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

Yosuke Watanabe, Colin E. Murdoch, Soichi Sano, Yasuo Ido, Markus M. Bachschmid, Richard A. Cohen, Reiko Matsui

Research output: Contribution to journalArticlepeer-review

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

Original languageEnglish
Pages (from-to)6011-6016
Number of pages6
JournalProceedings of the National Academy of Sciences
Volume113
Issue number21
Early online date9 May 2016
DOIs
Publication statusPublished - 24 May 2016

Bibliographical note

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.

Keywords

  • GSH-protein adducts
  • S-glutathionylation
  • hypoxia-inducible factor-1
  • glutaredoxin-1
  • ischemic limb revascularization

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