ACh Stimulation regulates N‐WASp and Arp2/3 Mediated Cortical Actin Polymerization in Airway Smooth Muscle (ASM) by RhoA‐mediated activation of paxillin and cdc42

Abstract

In ASM, ACh stimulation activates RhoA GTPase which mediates contraction by regulating actin polymerization rather than myosin light chain phosphorylation We investigated the mechanisms by which RhoA regulates actin polymerization in canine ASM tissues using the inactive RhoA mutant, RhoA T19N and C3 exoenzyme. Actin polymerization in ASM requires the phosphorylation of paxillin and the activation of cdc42, which catalyzes activation of the actin filament nucleating proteins N-WASp and the Arp2/3 complex. RhoA inactivation inhibited ACh induced paxillin phosphorylation and cdc42 activation and prevented the interaction of the Arp2/3 complex with N-WASp at the cell membrane. In contrast, the inhibition of Rho kinase did not inhibit paxillin phosphorylation. A non phosphorylatable paxillin mutant, paxillin F31/F118, inhibited ACh induced cdc42 activation but not RhoA activation. The inactive mutant cdc42 T17N inhibited actin polymerization and N-WASp activation but not ACh induced RhoA activation or paxillin phosphorylation. Inactivation of RhoA, cdc42, and paxillin all inhibit N-WASp mediated actin polymerization and tension development induced by ACh. We conclude that ACh induced RhoA activation initiates cortical actin polymerization in ASM by mediating the activation of paxillin, which catalyzes the activation of cdc42 and N-WASp, and the Arp2/3 complex at the ASM membrane. HL29289, HL074099