Going the Right Distance

Abstract

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 M 3 and M 4 acetylcholine receptors in keratinocyte migration. Human keratinocytes transfected with antibodies against M 3 small, interfering RNA (siRNA) migrated further into gels than did control cells, as did keratinocytes from mice lacking M 3 , whereas keratinocytes transfected with antibodies against M 4 siRNA (or from mice lacking M 4 ) showed decreased migration. In vivo experiments also indicated that M 4 enhanced and M 3 inhibited migration. Pharmacologic analysis indicated that the effects of M 3 (which acts through the Gα q/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 M 4 (which acts through Gα i/o ) were mediated through inhibition of the adenylate cyclase-cAMP (adenosine 3′,5′-monophosphate)-protein kinase A cascade. The M 3 and M 4 pathways converged on Rho and Rho kinase. Pharmacologic and siRNA analysis, as well as analysis of knockout mice, indicated that M 4 promoted expression of integrins that mediate adhesion to proteins over which keratinocytes migrate during wound healing, whereas M 3 promoted expression of integrins found at cell-cell contact sites. Further, antibodies to the "migratory integrins" reduced in vitro migration of keratinocytes lacking M 3 , whereas antibodies to the "sedentary integrins" reduced migration of keratinocytes lacking M 4 . Thus, the authors propose that opposing inhibitory and stimulatory pathways mediated through the M 3 and M 4 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 M 3 and M 4 muscarinic receptors. J. Cell Biol. 166 , 261-272 (2004). [Abstract] [Full Text]