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Contribution of guanine nucleotide exchange factor Vav2 to hyperhomocysteinemic glomerulosclerosis in rats.

ABSTRACT: We currently reported that Vav2, a member of the guanine nucleotide exchange factor-Vav subfamily, participates in homocysteine-induced increases in Rac1 activity and consequent activation of NADPH oxidase in rat mesangial cells. However, the physiological relevance of this cellular action of Vav2 remains unknown. The present study tested a hypothesis that Vav2 importantly mediates the injurious action of homocysteine on glomeruli and thereby contributes to the development of glomerulosclerosis during hyperhomocysteinemia. We found that, among Vav members, Vav2 was abundantly expressed in glomeruli. When Vav2 short hairpin RNA was transfected into the kidneys of Sprague-Dawley rats, hyperhomocysteinemia induced by folate-free diet failed to significantly enhance Rac1 activity and increase NADPH-dependent superoxide production. In these rats with silenced renal Vav2 gene, glomerular injury during hyperhomocysteinemia was markedly attenuated compared with those rats only receiving mock vector transfection, as shown by ameliorated albuminuria and extracellular matrix metabolism. In the rat kidneys with transfection of a dominant-active Vav2 variant (onco-Vav2), we found that overexpression of Vav2 led to significant increases in Rac1 activity, superoxide production, and glomerular injury, which was similar to that induced by hyperhomocysteinemia. However, this Vav2 overexpression was unable to further enhance homocysteine-induced glomerular injury. We concluded that Vav2-mediated activation of NADPH oxidase is an important initiating mechanism resulting in hyperhomocysteinemic glomerular injury through enhanced local oxidative stress.

SUBMITTER: Yi F

PROVIDER: S-EPMC2666012 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Contribution of guanine nucleotide exchange factor Vav2 to hyperhomocysteinemic glomerulosclerosis in rats.

Yi Fan F Xia Min M Li Ningjun N Zhang Chun C Tang Lin L Li Pin-Lan PL

Hypertension (Dallas, Tex. : 1979) 20081124 1

We currently reported that Vav2, a member of the guanine nucleotide exchange factor-Vav subfamily, participates in homocysteine-induced increases in Rac1 activity and consequent activation of NADPH oxidase in rat mesangial cells. However, the physiological relevance of this cellular action of Vav2 remains unknown. The present study tested a hypothesis that Vav2 importantly mediates the injurious action of homocysteine on glomeruli and thereby contributes to the development of glomerulosclerosis ...[more]

PMID: 19029489

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Project description:Aims/hypothesisRho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.MethodsCo-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).ResultsWestern blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.Conclusions/interpretationOur data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

Project description:Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.

Dataset Information

Contribution of guanine nucleotide exchange factor Vav2 to hyperhomocysteinemic glomerulosclerosis in rats.

Publications

Contribution of guanine nucleotide exchange factor Vav2 to hyperhomocysteinemic glomerulosclerosis in rats.

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