Glutaredoxin mediates Akt and eNOS activation by flow in a glutathione reductase-dependent manner.
OBJECTIVE
The glutathione (GSH)/glutaredoxin (Grx) system regulates activities of many redox sensitive enzymes. This system has been shown to protect cells from hydrogen peroxide-induced apoptosis by regulating the redox state of Akt. Grx can be regulated by redox state; the oxidized Grx is selectively recycled to the reduced form by GSH. Flow can maintain endothelial cells in a reduced state by activating glutathione reductase (GR) and increasing the GSH/GSSG ratio. Because steady laminar flow exerts an antioxidant effect, we hypothesized that Grx mediates flow induced Akt and eNOS phosphorylation in a GR dependent manner.


RESULTS
Exposure of endothelial cells (ECs) to physiological steady laminar flow (shear stress=12 dyn/cm2) for 5 minutes significantly increased Grx activity (1.9+/-0.2-fold), and also increased Akt and eNOS phosphorylation. Overexpression of GFP-GR in ECs significantly increased Grx activity by 1.6+/-0.1-fold. Pretreatment with the GR inhibitor 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU) for 30 minutes dramatically reduced Grx activity and inhibited the increase in Akt and eNOS phosphorylation induced by flow. Overexpression of wild-type Grx in ECs increased both Akt and eNOS phosphorylation. In contrast, a mutated Grx (C22S/C25S), which lacks thioltransferase activity, had no effect. Therefore, flow-induced Akt and eNOS phosphorylation depend on Grx thioltransferase activity. Downregulation of Grx by small interfering RNA decreased flow induced Akt and eNOS phosphorylation.


CONCLUSIONS
These data suggest that Grx is an important mediator for flow-induced Akt and eNOS activation, and Grx activity depends on GR-mediated changes in EC redox state.
Wang
Jing
J
University of Rochester, Cardiovascular Research Institute and Department of Medicine, 601 Elmwood Ave, Rochester, NY 14642, USA.
Pan
Shi
S
Berk
Bradford C
BC
D000818:Animals; D002330:Carmustine / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004780:Environment, Controlled; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013314:Stress, Mechanical; D014162:Transfection
D004791:Enzyme Inhibitors; C516005:GLRX protein, human; D054477:Glutaredoxins; D034741:RNA, Small Interfering; D010088:Oxidoreductases; D052250:Nitric Oxide Synthase Type III; D005980:Glutathione Reductase; D051057:Proto-Oncogene Proteins c-akt; D005978:Glutathione; D002330:Carmustine