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Contribution of Rac GTPase‐Mediated NAD(P)H Oxidase Activation to Reduction of MMP Activity in Hcys‐Treated Rat Mesangial Cells
Author(s) -
Yi Fan,
Muh Rachel W,
Li PinLan
Publication year - 2006
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.20.5.a1140
Subject(s) - nad+ kinase , gtpase , oxidase test , nad(p)h oxidase , chemistry , microbiology and biotechnology , western blot , nadph oxidase , superoxide , matrix metalloproteinase , biochemistry , enzyme , biology , gene
Rac GTPase importantly participates in the assembly of cytosolic subunits of NAD(P)H oxidase to the cell membrane during activation of this enzyme. We have demonstrated that this Rac‐mediated NAD(P)H oxidase activation is attributed to homocysteine (Hcys)‐induced superoxide (O 2 ·− ) production in rat mesangial (MG) cells. The present study was to further clarify the role of Rac‐NAD(P)H oxidase activity in Hcys‐induced reduction of matrix metalloproteinase‐1 (MMP‐1) activity in these cells. In Rac‐siRNA transfected rat MG cells, Rac GTPase protein was decreased by 70%, which was accompanied by 65% reduction of Hcys‐induced O 2 ·− production by Electron Spin Resonance (ESR) spectrometric analysis. Using fluorescence resonance energy transfer (FRET) marker‐labeled peptide as a substrate to measure MMP activity, we found that Hcys reduced MMP‐1 activity by 32% in normal MG cells. This Hcys‐induced reduction of MMP‐1 activity was significantly restored (to 87% of control) by Rac RNA interference. Western blot analyses showed that Rac RNA interference significantly inhibited Hycs‐induced enhancement of tissue inhibitor of metalloproteinase‐1 (TIMP‐1). It is concluded that Rac‐mediated NAD(P)H oxidase activation may promote the formation of TIMP‐1 and thereby decrease MMP‐1 activity, ultimately leading to deposition of collagen in rat MG cells (supported by NIH grants DK054927, and HL057244).