Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors

To test the hypothesis that keratinocyte (KC) migration is modulated by distinct muscarinic acetylcholine (ACh) receptor subtypes, we inactivated signaling through specific receptors in in vitro and in vivo models of reepithelialization by subtype-selective antagonists, small interfering RNA, and gene knockout in mice. KC migration and wound reepithelialization were facilitated by M4 and inhibited by M3. Additional studies showed that M4 increases expression of "migratory" integrins alpha5beta1, alphaVbeta5, and alphaVbeta6, whereas M3 up-regulates "sedentary" integrins alpha2beta1 and alpha3beta1. Inhibition of migration by M3 was mediated through Ca2+-dependent guanylyl cyclase-cyclic GMP-protein kinase G signaling pathway. The M4 effects resulted from inhibition of the inhibitory pathway involving the adenylyl cyclase-cyclic AMP-protein kinase A pathway. Both signaling pathways intersected at Rho, indicating that Rho kinase provides a common effector for M3 and M4 regulation of cell migration. These findings offer novel insights into the mechanisms of ACh-mediated modulation of KC migration and wound reepithelialization, and may aid the development of novel methods to promote wound healing.