JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells

To understand the role of redox-sensitive mechanisms in vascular smooth mus cle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC ), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and throm bin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase acti vation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist -induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and Ju nB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-deriv ed growth factor BB-induced ERK2 activation. In contrast, DPI only inhibite d agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotrans fection of VSMC with expression plasmids for c-Fos and members of the Jun f amily along with the AP-l-dependent reporter gene revealed that AP-1 with c -Fos and JunB composition exhibited a higher transactivating activity than AP-I with other compositions tested. All three agonists significantly stimu lated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox -sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase act ivation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesi s in VSMC. These results also suggest a role for NADH/NADPH oxidase activit y in some subset of early signaling events such as p38 MAP kinase activatio n and c-Fos and JunB induction, which appear to be important in agonist-ind uced AP-I activity and DNA synthesis in VSMC.