RESUMO
The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase beta-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3beta. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3beta, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3beta. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.
Assuntos
Proteínas F-Box/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Western Blotting , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Proteínas F-Box/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imunoprecipitação , Camundongos , Mutação , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Transfecção , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to gamma radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after gamma radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after gamma radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.
Assuntos
Apoptose/efeitos da radiação , Raios gama , Regulação da Expressão Gênica , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Dano ao DNA , DNA Complementar , Cães , Fase G2 , Genes Reporter , Humanos , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/análise , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/metabolismo , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Puromicina/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Seleção Genética , Fatores de Transcrição da Família Snail , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , TransfecçãoRESUMO
The Snail gene product is a transcriptional repressor of E-cadherin expression and an inducer of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. This report presents data indicating that Snail function is controlled by its intracellular location. The cytosolic distribution of Snail depended on export from the nucleus by a CRM1-dependent mechanism, and a nuclear export sequence (NES) was located in the regulatory domain of this protein. Export of Snail was controlled by phosphorylation of a Ser-rich sequence adjacent to this NES. Modification of this sequence released the restriction created by the zinc finger domain and allowed nuclear export of the protein. The phosphorylation and subcellular distribution of Snail are controlled by cell attachment to the extracellular matrix. Suspended cells presented higher levels of phosphorylated Snail and an augmented extranuclear localization with respect to cells attached to the plate. These findings show the existence in tumor cells of an effective and fine-tuning nontranscriptional mechanism of regulation of Snail activity dependent on the extracellular environment.