RESUMO
Hepatocyte nuclear factor-1ß (HNF-1ß) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1ß in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1ß in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1ß. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1ß. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1ß. Overexpression of zyxin stimulates the transcriptional activity of HNF-1ß, whereas small interfering RNA silencing of zyxin inhibits HNF-1ß-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1ß-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1ß, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.
Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Fator 1-beta Nuclear de Hepatócito/metabolismo , Rim/metabolismo , Zixina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Movimento Celular/efeitos dos fármacos , GMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Imunoprecipitação , Rim/citologia , Rim/efeitos dos fármacos , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais , Transcrição Gênica , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Zixina/genéticaRESUMO
Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1beta develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1-binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1beta and the structurally related HNF-1alpha bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1beta mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1beta mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1beta (MODY5) and PKHD1 (ARPKD). HNF-1beta directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.
Assuntos
Proteínas de Ligação a DNA/genética , Doenças Renais Císticas/genética , Mutação , Receptores de Superfície Celular/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Fator 1-beta Nuclear de Hepatócito , Humanos , Doenças Renais Císticas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Receptores de Superfície Celular/biossíntese , Fatores de Transcrição/metabolismoRESUMO
Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta produce maturity-onset diabetes of the young type 5 (MODY5) and are associated with congenital cystic abnormalities of the kidney. Transgenic mice expressing mutant HNF-1beta under the control of a kidney-specific promoter develop kidney cysts and renal failure, which is similar to the phenotype of humans with MODY5. Similarly, kidney-specific deletion of HNF-1beta using Cre/loxP recombination results in renal cyst formation. HNF-1beta directly regulates the Pkhd1 promoter. HNF-1beta mutant mice show decreased expression of Pkhd1, the gene that is mutated in humans with autosomal-recessive polycystic kidney disease (ARPKD). These studies demonstrate that HNF-1beta is required for the development of the mammalian kidney. They establish a previously unrecognized link between two renal cystic diseases, MODY5 and ARPKD, and suggest that the mechanism of cyst formation in humans with mutations of HNF-1beta involves down-regulation of PKHD1 gene transcription.