Distinct roles of Ca2+, calmodulin, and protein kinase C in H2O2-induced activation of ERK1/2, p38 MAPK, and protein kinase B signaling in vascular smooth muscle cells

We have shown earlier that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), two key mediators of growth-promoting and proliferative responses, are activated by hydrogen peroxide (H2O2) in A10 vascular smooth muscle cells (VSMC). In the present studies, using a series of pharmacological inhibitors, we explored the upstream mechanisms responsible for their activation in response to H2O2. H2O2 treatment of VSMC stimulated ERK1/2, p38 mitogen-activated protein kinase (MAPK), and PKB phosphorylation in a dose- and time-dependent fashion. BAPTA-AM and EGTA, chelators of intracellular and extracellular Ca2+, respectively, inhibited H2O2-stimulated ERK1/2, p38 MAPK, and PKB phosphorylation. Fluphenazine, an antagonist of the Ca2+-binding protein calmodulin, also suppressed the enhanced phosphorylation of ERK1/2, p38 MAPK, and PKB. In contrast, the protein kinase C (PKC) inhibitors Go 6983 and Ro 31-8220 attenuated H2O2-induced ERK1/2 phosphorylation, but had no effect on p38 MAPK and PKB phosphorylation. Taken together, these data demonstrate that the activation of Ca2+/calmodulin-dependent pathways represents a key component mediating the stimulatory action of H2O2 on ERK1/2, p38 MAPK, and PKB phosphorylation. On the other hand, PKC appears to be an upstream modulator of the increased ERK1/2 phosphorylation, but not of p38 MAPK and PKB in response to H2O2 in VSMC.