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Biochim Biophys Acta ; 1849(12): 1411-22, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26477491


Intestinal epithelial cells are exposed to luminal bacterial threat and require adequate defense mechanisms to ensure host protection and epithelium regeneration against possible deleterious damage. Differentiated intestinal epithelial cells produce antimicrobial and regenerative components that protect against such challenges. Few intestinal specific transcription factors have been identified to control the switching from repression to activation of this class of gene. Herein, we show that gene transcription of some regenerating islet-derived (REG) family members is dependent on the transcription factor GATA-4. Silencing of GATA-4 expression in cultured intestinal epithelial cells identified Reg3ß as a target gene using an unbiased approach of gene expression profiling. Co-transfection and RNA interference assays identified complex GATA-4-interactive transcriptional components required for the activation or repression of Reg3ß gene activity. Conditional deletion of Gata4 in the mouse intestinal epithelium supported its regulatory role for Reg1, Reg3α, Reg3ß and Reg3γ genes. Reg1 dramatic down-modulation of expression in Gata4 conditional null mice was associated with a significant decrease in intestinal epithelial cell migration. Altogether, these results identify a novel and complex role for GATA-4 in the regulation of REG family members gene expression.

Células Epiteliais/metabolismo , Fator de Transcrição GATA4/fisiologia , Regulação da Expressão Gênica/genética , Mucosa Intestinal/citologia , Família Multigênica , Transcrição Genética , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Fator de Transcrição CDX2 , Diferenciação Celular/genética , Linhagem Celular , Técnicas de Cocultura , Fator de Transcrição GATA4/classificação , Fator de Transcrição GATA4/deficiência , Fator de Transcrição GATA4/genética , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Lectinas Tipo C/metabolismo , Litostatina/metabolismo , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas Associadas a Pancreatite , Estrutura Terciária de Proteína , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
Diabetes ; 64(9): 3314-20, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25979074


Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic ß-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non-insulin-dependent diabetes.

Glicemia/metabolismo , Polipeptídeo Inibidor Gástrico/genética , Mucosa Gástrica/metabolismo , Grelina/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Jejuno/metabolismo , RNA Mensageiro/metabolismo , Animais , Glicemia/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/metabolismo , Grelina/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Oligopeptídeos/farmacologia , Receptores de Grelina/antagonistas & inibidores , Regulação para Cima
PLoS One ; 5(8): e12378, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20808783


BACKGROUND AND AIMS: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1alpha on the maintenance of adult small intestinal epithelial functions. METHODOLOGY/PRINCIPAL FINDINGS: An Hnf1alpha knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1alpha displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1alpha mutant mice. The intestinal epithelium of Hnf1alpha null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. CONCLUSIONS/SIGNIFICANCE: Together, these results unravel a functional role for Hnf1alpha in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

Diferenciação Celular , Fator 1-alfa Nuclear de Hepatócito/deficiência , Fator 1-alfa Nuclear de Hepatócito/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Animais , Transporte Biológico/genética , Proliferação de Células , Enterócitos/citologia , Enterócitos/metabolismo , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Glucose/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Homeostase/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR , Regulação para Cima
Am J Physiol Gastrointest Liver Physiol ; 297(1): G124-34, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19389805


Hepatocyte nuclear factor 4alpha (HNF4alpha) is a regulator of hepatocyte and pancreatic transcription. Hnf4alpha deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4alpha in the developing colon was much less important than in the liver. However, the precise role of Hnf4alpha in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4alpha to support an intestinal epithelial phenotype. First, Hnf4alpha potential to dictate this phenotype was assessed in nonintestinal cell lines in vitro. Forced expression of Hnf4alpha in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4alpha with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4alpha in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4alpha developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4alpha mutant mice displayed an increase in the number of goblet and enteroendocrine cells compared with controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.

Diferenciação Celular , Células Epiteliais/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Mucosa Intestinal/metabolismo , Envelhecimento/metabolismo , Animais , Fator de Transcrição CDX2 , Células CACO-2 , Proliferação de Células , Forma Celular , Células Epiteliais/diagnóstico por imagem , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Humanos , Integrases/genética , Mucosa Intestinal/ultraestrutura , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Fenótipo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Ultrassonografia
Am J Physiol Gastrointest Liver Physiol ; 294(2): G418-28, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18032476


Normal cellular models able to efficiently recapitulate intestinal epithelial cell differentiation in culture are not yet available. The aim of this work was to establish and genetically characterize a mesenchymal-epithelial coculture system to identify transcriptional regulators involved in this process. The deposition of rat intestinal epithelial cells on human intestinal mesenchymal cells led to the formation of clustered structures that expanded shortly after seeding. These structures were composed of polarized epithelial cells with brush borders and cell junction complexes. A rat GeneChip statistical analysis performed at different time points during this process identified hepatocyte nuclear factor-4alpha (HNF-4alpha) and hepatocyte nuclear factor-1alpha (HNF-1alpha) as being induced coincidently with the apparition of polarized epithelial structures. Stable introduction of HNF-4alpha in undifferentiated epithelial cells alone led to the rapid induction of HNF-1alpha and several intestinal-specific markers and metabolism-related genes for which mRNA was identified to be upregulated during epithelial differentiation. HNF-4alpha was capable to transactivate the calbindin 3 gene promoter, a process that was synergistically increased in the presence of HNF-1alpha. When HNF-4alpha-expressing cells were plated on mesenchymal cells, an epithelial monolayer formed rapidly with the apparition of dome structures that are characteristics of vectorial ion transport. Forced expression of HNF-1alpha alone did not result in dome structures formation. In sum, this novel coculture system functionally identified for the first time HNF-4alpha as an important modulator of intestinal epithelial differentiation and offers an innovative opportunity to investigate molecular mechanisms involved in this process.

Células Epiteliais/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/farmacologia , Intestinos/citologia , Adulto , Western Blotting , Fator de Transcrição CDX2 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Interpretação Estatística de Dados , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Marcadores Genéticos , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Proteínas de Homeodomínio/fisiologia , Humanos , Intestinos/efeitos dos fármacos , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa
FASEB J ; 21(14): 3853-65, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17622569


Intestinal epithelial integrity and polarity are maintained by cohesive interactions between cells via the formation of tight junctions. Irregularities in tight junctions have only recently been found to be associated with the initiation and progression of intestinal neoplasia. The claudin family of proteins is integral to the structure and function of the tight junction but little is known of the molecular events that regulate the expression of these components. The present report identifies cathepsin L, classically a lysosomal cysteine protease, as being induced during intestinal epithelial cell polarization and differentiation. Inhibition of intracellular cathepsin L activity results in the accumulation of disorganized cell layers and a decline in the expression of differentiation markers in cultured intestinal epithelial cells. This coincides with a rapid up-regulation of claudin-1 protein accumulation. Mutant mice defective in cathepsin L activity (furless) display an elevated level of intestinal claudin-1 and claudin-2 expression. Loss of cathepsin L activity leads to a marked increase in tumor multiplicity in the intestine of Apc(Min) mice. Given the traditionally viewed biological role of cathepsin L in the processing of lysosomal content as well as in pathological extracellular matrix remodeling, the results here demonstrate an as yet unsuspected intracellular role for this protease in normal intestinal epithelial polarization and initiation of neoplasia.

Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Neoplasias Intestinais/etiologia , Neoplasias Intestinais/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Animais , Sequência de Bases , Células CACO-2 , Catepsina L , Catepsinas/deficiência , Catepsinas/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Claudina-1 , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Neoplasias Intestinais/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Coelhos , Regulação para Cima/fisiologia