dos fármacos , Útero/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PR(cre) mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PR(cre/+)Wnt4(f/f) (Wnt4(d/d)) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63-positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4(d/d) mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.
Assuntos
Decídua/fisiologia , Útero/fisiologia , Proteínas Wnt/fisiologia , Animais , Apoptose/efeitos dos fármacos , Implantação do Embrião/fisiologia , Feminino , Camundongos , Gravidez , Progesterona/fisiologia , Transdução de Sinais/fisiologia , Útero/crescimento & desenvolvimento , Proteína Wnt4RESUMO
Previous work has identified Indian hedgehog (Ihh) as a major mediator of progesterone signaling during embryo implantation. Ihh acts through its downstream effector smoothened (Smo) to activate the GLI family of transcription factors. In order to gain a better understanding of Ihh action during embryo implantation, we expressed a Cre-recombinase-dependent constitutively activated SMO in the murine uterus using the Pgr(tm2(cre)Lyd) (PR(cre)) mouse model [Pgr(tm2(cre)Lyd+)Gt(ROSA)26Sor(tm1(Smo/EYFP)Amc)(+) (PR(cre/+)SmoM2(+))]. Female PR(cre/+)SmoM2(+) mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but their ova failed to be fertilized in vivo and their uterus failed to undergo the artificially induced decidual response. Examination of the PR(cre/+)SmoM2(+) uteri revealed numerous features such as uterine hypertrophy, the presence of a stratified luminal epithelial cell layer, a reduced number of uterine glands, and an endometrial stroma that had lost its normal morphologic characteristics. Microarray analysis of 3-mo-old PR(cre/+)SmoM2(+) uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of PR(cre/+)SmoM2(+) increased extracellular matrix production. Thus, constitutive activation of Smo in the mouse uterus alters postnatal uterine differentiation which interferes with early pregnancy. These results provide new insight into the role of Hedgehog signaling during embryo implantation.
Assuntos
Implantação do Embrião/fisiologia , Infertilidade Feminina/metabolismo , Progesterona/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Útero/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Decídua/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica/fisiologia , Proteínas Hedgehog/metabolismo , Metaboloma , Camundongos , Camundongos Mutantes , Gravidez , Análise Serial de Proteínas , Receptores de Progesterona/metabolismo , Transdução de Sinais/fisiologia , Receptor Smoothened , Útero/citologia , Útero/metabolismoRESUMO
Healthy uterine function depends on the balanced interaction of the ovarian steroids estrogen and progesterone (P4) signaling through their respective receptors. The expression of each receptor is regulated by the other through crucial cross talk between the epithelial and stromal compartments. Ablation of the progesterone receptor (PR) results in complete infertility in mice, and evidence increasingly demonstrates that the PR is a major mediator of epithelial-stromal cross talk and events leading to the disruption of this communication can lead to P4 resistance in the uterus. This resistance, through impaired P4 signaling, can be at the level of the PR itself, coregulators, and downstream effectors. The mechanisms underlying P4 resistance is of critical importance in women's health because this defect is seen in a wide variety of diseases including infertility, endometriosis, endometrial carcinoma, polycystic ovarian syndrome, and leiomyomas. By using mouse models of PR signaling, many of these mechanisms are beginning to be elucidated and aid in the development of effective therapies for treatment of uterine diseases.