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 α₅β₁, α_Vβ₅, and α_Vβ₆, whereas M₃ up-regulates “sedentary” integrins α₂β₁ and α₃β₁. 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.