RESUMO
AIMS/HYPOTHESIS: The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function. METHODS: Isolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1(-/-)) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells. RESULTS: BetaRac1(-/-) mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1(-/-) mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion. CONCLUSIONS/INTERPRETATION: In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.
Assuntos
Citoesqueleto de Actina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Neuropeptídeos/metabolismo , Via Secretória , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Hiperglicemia/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Camundongos Knockout , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Pâncreas/citologia , Pâncreas/metabolismo , Perfusão , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Técnicas de Cultura de Tecidos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Transcription factor Ets-1 expressed in endothelial cells promotes angiogenesis. Here, we transiently overexpressed Ets-1 in human umbilical vein endothelial cells (HUVECs) and comprehensively searched for potential downstream targets of Ets-1 by cDNA microarray analysis. The expression of several angiogenesis-related genes including neuropilin-1 was augmented by the overexpression of Ets-1. Quantitative real-time RT-PCR and Western blotting confirmed the increase in the levels of neuropilin-1 mRNA and protein. In contrast, dominant negative ets-1 decreased the levels of neuropilin-1 mRNA and protein. These results indicate that neuropilin-1 is a downstream target of Ets-1 in HUVECs.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/metabolismo , Linfocinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Veias Umbilicais/metabolismo , Sequência de Bases , Células Cultivadas , Primers do DNA , DNA Complementar , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Humanos , Linfocinas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas c-ets , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Transcription factor Ets-1 is induced in endothelial cells (ECs) by angiogenic factors, and promotes angiogenesis by inducing angiogenesis-related genes such as MMPs and integrin beta3. Here, we examined the effect of Ets-1 on apoptosis in ECs. Overexpression of Ets-1 in human umbilical vein endothelial cells (HUVECs) induced apoptosis under the serum-deprived condition. VEGF inhibited apoptosis and augmented the DNA binding of Ets-1 in HUVECs. The inhibition of transcriptional activity of endogenous Ets-1 by a dominant negative molecule intensified the anti-apoptotic effect of VEGF. Caspase inhibitors blocked apoptosis of HUVECs induced by Ets-1. DNA array analysis showed that Ets-1 up-regulated pro-apoptotic genes such as Bid, cytochrome p450, caspase-4, p27, and p21 more than 2 fold, and down-regualted anti-apoptotic genes such as DAD-1, AXL, Cox-2, IAP-2, and MDM-2 less than 0.5 fold in HUVECs. These results indicate that Ets-1 itself is pro-apoptotic to ECs by modulating the expression of apoptosis-related genes.
Assuntos
Apoptose/fisiologia , Endotélio Vascular/citologia , Neovascularização Fisiológica/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/metabolismo , Expressão Gênica/fisiologia , Vetores Genéticos , Humanos , Linfocinas/farmacologia , Oligopeptídeos/farmacologia , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Angiogenesis is a complex phenomenon that requires at least migration, proliferation, and tubular morphogenesis of endothelial cells (ECs). Some genes are expressed in ECs during these processes, and therefore the regulation of gene expression in ECs is critical. Increasing evidence suggests that the Ets family of transcription factors plays an important role in angiogenesis. We observed that Ets-1, a prototype of the Ets family of transcription factors, promoted angiogenesis by inducing the expression of matrix metalloproteinases and integrin beta3 in ECs, and the elimination of the transactivation activity of Ets-1 by a dominant negative molecule inhibited angiogenesis. Apoptosis, a term used to describe the terminal morphological and biochemical events seen in programmed cell death, is critical for the development or reconstitution of multicellular organs. Apoptosis of ECs is observed at the initiation of angiogenesis, at the branching or communication with newly formed vessels, and at the regression of neo-vessels. The Ets family of transcription factors is generally thought to be anti-apoptotic. However, there are conflicting reports on the role of Ets-1 in apoptosis. We examined the role of Ets-1 in apoptosis of ECs and found that Ets-1 was pro-apoptotic to ECs by modulating the expression of several apoptosis-related genes.
