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1.
Endocrinology ; 161(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31875883

RESUMO

Uterine receptivity is critical for establishing and maintaining pregnancy. For the endometrium to become receptive, stromal cells must differentiate into decidual cells capable of secreting factors necessary for embryo survival and placental development. Although there are multiple reports of autophagy induction correlated with endometrial stromal cell (ESC) decidualization, the role of autophagy in decidualization has remained elusive. To determine the role of autophagy in decidualization, we utilized 2 genetic models carrying mutations to the autophagy gene Atg16L1. Although the hypomorphic Atg16L1 mouse was fertile and displayed proper decidualization, conditional knockout in the reproductive tract of female mice reduced fertility by decreasing the implantation rate. In the absence of Atg16L1, ESCs failed to properly decidualize and fewer blastocysts were able to implant. Additionally, small interfering RNA knock down of Atg16L1 was detrimental to the decidualization response of human ESCs. We conclude that Atg16L1 is necessary for decidualization, implantation, and overall fertility in mice. Furthermore, considering its requirement for human endometrial decidualization, these data suggest Atg16L1 may be a potential mediator of implantation success in women.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Decídua/metabolismo , Endométrio/metabolismo , Mutação , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Decídua/citologia , Implantação do Embrião/genética , Endométrio/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Interferência de RNA , Células Estromais/citologia , Células Estromais/metabolismo
2.
J Biol Chem ; 294(25): 9746-9759, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31073032

RESUMO

Insulin-like growth factor 1 (IGF1) is primarily synthesized in and secreted from the liver; however, estrogen (E2), through E2 receptor α (ERα), increases uterine Igf1 mRNA levels. Previous ChIP-seq analyses of the murine uterus have revealed a potential enhancer region distal from the Igf1 transcription start site (TSS) with multiple E2-dependent ERα-binding regions. Here, we show E2-dependent super enhancer-associated characteristics and suggest contact between the distal enhancer and the Igf1 TSS. We hypothesized that this distal super-enhancer region controls E2-responsive induction of uterine Igf1 transcripts. We deleted 430 bp, encompassing one of the ERα-binding sites, thereby disrupting interactions of the enhancer with gene-regulatory factors. As a result, E2-mediated induction of mouse uterine Igf1 mRNA is completely eliminated, whereas hepatic Igf1 expression remains unaffected. This highlights the central role of a distal enhancer in the assembly of the factors necessary for E2-dependent interaction with the Igf1 TSS and induction of uterus-specific Igf1 transcription. Of note, loss of the enhancer did not affect fertility or uterine growth responses. Deletion of uterine Igf1 in a PgrCre;Igf1f/f model decreased female fertility but did not impact the E2-induced uterine growth response. Moreover, E2-dependent activation of uterine IGF1 signaling was not impaired by disrupting the distal enhancer or by deleting the coding transcript. This indicated a role for systemic IGF1, suggested that other growth mediators drive uterine response to E2, and suggested that uterine-derived IGF1 is essential for reproductive success. Our findings elucidate the role of a super enhancer in Igf1 regulation and uterine growth.


Assuntos
Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/fisiologia , Transcrição Genética/efeitos dos fármacos , Útero/metabolismo , Animais , Feminino , Camundongos , Camundongos Knockout , Útero/efeitos dos fármacos
3.
Elife ; 3: e01694, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24737860

RESUMO

Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR. In response to ER stress, LRH-1 induces expression of the kinase Plk3, which phosphorylates and activates the transcription factor ATF2. Plk3-null mice also cannot resolve ER stress, and restoring Plk3 expression in Lrh-1-null cells rescues ER stress resolution. Reduced or heightened ATF2 activity also sensitizes or desensitizes cells to ER stress, respectively. LRH-1 agonist treatment increases ER stress resistance and decreases cell death. We conclude that LRH-1 initiates a novel pathway of ER stress resolution that is independent of the UPR, yet equivalently required. Targeting LRH-1 may be beneficial in human disorders associated with chronic ER stress. DOI: http://dx.doi.org/10.7554/eLife.01694.001.


Assuntos
Estresse do Retículo Endoplasmático , Fígado/fisiopatologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Morte Celular , Células Cultivadas , Hepatócitos/fisiologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/genética
4.
Biol Reprod ; 90(4): 75, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24571987

RESUMO

Recent data from human and mouse studies strongly support an indispensable role for steroid receptor coactivator-2 (SRC-2)-a member of the p160/SRC family of coregulators-in progesterone-dependent endometrial stromal cell decidualization, an essential cellular transformation process that regulates invasion of the developing embryo into the maternal compartment. To identify the key progesterone-induced transcriptional changes that are dependent on SRC-2 and required for endometrial decidualization, we performed comparative genome-wide transcriptional profiling of endometrial tissue RNA from ovariectomized SRC-2(flox/flox) (SRC-2(f/f) [control]) and PR(cre/+)/SRC-2(flox/flox) (SRC-2(d/d) [SRC-2-depleted]) mice, acutely treated with vehicle or progesterone. Although data mining revealed that only a small subset of the total progesterone-dependent transcriptional changes is dependent on SRC-2 (∼13%), key genes previously reported to mediate progesterone-driven endometrial stromal cell decidualization are present within this subset. Along with providing a more detailed molecular portrait of the decidual transcriptional program governed by SRC-2, the degree of functional diversity of these progesterone mediators underscores the pleiotropic regulatory role of SRC-2 in this tissue. To showcase the utility of this powerful informational resource to uncover novel signaling paradigms, we stratified the total SRC-2-dependent subset of progesterone-induced transcriptional changes in terms of novel gene expression and identified transcription factor 23 (Tcf23), a basic-helix-loop-helix transcription factor, as a new progesterone-induced target gene that requires SRC-2 for full induction. Importantly, using primary human endometrial stromal cells in culture, we demonstrate that TCF23 function is essential for progesterone-dependent decidualization, providing crucial translational support for this transcription factor as a new decidual mediator of progesterone action.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Decídua/citologia , Coativador 2 de Receptor Nuclear/genética , Células Estromais/citologia , Animais , Decídua/fisiologia , Feminino , Humanos , Camundongos , Camundongos Mutantes , Coativador 2 de Receptor Nuclear/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Progesterona/metabolismo , RNA Interferente Pequeno/genética , Células Estromais/fisiologia , Transcrição Genética/fisiologia , Transcriptoma/fisiologia , Útero/citologia , Útero/fisiologia
5.
Mol Endocrinol ; 27(12): 2041-54, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24176914

RESUMO

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2) is an orphan nuclear receptor involved in cell-fate specification, organogenesis, angiogenesis, and metabolism. Ablation of COUP-TFII in the mouse uterus causes infertility due to defects in embryo attachment and impaired uterine stromal cell decidualization. Although the function of COUP-TFII in uterine decidualization has been described in mice, its role in the human uterus remains unknown. We observed that, as in mice, COUP-TFII is robustly expressed in the endometrial stroma of healthy women, and its expression is reduced in the ectopic lesions of women with endometriosis. To interrogate the role of COUP-TFII in human endometrial function, we used a small interfering RNA-mediated loss of function approach in primary human endometrial stromal cells. Attenuation of COUP-TFII expression did not completely block decidualization; rather it had a selective effect on gene expression. To better elucidate the role of COUP-TFII in endometrial stroma cell biology, the COUP-TFII transcriptome was defined by pairing microarray comparison with chromatin immunoprecipitation followed by deep sequencing. Gene ontology analysis demonstrates that COUP-TFII regulates a subset of genes in endometrial stroma cell decidualization such as those involved in cell adhesion, angiogenesis, and inflammation. Importantly this analysis shows that COUP-TFII plays a role in controlling the expression of inflammatory cytokines. The determination that COUP-TFII plays a role in inflammation may add insight into the role of COUP-TFII in embryo implantation and in endometrial diseases such as endometriosis.


Assuntos
Fator II de Transcrição COUP/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/patologia , Adolescente , Adulto , Animais , Sítios de Ligação , Coristoma/genética , Coristoma/patologia , Imunoprecipitação da Cromatina , Decídua/metabolismo , Feminino , Genoma Humano/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Células Estromais/metabolismo , Células Estromais/patologia , Adulto Jovem
6.
EMBO Mol Med ; 5(9): 1415-30, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23853117

RESUMO

The features and regulation of uterine angiogenesis and vascular remodelling during pregnancy are poorly defined. Here we show that dynamic and variable decidual angiogenesis (sprouting, intussusception and networking), and active vigorous vascular remodelling such as enlargement and elongation of 'vascular sinus folding' (VSF) and mural cell drop-out occur distinctly in a spatiotemporal manner in the rapidly growing mouse uterus during early pregnancy - just after implantation but before placentation. Decidual angiogenesis is mainly regulated through VEGF-A secreted from the progesterone receptor (PR)-expressing decidual stromal cells which are largely distributed in the anti-mesometrial region (AMR). In comparison, P4 -PR-regulated VEGF-A-VEGFR2 signalling, ligand-independent VEGFR3 signalling and uterine natural killer (uNK) cells positively and coordinately regulate enlargement and elongation of VSF. During the postpartum period, Tie2 signalling could be involved in vascular maturation at the endometrium in a ligand-independent manner, with marked reduction of VEGF-A, VEGFR2 and PR expressions. Overall, we show that two key vascular growth factor receptors - VEGFR2 and Tie2 - strikingly but differentially regulate decidual angiogenesis and vascular remodelling in rapidly growing and regressing uteri in an organotypic manner.


Assuntos
Decídua/efeitos dos fármacos , Decídua/fisiologia , Neovascularização Fisiológica , Progesterona/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Gravidez
7.
Dev Cell ; 24(4): 345-58, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23449471

RESUMO

The mechanisms that govern the maintenance and differentiation of tissue-specific progenitors in development and tissue regeneration are poorly understood. We show that development of Sox2+ progenitors in the lung endoderm is regulated by histone deacetylases 1 and 2 (Hdac1/2). Hdac1/2 deficiency leads to a loss of Sox2 expression and a block in proximal airway development. This is mediated in part by derepression of Bmp4 and the tumor suppressor Rb1, which are direct transcriptional targets of Hdac1/2. In contrast to development, postnatal loss of Hdac1/2 in airway epithelium does not affect the expression of Sox2 or Bmp4. However, postnatal loss of Hdac1/2 leads to increased expression of the cell-cycle regulators Rb1, p21/Cdkn1a, and p16/Ink4a, resulting in a loss of cell-cycle progression and defective regeneration of Sox2+ lung epithelium. Thus, Hdac1/2 have both common and unique targets that differentially regulate tissue-specific progenitor activity during development and regeneration.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilase 1/fisiologia , Histona Desacetilase 2/fisiologia , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Embrião de Mamíferos/citologia , Endoderma/metabolismo , Perfilação da Expressão Gênica , Proteínas Hedgehog/fisiologia , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Regeneração , Proteína do Retinoblastoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Células-Tronco/metabolismo
8.
Am J Respir Cell Mol Biol ; 48(2): 188-97, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23087054

RESUMO

Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 µm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 µg/cm(2)) caused substantial necrosis. At low doses (20 µg/cm(2)), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α-smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma.


Assuntos
Poluentes Atmosféricos/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Bronquíolos/citologia , Bronquíolos/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Camundongos , Estresse Oxidativo , Tamanho da Partícula
9.
Curr Protoc Mouse Biol ; 2: 245-262, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23024927

RESUMO

The generation of transgenic mice by DNA microinjection is a powerful tool to investigate the molecular regulation of gene expression, development, and disease. The power of this technology is that foreign DNA can be introduced into every cell of a developing organism and the phenotypic impact of this genetic modification can be investigated in a system under the constraints of normal development and physiology. The generation of transgenic mice requires the preparation of the transgene DNA construction, collection of one-cell fertilized mouse embryos, injection of the transgene into mouse embryos, and transfer of the surviving embryos. Mice born from such manipulations are then screened for the presence of the transgene. The execution of these procedures requires a highly efficient system otherwise the cost of the generation of these mice can be cost prohibitive. However, the production of these animals can serve as an invaluable research resource.

10.
PLoS One ; 7(8): e40452, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22905092

RESUMO

Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Immunohistochemistry of human pancreatic and islet neoplasia specimens revealed marked PDX-1 overexpression, suggesting PDX-1 as a "drugable" target within these diseases. To do so, a novel RNA interference effector platform, bifunctional shRNA(PDX-1), was developed and studied in mouse and human cell lines as well as in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Systemic delivery of bi-shRNA(humanPDX-1) lipoplexes resulted in marked reduction of tumor volume and improved survival in a human pancreatic cancer xenograft mouse model. bi-shRNA(mousePDX-1) lipoplexes prevented death from hyperinsulinemia and hypoglycemia in an insulinoma mouse model. shRNA(mousePDX-1) lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of islet neoplasia. PDX-1 was overexpressed in pancreatic neuroendocrine tumors and nesidioblastosis. These data demonstrate that PDX-1 RNAi therapy controls hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia, therefore, PDX-1 is a potential therapeutic target for these pancreatic diseases.


Assuntos
Proteínas de Homeodomínio/metabolismo , Insulinoma/terapia , Ilhotas Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Interferência de RNA , Transativadores/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Teste de Tolerância a Glucose , Humanos , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas , Insulina/metabolismo , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Pâncreas/metabolismo
11.
Mol Endocrinol ; 26(7): 1225-34, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22669743

RESUMO

Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin and its analogs on insulin expression/secretion and islet cell proliferation. We provide biochemical and genetic evidence that SSTR5 exerted its physiological actions via down-regulating pancreatic and duodenal homeobox-1 (PDX-1), a ß-cell-specific homeodomain-containing transcription factor. Cotransfection of SSTR5 with PDX-1 resulted in dose-dependent inhibition of PDX-1 expression in human embryonic kidney 293 cells. SSTR5 agonist RPL-1980 inhibited PDX-1 expression and abolished glucagon-like peptide 1-stimulated PDX-1 expression in mouse insulinoma ß-TC-6 cells. SSTR5 knockdown by short hairpin RNA led to increased PDX-1 expression that was accompanied by enhanced insulin secretion stimulated by high glucose in ß-TC6 cells and alternated expressions of cell cycle proteins that favor cell proliferation in mouse insulinoma MIN6 cells. Quantitative RT-PCR analysis showed that cotransfected SSTR5 inhibited PDX-1 mRNA expression, whereas knockdown of SSTR5 increased PDX-1 mRNA expression. In addition, we found that cotransfected wild-type SSTR5 increased PDX-1 ubiquitination in human embryonic kidney 293 cells, whereas SSTR5 P335L, a hypofunctional single nucleotide polymorphism of SSTR5, inhibited PDX-1 ubiquitination. SSTR5 knockout resulted in increased expression of PDX-1, insulin, and proliferating cell nuclear antigen in the islets of sstr(-/-) mice. Immunohistochemistry analysis showed that SSTR5 P335L was associated with elevated expression of PDX-1 in human pancreatic neuroendocrine tumor. Taken together, our studies demonstrated that SSTR5 is a negative regulator for PDX-1 expression and that SSTR5 may mediate the inhibitory effects of somatostatin and its analogs on insulin expression/secretion and cell proliferation via down-regulating PDX-1 at both transcriptional and posttranslational levels.


Assuntos
Proteínas de Homeodomínio/metabolismo , Insulina/biossíntese , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Insulina/metabolismo , Secreção de Insulina , Insulinoma , Camundongos , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Antígeno Nuclear de Célula em Proliferação/biossíntese , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptores de Somatostatina/genética , Transativadores/genética , Ubiquitinação
12.
Transgenic Res ; 21(5): 1117-23, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22350718

RESUMO

Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreER(T2) inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreER(T2), the K8-CreER(T2) mice were bred with a Gt(ROSA 26)( ACTB-tdTomato-EGFP ) fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreER(T2) and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreER(T2) can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.


Assuntos
Cromossomos Artificiais Bacterianos/metabolismo , Células Epiteliais/metabolismo , Técnicas de Transferência de Genes , Integrases/metabolismo , Queratina-8/metabolismo , Animais , Ativação Enzimática , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica , Marcação de Genes/métodos , Genes Reporter , Técnicas de Genotipagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Integrases/genética , Queratina-8/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Tamoxifeno/metabolismo , Tamoxifeno/farmacologia , Transgenes
13.
FASEB J ; 26(3): 1218-27, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22155565

RESUMO

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/metabolismo
14.
Cancer Res ; 71(24): 7694-704, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22025562

RESUMO

Downregulation of the proapoptotic p53 target gene glioma pathogenesis-related protein 1 (GLIPR1) occurs frequently in prostate cancer, but the functional meaning of this event is obscure. Here, we report the discovery of functional relationship between GLIPR1 and c-Myc in prostate cancer where c-Myc is often upregulated. We found that the expression of GLIPR1 and c-Myc were inversely correlated in human prostate cancer. Restoration of GLIPR1 expression in prostate cancer cells downregulated c-myc levels, inhibiting cell-cycle progression. Downregulation was linked to a reduction in ß-catenin/TCF4-mediated transcription of the c-myc gene, which was caused by GLIPR1-mediated redistribution of casein kinase 1α (CK1α) from the Golgi apparatus to the cytoplasm where CK1α could phosphorylate ß-catenin and mediate its destruction. In parallel, GLIPR1 also promoted c-Myc protein ubiquitination and degradation by glycogen synthase kinase-3α- and/or CK1α-mediated c-Myc phosphorylation. Notably, genetic ablation of the mouse homolog of Glipr1 cooperated with c-myc overexpression to induce prostatic intraepithelial neoplasia and prostate cancer. Together, our findings provide evidence for CK1α-mediated destruction of c-Myc and identify c-Myc S252 as a crucial CK1α phosphorylation site for c-Myc degradation. Furthermore, they reveal parallel mechanisms of c-myc downregulation by GLIPR1 that when ablated in the prostate are sufficient to drive c-Myc expression and malignant development.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Apoptose/genética , Western Blotting , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação
15.
J Clin Invest ; 121(5): 1935-45, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21490395

RESUMO

Although mutations in Kras are present in 21% of lung tumors, there is a high level of heterogeneity in phenotype and outcome among patients with lung cancer bearing similar mutations, suggesting that other pathways are important. Wnt/ß-catenin signaling is a known oncogenic pathway that plays a well-defined role in colon and skin cancer; however, its role in lung cancer is unclear. We have shown here that activation of Wnt/ß-catenin in the bronchiolar epithelium of the adult mouse lung does not itself promote tumor development. However, concurrent activation of Wnt/ß-catenin signaling and expression of a constitutively active Kras mutant (KrasG12D) led to a dramatic increase in both overall tumor number and size compared with KrasG12D alone. Activation of Wnt/ß-catenin signaling altered the KrasG12D tumor phenotype, resulting in a phenotypic switch from bronchiolar epithelium to the highly proliferative distal progenitors found in the embryonic lung. This was associated with decreased E-cadherin expression at the cell surface, which may underlie the increased metastasis of tumors with active Wnt/ß-catenin signaling. Together, these data suggest that activation of Wnt/ß-catenin signaling can combine with other oncogenic pathways in lung epithelium to produce a more aggressive tumor phenotype by imposing an embryonic distal progenitor phenotype and by decreasing E-cadherin expression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Brônquios/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Epiteliais/citologia , Humanos , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Transdução de Sinais
16.
World J Surg ; 35(8): 1715-24, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21249361

RESUMO

BACKGROUND: Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin on insulin expression/secretion and cell proliferation. A number of single nucleotide polymorphisms (SNPs) of SSTR5 have been identified, including P335L, a nonsynonymous SNP located in the protein C-terminal region and encrypted by the codon CCG (proline) or the codon CTG (leucine). In the present study we sought to determine the distribution of the SSTR5 P335L SNP in a cohort of pancreatic cancer patients and whether the P335L SNP affected cellular function of SSTR5 in human pancreatic cancer. METHODS: The P335L germline genotype of 246 patients with pancreatic cancer (213 Caucasians, 16 Hispanics, and 17 African Americans) and 17 human pancreatic cell lines was determined with the TaqMan SNP Genotyping assay. Human SSTR5 leucine variant (L335) was generated by performing site-directed mutagenesis using SSTR5 proline variant (P335) as a template. Transient transfections were performed in HEK293, Mia PaCa-2, and ß-TC-6 cells using Lipofectamine 2000. The expression of SSTR5 L335 was determined with a mouse monoclonal anti-SSTR5 L335 antibody generated in our laboratory. The cell proliferation rate was measured by performing MTS assays. Insulin concentration was measured by performing ELISA assays. RESULTS: Genotyping of the patients' blood indicated that the frequency of the T allele (CT and TT genotypes) in codon 335 of SSTR5 in Caucasians, Hispanics, and African Americans was 52, 69, and 35%, respectively, which was race-dependent. Statistical analysis indicated that association between the frequency of the T allele and the existence of pancreatic cancer in each race missed significance perhaps due to limited sample size. In 17 tested human pancreatic cancer cell lines, 5 (Capan-2, HPAF-II, Panc03.27, Panc-1, and -3) were homozygous (TT genotype) and 9, including Mia PaCa-2, were heterozygous (CT genotype). Overexpression of SSTR5 L335 in Mia PaCa-2 cells enhanced cell proliferation compared to overexpression of SSTR5 P335. Overexpression of SSTR5 P335 enhanced the inhibitory effect of SSTR5 agonist RPL-1980 on cell proliferation of Mia PaCa-2 cells and glucose-stimulated insulin secretion from mouse insulinoma cells, while overexpression of SSTR5 L335 blocked the inhibitory effect of RPL-1980. Overexpression of SSTR5 L335 enhanced PDX-1 expression in Mia PaCa-2 cells. A specific monoclonal antibody was generated to detect SSTR5 P335L. CONCLUSION: SSTR5 P335L SNP widely exists in the human population, in patients with pancreatic cancer, and is race-dependent. The SNP is also present in selected human pancreatic cancer cell lines. In contrast to SSTR5 P335, overexpression of the SSTR5 L335 variant resulted in cellular proliferation and PDX-1 overexpression in human pancreatic cancer cells. Its overexpression blocked the inhibitory effect of an SSTR5-specific analog on human pancreatic cancer cell proliferation and on glucose-stimulated insulin secretion from mouse insulinoma cells. These data suggest that SSTR5 P335L is a hypofunctional protein with a potentially harmful effect on function, as well as potential latent effect, and therefore it could affect the clinical response to somatostatin analog therapy for patients with pancreatic cancer.


Assuntos
Adenocarcinoma/genética , Alelos , Genótipo , Neoplasias Pancreáticas/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Somatostatina/genética , Adenocarcinoma/etnologia , Afro-Americanos/genética , Animais , Anticorpos Monoclonais/genética , Linhagem Celular Tumoral , Proliferação de Células , Códon/genética , Grupo com Ancestrais do Continente Europeu/genética , Regulação Neoplásica da Expressão Gênica/genética , Frequência do Gene , Hispano-Americanos/genética , Humanos , Insulina/metabolismo , Secreção de Insulina , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Pancreáticas/etnologia , Receptores de Somatostatina/imunologia
17.
Mol Endocrinol ; 24(11): 2099-113, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20829392

RESUMO

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.


Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Progesterona/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Lactação/efeitos dos fármacos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Morfogênese/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Paridade/efeitos dos fármacos , Gravidez , Progesterona/farmacologia , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt4
18.
Mol Endocrinol ; 24(6): 1297-304, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20382891

RESUMO

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/patologia
19.
Mol Endocrinol ; 24(6): 1251-66, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20363875

RESUMO

Bone morphogenetic proteins (BMPs) have diverse roles in development and reproduction. Although several BMPs are produced by oocytes, thecal cells, and granulosa cells of developing follicles, the in vivo functions of most of these ligands are unknown. BMP signals are transduced by multiple type I and type II TGFbeta family receptors, and of the type I receptors, BMP receptor 1A (BMPR1A) and BMP receptor 1B (BMPR1B) are known to be expressed in rodent granulosa cells. Female mice homozygous null for Bmpr1b are sterile due to compromised cumulus expansion, but the function of BMPR1A in the ovary is unknown. To further decipher a role for BMP signaling in mouse granulosa cells, we deleted Bmpr1a in the granulosa cells of the ovary and found Bmpr1a conditional knockout females to be subfertile with reduced spontaneous ovulation. To explore the redundant functions of BMP receptor signaling in the ovary, we generated Bmpr1a Bmpr1b double-mutant mice, which developed granulosa cell tumors that have evidence of increased TGFbeta and hedgehog signaling. Thus, similar to SMAD1 and SMAD5, which have redundant roles in suppressing granulosa cell tumor development in mice, two type I BMP receptors, BMPR1A and BMPR1B, function together to prevent ovarian tumorigenesis. These studies support a role for a functional BMP signaling axis as a tumor suppressor pathway in the ovary, with BMPR1A and BMPR1B acting downstream of BMP ligands and upstream of BMP receptor SMADs.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Fertilidade/fisiologia , Células da Granulosa/metabolismo , Neoplasias Ovarianas/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Células do Cúmulo/metabolismo , Células do Cúmulo/patologia , Ciclo Estral/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células da Granulosa/patologia , Proteínas Hedgehog/metabolismo , Hormônios/sangue , Camundongos , Camundongos Knockout , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/fisiopatologia , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/fisiopatologia , Transdução de Sinais
20.
Endocrinology ; 151(6): 2923-32, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20308529

RESUMO

The nuclear receptor cofactor receptor-interacting protein 140 (RIP140) is essential for cumulus cell-oocyte complex (COC) expansion, follicular rupture, and oocyte release during ovulation. The expression of many genes necessary for COC expansion is impaired in the absence of RIP140, but the studies herein document that their expression can be restored and COC expansion rescued by treatment with the epidermal growth factor (EGF)-like factor amphiregulin (AREG) both in vitro and in vivo. We demonstrate by several approaches that RIP140 is required for the expression of the EGF-like factors in granulosa cells, but the dependence of genes involved in cumulus expansion, including Ptgs2 Has2, Tnfaip6, and Ptx3, is indirect because they are induced by AREG. Treatment of granulosa cells with forskolin to mimic the effects of LH increases AREG promoter activity in a RIP140-dependent manner that 1) requires an intact cAMP response element in the proximal promoter region of the Areg gene and 2) involves its actions as a coactivator for cAMP response element-binding protein/c-Jun transcription factors. Although human chorionic gonadotropin and AREG coadministration is sufficient to restore ovulation fully in RIP140 heterozygous mice in vivo, both follicular rupture and ovulation remain impaired in the RIP140 null mice. Thus, we conclude that although the level of RIP140 expression in the ovary is a crucial factor required for the transient expression of EGF-like factors necessary for cumulus expansion, it also plays a role in other signaling pathways that induce follicular rupture.


Assuntos
Células do Cúmulo/citologia , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Oócitos/citologia , Ovário/metabolismo , Anfirregulina , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Colforsina/farmacologia , Células do Cúmulo/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Família de Proteínas EGF , Feminino , Genes jun/genética , Glicoproteínas/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Knockout , Correpressor 1 de Receptor Nuclear/genética , Oócitos/efeitos dos fármacos , Ovário/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Interferência de RNA
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