Akt-mediated activation of HIF-1 in pulmonary vascular endothelial cells by S-nitrosoglutathione.
S-nitrosoglutathione (GSNO) stabilizes the alpha-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-1alpha 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-1alpha stabilization were blocked by acivicin, an inhibitor of gamma-glutamyl transpeptidase (gammaGT), a transmembrane protein that can translate extracellular GSNO to intracellular S-nitrosocysteinylglycine. Dithiothreitol blocked GSNO-induced Akt kinase activity and HIF-1alpha 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 gammaGT-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 Po(2), in the pulmonary vasculature.
Carver
D Jeannean
DJ
Department of Pediatrics, Divisions of Critical Care and Respiratory Medicine, University of Virginia School of Medicine, P.O. Box 801366, Charlottesville, VA 22908, USA.
Gaston
Benjamin
B
Deronde
Kimberly
K
Palmer
Lisa A
LA
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D051059:PTEN Phosphohydrolase / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000378:metabolism / Q000494:pharmacology*
D051793:Hypoxia-Inducible Factor 1; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D026422:S-Nitrosoglutathione