Abstract 3653: JNK3 Is Required For SDF1α-induced Endothelial Cell Migration: The Regulation Of Mkp7 By eNOS Activation
SDF-1α is a pro-angiogenic chemokine that exerts its actions through the induction of endothelial cell migration as well as the promotion of endothelial progenitor cell mobilization and homing processes. However, the mechanisms by which SDF-1α triggers these endothelial cell activation responses is unclear. In this study, we show that both eNOS and JNK are targets of SDF-1α-induced activation in bovine aortic endothelial cells. Results from a Boyden chamber assay demonstrated that endothelial cell migration induced by 50 ng/ml SDF-1α was reduced by L-NAME and SP600125, inhibitors of eNOS and JNK respectively. SDF-1α (50 ng/ml) treatment of cells transfected with various isoforms of JNK resulted in phosphorylation of cells expressing JNK3, but not those expressing JNK1 or 2; indicating that JNK3 is the major isoform activated by SDF-1α. Interestingly, the activation of JNK3 was inhibited by the eNOS inhibitor L-NMMA, suggesting that JNK3 acts downstream of eNOS in the SDF-1α signaling casade. An in vitro nitrosylation assay (using the biotin switch method) demonstrated that MKP7, a JNK3 phosphatase, was nitrosylated by the nitric oxide donor nitrosoglutathione (GSNO) and SDF-1α, leading to a significant inhibition of MKP7’s phosphatase activity on JNK3. A Cys244→Ser mutant of MKP7 exhibited decreased nitrosylation by GSNO compared with wildtype MKP7, indicating that the Cys244 residue was the major cysteine targeted for nitrosylation. Despite this decrease in nitrosylation, the Cys244→Ser mutant also possessed decreased phosphatase activity compared to wild type MKP7, presumably because Cys244 is a key amino acid located in the catalytic domain of MKP7 and its modification leads to kinase death. Interestingly, SDF-1→-induced cell migration was not inhibited by the Cys244→Ser mutant of MKP7, whereas wild type MKP7 significantly decreased SDF-1á-induced cell migration (from 125±9 to 84±12 cells per field). Collectively, these results demonstrate that MKP7 acts as a key player in the coordination of SDF-1á’s effects on endothelial cell migration activated by both eNOS and JNK3. The salient roles of MKP7 and JNK3 in SDF-1á-induced cell migration provide novel and unexpected therapeutic targets for angiogenesis-dependent diseases.