Induction of endothelial NO synthase by hydrogen peroxide via a Ca2+/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway

We have recently demonstrated that hydrogen peroxide (H2O2) is an extremely potent stimulus of endothelial NO synthase (eNOS) gene expression. The present study was designed to identify the signaling mechanisms mediating this response. Induction of eNOS expression by H2O2 was found to be Ca2+ dependent, inasmuch as it was blocked by BAPTA-AM. Further studies have indicated that Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) plays a critical role in mediating this response. Immunocytochemical staining with an anti-CaM kinase II antibody confirmed the expression of CaM kinase II in cultured bovine aortic endothelial cells. H2O2 induced autophosphorylation of CaM kinase II and increased the activity of the enzyme, as assessed by an in-gel kinase assay. A specific inhibitor for CaM kinase II, KN93, and a calmodulin antagonist, W-7, attenuated eNOS induction by H2O2. Further studies have indicated that janus kinase 2 is important in mediating increased eNOS expression in response to H2O2 and likely is downstream from CaM kinase II. In conclusion, these data provide the first evidence that CaM kinase II plays a critical role in endothelial redox signaling. Regulation of eNOS via this pathway may represent an important vascular adaptation to oxidant stress.