Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Abstract

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 serine mutant is resistant to 2-AAPA–induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles

Publication DOI: https://doi.org/10.1073/pnas.1524198113
Divisions: Aston Medical School
Life & Health Sciences > Biosciences
Additional Information: Watanabe, Y., Murdoch, C. E., Sano, S., Ido, Y., Bachschmid, M. M., Cohen, R. A., & Matsui, R. (2016). Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization. Proceedings of the National Academy of Sciences, In press. 10.1073/pnas.1524198113 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1524198113/-/DCSupplemental.
Uncontrolled Keywords: GSH-protein adducts,S-glutathionylation,hypoxia-inducible factor-1,glutaredoxin-1,ischemic limb revascularization
Full Text Link: https://www.ncb ... les/PMC4889374/
Related URLs: http://www.pnas ... 5/06/1524198113 (Publisher URL)
PURE Output Type: Article
Published Date: 2016-05-24
Published Online Date: 2016-05-09
Accepted Date: 2016-04-15
Submitted Date: 2015-12-14
Authors: Watanabe, Yosuke
Murdoch, Colin E. ( 0000-0002-0274-819X)
Sano, Soichi
Ido, Yasuo
Bachschmid, Markus M.
Cohen, Richard A.
Matsui, Reiko

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