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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1283.)
© 2007 American Heart Association, Inc.

Vascular Biology

Glutaredoxin Mediates Akt and eNOS Activation by Flow in a Glutathione Reductase-Dependent Manner

Jing Wang; Shi Pan; Bradford C. Berk

From the Cardiovascular Research Institute and Department of Medicine, University of Rochester, NY.

Correspondence to Shi Pan, PhD, University of Rochester, Cardiovascular Research Institute, 601 Elmwood Ave, Rochester, NY 14642. E-mail Shi_Pan{at}urmc.rochester.edu

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.

Methods and 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.

The role of a thioltransferase, glutaredoxin was studied in flow stimulation of the Akt-eNOS signaling pathway in endothelial cells. Flow activates glutaredoxin via a mechanism dependent on glutathione reductase. Glutaredoxin maintains Akt in the reduced form, which enables its activation, and stimulation of the eNOS–NO signaling pathway.

Key Words: Grx • Akt • eNOS • GR • endothelial cells

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