ABSTRACT
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.
Subject(s)
Actin Cytoskeleton/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Neuropeptides/metabolism , Secretory Pathway , rac1 GTP-Binding Protein/metabolism , Animals , Cell Line , Hyperglycemia/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Male , Mice , Mice, Knockout , Neuropeptides/antagonists & inhibitors , Neuropeptides/genetics , Pancreas/cytology , Pancreas/metabolism , Perfusion , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering , Rats , Tissue Culture Techniques , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/geneticsABSTRACT
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.
Subject(s)
Endothelial Growth Factors/metabolism , Endothelium, Vascular/metabolism , Lymphokines/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Umbilical Veins/metabolism , Base Sequence , Cells, Cultured , DNA Primers , DNA, Complementary , Endothelial Growth Factors/genetics , Endothelium, Vascular/cytology , Humans , Lymphokines/genetics , Oligonucleotide Array Sequence Analysis , Protein Binding , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-ets , Umbilical Veins/cytology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
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.
Subject(s)
Apoptosis/physiology , Endothelium, Vascular/cytology , Neovascularization, Physiologic/physiology , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Adenoviridae/genetics , Apoptosis/drug effects , Caspase Inhibitors , Caspases/metabolism , Cell Division/drug effects , Cell Division/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/metabolism , Gene Expression/physiology , Genetic Vectors , Humans , Lymphokines/pharmacology , Oligopeptides/pharmacology , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ets , Transcription Factors/genetics , Transcription, Genetic/physiology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
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.
