Keratinocytes migrate over injured skin to form new epidermis. Acetylcholine, acting through muscarinic receptors, influences keratinocyte migration and wound healing; however, the detailed mechanisms remain incompletely understood. Chernyavsky et al. used a model of wound healing in which keratinocytes migrated into agarose gels, a monolayer wounding assay, and in vivo knockout experiments to investigate the role of M3 and M4 acetylcholine receptors in keratinocyte migration. Human keratinocytes transfected with antibodies against M3 small, interfering RNA (siRNA) migrated further into gels than did control cells, as did keratinocytes from mice lacking M3, whereas keratinocytes transfected with antibodies against M4 siRNA (or from mice lacking M4) showed decreased migration. In vivo experiments also indicated that M4 enhanced and M3 inhibited migration. Pharmacologic analysis indicated that the effects of M3 (which acts through the Gαq/11 family of heterotrimeric guanosine triphosphate-binding proteins) involved calcium mobilization and subsequent activation of a guanylate kinase-cGMP (guanosine 3′,5′-monophosphate)-protein kinase G signaling pathway. The effects of M4 (which acts through Gαi/o) were mediated through inhibition of the adenylate cyclase-cAMP (adenosine 3′,5′-monophosphate)-protein kinase A cascade. The M3 and M4 pathways converged on Rho and Rho kinase. Pharmacologic and siRNA analysis, as well as analysis of knockout mice, indicated that M4 promoted expression of integrins that mediate adhesion to proteins over which keratinocytes migrate during wound healing, whereas M3 promoted expression of integrins found at cell-cell contact sites. Further, antibodies to the "migratory integrins" reduced in vitro migration of keratinocytes lacking M3, whereas antibodies to the "sedentary integrins" reduced migration of keratinocytes lacking M4. Thus, the authors propose that opposing inhibitory and stimulatory pathways mediated through the M3 and M4 receptors regulate keratinocyte migration during wound healing.
A. I. Chernyavsky, J. Arredondo, J. Weiss, E. Karlsson, S. A. Grando, Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors. J. Cell Biol. 166, 261-272 (2004). [Abstract] [Full Text]