Premium
Vav1‐Dependent Activation of Rac1/NAD(P)H Oxidase Signaling Induced by Homocysteine in Rat Mesangial Cells
Author(s) -
Chen QiZheng,
Yi Fan,
Jin Si,
Li PinLan
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a431-a
Subject(s) - gtpase , rac1 , guanine nucleotide exchange factor , cdc42 , nad+ kinase , rac gtp binding proteins , microbiology and biotechnology , phosphorylation , nadph oxidase , signal transduction , transfection , small gtpase , ceramide , kinase , chemistry , superoxide , oxidase test , biochemistry , biology , reactive oxygen species , enzyme , apoptosis , gene
In our previous studies, homocysteine (Hcys) has been reported to stimulate de novo ceramide synthesis and thereby induce NAD(P)H oxidase activation by increase in Rac GTPase activity in rat mesangial (RMG) cells. However, the mechanism by which Hcys‐enhances Rac GTPase activity is still unknown. Given Vav1, a member of guanine exchange factors (GEFs), plays an important role in the activation of Rho/Rac GTPase activity, the present study tested the hypothesis that Rac1/NAD(P)H oxidase signaling induced by Hcys is associated with Vav1 in RMG cells. Real‐time RT PCR and Western blot analysis confirmed that Vav1 was present in RMG cells. By Rac GTPase pull down assay, it was found that NSC23766 , an inhibitor by interfering Rac1 interaction with the Rac‐specific GEF, blocked the Hcys‐induced Rac GTPase activity by 42%. In Vav1‐siRNA transfected RMG cells, Hcys‐induced Rac GTPase activity decreased by 52%, which was accompanied by 55% reduction of Hcys‐induced superoxide (O 2 − ) production, as measured by ESR spectrometry. By immunofluorescence assay, it was found that Hcys stimulated Vav1 phosphorylated and then translocated to cell membrane. Similar results were obtained by C16‐ceramide treatments of RMG cells. Based on these results, we conclude that Rac1/NADPH oxidase signaling induced by Hcys is Vav1‐depentent in RMG cells, which involves its phosphorylation and subsequently translocation, an essential step for the activation of this GEF (supported by NIH Grants DK054927, HL57244, and HL70726).