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Akt-Mediated Activation of HIF-1 in Pulmonary Vascular Endothelial Cells by S-Nitrosoglutathione

Department of Pediatrics, Divisions of Critical Care and Respiratory Medicine; and Department of Anesthesiology, University of Virginia School of Medicine, Charlottesville, Virginia

Correspondence and requests for reprints should be addressed to Lisa A. Palmer, Ph.D., Department of Pediatrics, University of Virginia, P.O. Box 801366, Charlottesville, VA 22908. E-mail: lap5w{at}virginia.edu

Abstract

S-nitrosoglutathione (GSNO) stabilizes the -subunit of hypoxia inducible factor-1 (HIF-1) in normoxic cells, but not in the presence of PI3K inhibitors. In this report, the biochemical pathway by which GSNO alters PI3K/Akt activity to modify HIF-1 expression was characterized in Cos cells and primary pulmonary vascular endothelial cells. GSNO increased Akt kinase activity—and downstream HIF-1 protein accumulation and DNA-binding activity—in a dose- and time-dependent manner. The PI3K inhibitors, wortmannin and LY294002, blocked these responses. Neither glutathione nor 8-bromo-cyclic GMP mimicked the GSNO-induced increases in Akt kinase activity. GSNO-induced Akt kinase activity and downstream HIF-1 stabilization were blocked by acivicin, an inhibitor of –glutamyl transpeptidase (GT), a transmembrane protein that can translate extracellular GSNO to intracellular S-nitrosocysteinylglycine. Dithiothreitol blocked GSNO-induced Akt kinase activity and HIF-1 stabilization. Moreover, the 3'-phosphatase of phosphoinositides, PTEN (phosphatase and tensin homolog deleted on chromosome ten) was S-nitrosylated by GSNO in pulmonary arterial endothelial cells, which was reversed by dithiothreitol and ultraviolet light. Interestingly, the abundance of S-nitrosylated PTEN also correlated inversely with PTEN activity. Taken together, these results suggest that GSNO induction of Akt appears to be mediated by S-nitrosylation chemistry rather than classic NO signaling through guanylate cyclase/cGMP. We speculate that GT-dependent activation of Akt and subsequent activation of HIF-1 in vascular beds may be relevant to the regulation of HIF-1–dependent gene expression in conditions associated with oxyhemoglobin deoxygenation, as opposed to profoundly low PO₂, in the pulmonary vasculature.

Key Words: phosphatidylinositol 3-kinase • hypoxia-inducible factor-1 • S-nitrosothiol • -glutamyl transpeptidase • PTEN

CLINICAL RELEVANCE S-nitrosoglutathione, formed as NO moves away from erythrocytes in response to hemoglobin desaturation, may signal hypoxia-inducible factor-1–mediated physiologic and gene regulatory events in pulmonary endothelial cells without profound hypoxia, through a thiol-based reaction.

 

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