RESUMO
HER2-positive (HER2+) breast cancers (BrCs) contain approximately equal numbers of ERα+HER2+ and ERα-HER2+ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα+HER2+ BrCs could lose ERα to become ERα-HER2+ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERαCreRFP-T mice that produce an RFP-marked ERα+ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα+RFP+Erbb2+ and ERα-RFP-Erbb2+ MGECs. Early hyperplasia developed mostly from ERα+RFP+Erbb2+ cells and ERα-RFP-Erbb2+ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα+RFP+Erbb2+ cells, 15% fast-proliferating ERα-RFP+Erbb2+ cells derived from ERα+RFP+Erbb2+ cells, and 20% fast-proliferating ERα-RFP-Erbb2+ cells. The advanced tumors had mostly ERα-RFP+Erbb2+ and ERα-RFP-Erbb2+ cells and only a very small population of ERα+RFP+Erbb2+ cells. In ERα-RFP+Erbb2+ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα-RFP+Erbb2+ cells, a few ERα-RFP-Erbb2+ cells, and no ERα+RFP+Erbb2+ cells. The high metastatic capacity of ERα-RFP+Erbb2+ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα+RFP+Erbb2+ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα-RFP+Erbb2+ cells. The ERα-Erbb2+ BrCs with an ERα+ origin are more aggressive than those ERα-Erbb2+ BrCs with an ERα- origin, and thus, they should be distinguished and treated differently in the future.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/secundário , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Proliferação de Células , Transformação Celular Neoplásica , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas , Receptor ErbB-2/metabolismo , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Adenocarcinoma/patologia , Neoplasia de Células Basais/patologia , Neoplasias da Próstata/patologia , Transativadores/metabolismo , Adenocarcinoma/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Queratina-14/metabolismo , Masculino , Camundongos , Camundongos SCID , Camundongos Transgênicos , Neoplasia de Células Basais/metabolismo , Neoplasias da Próstata/metabolismo , Ratos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transativadores/genética , Transplante HeterólogoRESUMO
Puberty is characterized by dynamic tissue remodeling in the mammary gland involving ductal elongation, resolution into the mature epithelial bilayer, and lumen formation. To decipher the cellular mechanisms underlying these processes, we studied the fate of putative stem cells, termed cap cells, present in terminal end buds of pubertal mice. Employing a p63CreERT2-based lineage-tracing strategy, we identified a unipotent fate for proliferative cap cells that only generated cells with basal features. Furthermore, we observed that dislocated "cap-in-body" cells underwent apoptosis, which aided lumen formation during ductal development. Basal lineage-specific profiling and genetic loss-of-function experiments revealed a critical role for FOXO transcription factors in mediating these proliferative versus apoptotic fates. Importantly, these studies revealed a mode of WNT signaling-mediated FOXO1 inhibition, potentially mediated through AKT. Together, these data suggest that the WNT pathway confers proliferative and survival advantages on cap cells via regulation of FOXO1 localization.
Assuntos
Apoptose/fisiologia , Mama/metabolismo , Proteína Forkhead Box O1/metabolismo , Homeostase/fisiologia , Células-Tronco/citologia , Via de Sinalização Wnt/fisiologia , Animais , Mama/citologia , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos TransgênicosRESUMO
In several organ systems, the transitional zone between different types of epithelium is a hotspot for pre-neoplastic metaplasia and malignancy, but the cells of origin for these metaplastic epithelia and subsequent malignancies remain unknown. In the case of Barrett's oesophagus, intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells. On the basis of a number of experimental models, several alternative cell types have been proposed as the source of this metaplasia but in all cases the evidence is inconclusive: no model completely mimics Barrett's oesophagus in terms of the presence of intestinal goblet cells. Here we describe a transitional columnar epithelium with distinct basal progenitor cells (p63+KRT5+KRT7+) at the squamous-columnar junction of the upper gastrointestinal tract in a mouse model. We use multiple models and lineage tracing strategies to show that this squamous-columnar junction basal cell population serves as a source of progenitors for the transitional epithelium. On ectopic expression of CDX2, these transitional basal progenitors differentiate into intestinal-like epithelium (including goblet cells) and thereby reproduce Barrett's metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues (including the anorectal junction) as well as in the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (believed to be a precursor of Barrett's oesophagus) are both characterized by the expansion of the transitional basal progenitor cells. Our findings reveal a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63+KRT5+KRT7+ basal cells in this zone are the cells of origin for multi-layered epithelium and Barrett's oesophagus.
Assuntos
Esôfago de Barrett/patologia , Linhagem da Célula , Células Epiteliais/patologia , Epitélio/patologia , Junção Esofagogástrica/patologia , Células-Tronco/patologia , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Rastreamento de Células , Esofagite/metabolismo , Esofagite/patologia , Junção Esofagogástrica/metabolismo , Refluxo Gastroesofágico , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Humanos , Queratina-5/metabolismo , Queratina-7/metabolismo , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Fosfoproteínas/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismoRESUMO
Twist1 is an epithelial-mesenchymal transition (EMT)-inducing transcription factor (TF) that promotes cell migration and invasion. To determine the intrinsic role of Twist1 in EMT and breast cancer initiation, growth, and metastasis, we developed mouse models with an oncogene-induced mammary tumor containing wild-type (WT) Twist1 or tumor cell-specific Twist1 knockout (Twist1TKO). Twist1 knockout showed no effects on tumor initiation and growth. In both models with early-stage tumor cells, Twist1, and mesenchymal markers were not expressed, and lung metastasis was absent. Twist1 expression was detected in â¼6% of the advanced WT tumor cells. Most of these Twist1+ cells coexpressed several other EMT-inducing TFs (Snail, Slug, Zeb2), lost ERα and luminal marker K8, acquired basal cell markers (K5, p63), and exhibited a partial EMT plasticity (E-cadherin+/vimentin+). In advanced Twist1TKO tumor cells, Twist1 knockout largely diminished the expression of the aforementioned EMT-inducing TFs and basal and mesenchymal markers, but maintained the expression of the luminal markers. Circulating tumor cells (CTCs) were commonly detected in mice with advanced WT tumors, but not in mice with advanced Twist1TKO tumors. Nearly all WT CTCs coexpressed Twist1 with other EMT-inducing TFs and both epithelial and mesenchymal markers. Mice with advanced WT tumors developed extensive lung metastasis consisting of luminal tumor cells with silenced Twist1 and mesenchymal marker expression. Mice with advanced Twist1TKO tumors developed very little lung metastasis. Therefore, Twist1 is required for the expression of other EMT-inducing TFs in a small subset of tumor cells. Together, they induce partial EMT, basal-like tumor progression, intravasation, and metastasis.
Assuntos
Plasticidade Celular , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/ß-catenin activity in Sox2pos LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.
Assuntos
Linhagem da Célula , Proliferação de Células , Transdiferenciação Celular , Células Epiteliais/metabolismo , Hipóxia/metabolismo , Influenza Humana/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Oxigênio/metabolismo , Alvéolos Pulmonares/metabolismo , Regeneração , Animais , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Perfilação da Expressão Gênica , Genótipo , Humanos , Hipóxia/genética , Hipóxia/patologia , Hipóxia/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/genética , Influenza Humana/patologia , Influenza Humana/virologia , Queratina-5/genética , Queratina-5/metabolismo , Masculino , Camundongos Transgênicos , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/virologia , Receptores Notch/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Análise de Célula Única , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização WntRESUMO
Neuroendocrine tumor cells (NETCs) are commonly observed in prostate cancer. Their presence is associated with castration resistance, metastasis and poor prognosis. Cellular and molecular mechanisms for NETC initiation and growth are unknown. TRAMP mice develop heterogeneous adenocarcinomas induced by expression of the SV40-T/t oncogene in prostate epithelial cells. Here, we demonstrate prostate tumors in TRAMP mice with a mixed genetic background are characterized mostly by atypical hyperplasia (AH) containing steroid receptor coactiator-3-positive, androgen receptor-positive and synaptophysin-negative (SRC-3+/AR+/Syp-) cells. Few SRC-3+/AR-/Syp+ NETCs are present in their prostates. We generated TRAMP mice in which SRC-3 was specifically ablated in AR+/Syp- prostatic epithelial cells (termed PE3KOT mice). In these animals, we observed a substantial reduction in SRC-3-/AR+/Syp- AH tumor growth. There was a corresponding increase in SRC-3-/AR+/Syp- phyllodes lesions, suggesting SRC-3 knockout can convert aggressive AH tumors with mostly epithelial tumor cells into less aggressive phyllodes lesions with mostly stromal tissue. Surprisingly, PE3KOT mice developed many more SRC-3+/AR-/Syp+ NETCs versus control TRAMP mice, indicating SRC-3 expression was retained in NETCs. In contrast, TRAMP mice with global SRC-3 knockout did not develop any NETC, indicating SRC-3 is required for developing NETC. Analysis of cell-differentiating markers revealed that these NETCs might not be derived from the mature AR-/Syp+ neuroendocrine cells or the AR+/Syp- luminal epithelial tumor cells. Instead, these NETCs might originate from the SV40-T/t-transformed intermediate/progenitor epithelial cells. In summary, SRC-3 is required for both AR+/Syp- AH tumor growth and AR-/Syp+ NETC development, suggesting SRC-3 is a target for inhibiting aggressive prostate cancer containing NETCs.
Assuntos
Tumores Neuroendócrinos/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Técnicas de Inativação de Genes , Técnicas Histológicas , Imuno-Histoquímica , Masculino , Camundongos , Coativador 3 de Receptor Nuclear/genética , Sinaptofisina/metabolismoRESUMO
The prostate epithelium is composed of basal (BC), luminal (LEC), and neuroendocrine (NEC) cells. It is unclear how many subtypes of BCs in the prostate and which subtype of BCs contains the main stem cell niche in the adult prostate. Here we report seven BC subpopulations according to their p63, cytokeratin 14 (K14) and K5 expression patterns, including p63-positive/K14-negative/K5-negative (p63+/K14-/K5-), p63-/K14+/K5-, p63-/K14-/K5+, p63+/K14+/K5-, p63+/K14-/K5+, p63-/K14+/K5+, and p63+/K14+/K5+ BCs. We generated a p63-CreERT2 knock-in mouse line that expresses tamoxifen-inducible Cre activity in the p63-expressing cells, including the prostate BCs. We then crossbred this line with ROSA26R mice, and generated p63-CreERT2×ROSA26R bi-genic mice harboring the Cre-activated ß-galactosidase reporter gene. We treated these bi-genic mice with tamoxifen to mark the p63+ BCs at different ages or under different hormonal conditions, and then traced the lineage differentiation of these genetically labeled BCs. We discovered that these p63+ BCs contain self-renewable stem cells in culture and efficiently differentiated into LECs, NECs and BCs in the postnatal, adult and re-generating mouse prostates. Therefore, BC population contains heterogeneous BCs that express different combinations of the p63, K14 and K5 differentiation markers. Because K14+ and K5+ BCs were previously shown to be extremely inefficient to produce LECs in adulthood, we propose that the p63+/K5-/K14- subpopulation of BCs contains most stem-like cells, especially in adult animals.
Assuntos
Células Epiteliais/classificação , Regulação Neoplásica da Expressão Gênica/fisiologia , Fosfoproteínas/metabolismo , Neoplasias da Próstata/patologia , Transativadores/metabolismo , Androgênios/farmacologia , Animais , Diferenciação Celular , Células Epiteliais/citologia , Epitélio/fisiologia , Antagonistas de Estrogênios/farmacologia , Homeostase , Masculino , Camundongos , Células-Tronco Multipotentes/classificação , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/fisiologia , Fosfoproteínas/genética , Tamoxifeno/farmacologia , Transativadores/genéticaRESUMO
CONTEXT: Endometriosis is one of the most common gynecological diseases in women with a prevalence rate of approximately 10%. Chronic pelvic inflammation has been observed in patients with endometriosis and is associated with disease severity. However, how pelvic inflammation promotes endometriosis progression remains unknown. OBJECTIVE: The objective of the study was to investigate the regulatory network of proinflammatory cytokines in endometriosis progression. DESIGN, SETTINGS, AND PATIENTS: Immunostaining of human endometrial (n = 21) and endometriotic (n = 36) sections, quantitative RT-PCR, Western blotting, chromatin immunoprecipitation, and luciferase reporter assays in primary culture human endometrial stromal cells were performed. Autologous transplantation of uterine endometrium from control chicken ovalbumin upstream promoter-transcription factor II [(COUP-TFII) flox/flox] and uterus-specific COUP-TFII knockout mice was performed. RESULTS: Expression of COUP-TFII was significantly reduced in endometriotic stroma. Reduction of COUP-TFII in endometriotic stromal cells was mediated by proinflammatory cytokines including IL-1ß, TNF-α, and TGF-ß1 via a common effector, microRNA-302a. Treatment with these proinflammatory cytokines increased the expression of microRNA-302a, which targets the 3'untranslated region of COUP-TFII to cause its down-regulation. Intriguingly, down-regulation of COUP-TFII in endometrial stromal cells resulted in de-repression of cyclooxygenase-2 (COX-2). Further investigation demonstrated that COUP-TFII directly binds to COX-2 promoter to inhibit its transcription. Forced expression of COUP-TFII inhibited IL-1ß-induced COX-2 up-regulation, whereas the knockdown of COUP-TFII augmented this effect. CONCLUSION: Because overexpression of COX-2 has been demonstrated to be a master regulator in endometriosis progression, our data demonstrate the critical function of proinflammatory cytokines and the COUP-TFII regulatory gene network in the progression of endometriosis.
Assuntos
Fator II de Transcrição COUP/genética , Citocinas/fisiologia , Endometriose/genética , Mediadores da Inflamação/fisiologia , Doenças Uterinas/genética , Animais , Fator II de Transcrição COUP/metabolismo , Células Cultivadas , Citocinas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Endometriose/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Mediadores da Inflamação/farmacologia , Camundongos , Camundongos Knockout , Células Estromais/metabolismo , Células Estromais/patologia , Doenças Uterinas/metabolismoRESUMO
Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal-epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6(f/f)) mice were crossed to Wnt7a-Cre mice (Wnt7a(cre+)Mig-6(f/f)). Wnt7a(cre+)Mig-6(f/f) mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7a(cre+)Mig-6(f/f) mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7a(cre+)Mig-6(f/f) mice was prevented by progesterone treatment, whereas this treatment had no effect in PR(cre/+)Mig-6(f/f) mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/fisiologia , Proteína 3 com Repetições IAP de Baculovírus , Processos de Crescimento Celular/fisiologia , Hiperplasia Endometrial/genética , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/prevenção & controle , Células Epiteliais/patologia , Epitélio/patologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Progesterona/genética , Progesterona/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases , Útero/metabolismo , Útero/patologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Nuclear receptors and coregulators orchestrate diverse aspects of biological functions and inappropriate expression of these factors often associates with human diseases. The present study describes a conditional overexpression system consisting of a minigene located at the Rosa26 locus in the genome of mouse embryonic stem (ES) cells. Before activation, the minigene is silent due to a floxed STOP cassette inserted between the promoter and the transgene. Upon cre-mediated excision of the STOP cassette, the minigene constitutively expresses the tagged transgene driven by the ubiquitous CAGGS promoter. Thus, this system can be used to express target gene in any tissue in a spatial and/or temporal manner if respective cre mouse lines are available. Serving as proof of principle, the CAG-S-hCOUP-TFI allele was generated in ES cells and subsequently in mice. This allele was capable of conditionally overexpressing human chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in all tissues tested upon activation by cre drivers. This allele was further subjected to address functionality of expressed COUP-TFI and the functional similarity between COUP-TFI and COUP-TFII. Expression of COUP-TFI in COUP-TFII-ablated uterus suppressed aberrant estrogen receptor-alpha activities and rescued implantation and decidualization defects of COUP-TFII mutants, suggesting that COUP-TFI and COUP-TFII are able to functionally compensate for each other in the uterus. A toolbox currently under construction will contain ES cell lines for overexpressing all 48 nuclear receptors and selected 10 coregulators. Upon completion, it will be a very valuable resource for the scientific community. Several ES cells are currently available for distribution.
Assuntos
Células-Tronco Embrionárias/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Alelos , Animais , Fator I de Transcrição COUP/metabolismo , Fator II de Transcrição COUP/deficiência , Fator II de Transcrição COUP/metabolismo , Linhagem Celular , Feminino , Humanos , Camundongos , Útero/metabolismo , Útero/patologiaRESUMO
Synchrony between embryo competency and uterine receptivity is essential for successful implantation. Mice with ablation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) in the uterus (PR(Cre/+);COUP-TFII(flox/flox)) exhibit implantation defects and increased estrogen receptor (ER)alpha activity in the luminal epithelium, suggesting high ERalpha activity may disrupt the window of uterine receptivity. To determine whether increased ERalpha activity in the PR(Cre/+);COUP-TFII(flox/flox) uterus is the cause of defective implantation, we assessed whether inhibition of ERalpha activity could rescue the PR(Cre/+);COUP-TFII(flox/flox) uterine implantation defect. ICI 182,780 (ICI), a pure ERalpha antagonist, was administered to PR(Cre/+);COUP-TFII(flox/flox) mutant and COUP-TFII(flox/flox) control mice during the receptive period, and the number of implantation sites was examined. COUP-TFII(flox/flox) control mice treated with oil or ICI showed the normal number of implantation sites. As expected, no implantation sites were observed in PR(Cre/+);COUP-TFII(flox/flox) mutant mice treated with oil, consistent with previous observations. In contrast, implantation sites were greatly increased in ICI-treated PR(Cre/+);COUP-TFII(flox/flox) mutant mice, albeit at a reduced number in comparison with the control mice. ICI treatment was also able to restore the expression of Wnt4 and bone morphogenetic protein 2, important for endometrial decidualization in the PR(Cre/+);COUP-TFII(flox/flox) mutant mice. To confirm that the rescue of embryo attachment and decidualization is a consequence of a reduced ERalpha activity upon ICI treatment, we showed a reduction of the expression of ERalpha target genes in PR(Cre/+);COUP-TFII(flox/flox) mutant mice. Because COUP-TFII was also shown in our laboratory to be important for placentation during pregnancy, we asked whether ICI treatment could also rescue the placentation defect to allow full-term pregnancy in these mice. We found that whereas mice were born in COUP-TFII(flox/flox) control mice given ICI, no pups were born in the PR(Cre/+);COUP-TFII(flox/flox) mutant mice, suggesting that the increased ERalpha activity is not the reason for placentation defects. These results demonstrate that during the periimplantation period, COUP-TFII regulates embryo attachment and decidualization through controlling ERalpha activity. However, COUP-TFII expression is still required in the postimplantation period to facilitate placentation.
Assuntos
Fator II de Transcrição COUP/fisiologia , Implantação do Embrião/fisiologia , Receptor alfa de Estrogênio/metabolismo , Animais , Fator II de Transcrição COUP/genética , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/genética , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Fulvestranto , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) effects. E2 acts to stimulate the proliferation of uterine epithelial cells, while P4 action inhibits E2-mediated proliferation of the epithelium. P4 through its cognate receptor, the P4 receptor (Pgr), has important roles in the establishment and maintenance of pregnancy. In previous studies, we have identified ERBB receptor feedback inhibitor 1 (Errfi1) as a downstream target of Pgr action in the uterus. Herein, we show that Errfi1 mRNA expression was significantly increased in the uterus after Day 2.5 of gestation. Its expression is also induced in the uterus by acute E2 treatment, and this induction is synergistically induced by chronic E2 and P4 treatment. Although it is known that conditional ablation of Errfi1 in the Pgr-positive cells (Errfi1(d/d)) results in infertility, the function of Errfi1 in reproductive biology has remained elusive. Using Errfi1(d/d) mice, we have identified Errfi1 as an important mediator of uterine implantation. Epithelial ESR1 and target genes were significantly increased in the uteri of Errfi1(d/d) mice. Our results identify a new signaling paradigm of steroid hormone regulation in female reproductive biology that adds insight into the underlying dysregulation of hormonal signaling in human reproductive disorders such as endometriosis and endometrial cancer.
Assuntos
Implantação do Embrião/genética , Receptor alfa de Estrogênio/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Animais , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/fisiologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Hormônios Esteroides Gonadais/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Útero/efeitos dos fármacos , Útero/metabolismo , Útero/fisiologiaRESUMO
ASC-2, a multifunctional coactivator, forms a steady-state complex, named ASCOM (for ASC-2 COMplex), that contains the histone H3-lysine-4 (H3K4)-methyltransferase MLL3 or its paralogue MLL4. Somewhat surprisingly, given prior indications of redundancy between MLL3 and MLL4, targeted inactivation of the MLL3 H3K4-methylation activity in mice is found to result in ureter epithelial tumors. Interestingly, this phenotype is exacerbated in a p53(+/-) background and the tumorigenic cells are heavily immunostained for gammaH2AX, indicating a contribution of MLL3 to the DNA damage response pathway through p53. Consistent with the in vivo observations, and the demonstration of a direct interaction between p53 and ASCOM, cell-based assays have revealed that ASCOM, through ASC-2 and MLL3/4, acts as a p53 coactivator and is required for H3K4-trimethyation and expression of endogenous p53-target genes in response to the DNA damaging agent doxorubicin. In support of redundant functions for MLL3 and MLL4 for some events, siRNA-mediated down-regulation of both MLL3 and MLL4 is required to suppress doxorubicin-inducible expression of several p53-target genes. Importantly, this study identifies a specific H3K4 methytransferase complex, ASCOM, as a physiologically relevant coactivator for p53 and implicates ASCOM in the p53 tumor suppression pathway in vivo.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Transformação Celular Neoplásica , Dano ao DNA/genética , Reparo do DNA/genética , Ativação Enzimática , Histona-Lisina N-Metiltransferase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Coativadores de Receptor Nuclear , Proteína Supressora de Tumor p53/genética , Neoplasias Ureterais/enzimologia , Neoplasias Ureterais/genética , Neoplasias Ureterais/patologiaRESUMO
Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone-Indian hedgehog-Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.
Assuntos
Fator II de Transcrição COUP/fisiologia , Implantação do Embrião/fisiologia , Progesterona/fisiologia , Receptores de Estrogênio/antagonistas & inibidores , Útero/metabolismo , Animais , Fator II de Transcrição COUP/deficiência , Fator II de Transcrição COUP/genética , Comunicação Celular/genética , Epitélio/metabolismo , Epitélio/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Receptor Cross-Talk/fisiologia , Receptores de Estrogênio/metabolismo , Células Estromais/metabolismo , Células Estromais/fisiologiaRESUMO
Activating signal cointegrator-2 (ASC-2), a coactivator of multiple transcription factors that include retinoic acid receptor (RAR), associates with histone H3-K4 methyltranferases (H3K4MTs) MLL3 and MLL4 in mixed-lineage leukemia. Here, we show that mice expressing a SET domain mutant of MLL3 share phenotypes with isogenic ASC2+/- mice and that expression and H3-K4 trimethylation of RAR target gene RAR-beta2 are impaired in ASC-2-null mouse embryo fibroblasts (MEFs) or in MEFs expressing siRNAs against both MLL3 and MLL4. We also show that MLL3 and MLL4 are found in distinct ASC-2-containing complexes rather than in a common ASC-2 complex, and they are recruited to RAR-beta2 by ASC-2. In contrast, RAR-beta2 expression is intact in MEFs devoid of menin, a component of MLL1 and MLL2 H3K4MT complexes. These results suggest that ASC-2 confers target gene specificity to MLL3 and MLL4 H3K4MT complexes and that recruitment of H3K4MTs to their target genes generally involves interactions between integral components of H3K4MT complexes and transcription factors.
Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Supressores de Tumor , Histona-Lisina N-Metiltransferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Substâncias Macromoleculares , Metilação , Camundongos , Coativadores de Receptor Nuclear , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/fisiologia , Selectina E/metabolismo , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Fatores de Transcrição/fisiologia , Transporte Ativo do Núcleo Celular , Western Blotting , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Genes Dominantes , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Immunoblotting , Imunoprecipitação , Leupeptinas/farmacologia , Luciferases/metabolismo , Microscopia Confocal , Modelos Biológicos , Inibidor de NF-kappaB alfa , Peptídeos/química , Plasmídeos/metabolismo , Prolina/análogos & derivados , Prolina/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Acetato de Tetradecanoilforbol/farmacologia , Tiocarbamatos/farmacologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Insulin-induced gene-1 (Insig-1) and its homolog Insig-2 encode closely related proteins of the endoplasmic reticulum that block proteolytic activation of sterol regulatory element binding proteins, membrane-bound transcription factors that activate synthesis of cholesterol and fatty acids in animal cells. These proteins also restrict lipogenesis in mature adipocytes and block differentiation of preadipocytes. Herein, we identified a novel 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] response element in the promoter region of Insig-2 gene, which specifically binds to the heterodimer of retinoid X receptor and vitamin D receptor (VDR) and directs VDR-mediated transcriptional activation in a 1,25-(OH)2D3-dependent manner. Interestingly, 1,25-(OH)2D3 is known to directly suppress the expression of peroxisome proliferator-activated receptor gamma2 protein and inhibits adipocyte differentiation of 3T3-L1 preadipocytes and murine bone marrow stromal cells. Consistent with an idea that the antiadipogenic action of 1,25-(OH)2D3 may also involve up-regulation of Insig-2, we found that 1,25-(OH)2D3 transiently but strongly induces Insig-2 expression in 3T3-L1 cells. This novel regulatory circuit may also play important roles in other lipogenic cell types that express VDR, and collectively our results suggest an intriguing, new linkage between 1,25-(OH)2D3 and lipogenesis.
Assuntos
Adipócitos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Elementos de Resposta , Vitamina D/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Sequência de Bases , Células da Medula Óssea/citologia , Calcitriol/metabolismo , Diferenciação Celular , Primers do DNA/química , Dimerização , Relação Dose-Resposta a Droga , Ligantes , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , PPAR gama/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Receptores de Calcitriol/metabolismo , Receptores X de Retinoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , TransfecçãoRESUMO
The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.