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 M-4 and inhibited by M-3. Additional studies showed that M-4 increases expression of "migratory" integrins alpha(5)beta(1), alpha(v)beta(5), and alpha(v)beta(6), whereas M-3 up-regulates "sedentary" integrins alpha(2)beta(1) and alpha(3)beta(1). Inhibition of migration by M-3 was mediated through Ca2+-dependent guanylyl cyclase-cyclic GMP-protein kinase G signaling pathway. The M-4 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 M-3 and M-4 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.