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J. Cell Biol. 166 (2): 261-272

Copyright © 2004 by the Rockefeller University Press.


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

Alex I. Chernyavsky1, Juan Arredondo1, Jürgen Wess2, Evert Karlsson3, , and Sergei A. Grando1

1 Department of Dermatology, University of California, Davis, Sacramento, CA 95817
2 Laboratory of Bioorganic Chemistry, Molecular Signaling Section, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
3 Section of Experimental Geriatrics, Neurotec, Karolinska Institute, 141 86 Huddinge, Sweden

Address correspondence to Sergei A. Grando, Dept. of Dermatology, University of California, Davis, 4860 Y St., #3400, Sacramento, CA 95817. Tel.: (916) 734-6057. Fax: (916) 734-6793. email: sagrando{at}

Abstract: 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 {alpha}5ß1, {alpha}Vß5, and {alpha}Vß6, whereas M3 up-regulates "sedentary" integrins {alpha}2ß1 and {alpha}3ß1. 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.

Key Words: integrins; cyclic GMP; cyclic AMP; knockout mice; acetylcholine

A.I. Chernyavsky and J. Arredondo contributed equally to this paper.

Abbreviations used in this paper: AC, adenylyl cyclase; ACh, acetylcholine; AGKOS, agarose gel keratinocyte outgrowth system; [Ca2+]i, intracellular-free calcium; cGMP, cyclic GMP; GC, guanylyl cyclase; GPCR, G protein–coupled receptor; IF, immunofluorescence; KC, keratinocyte; KGM, KC growth medium; KO, knockout; mAChR, muscarinic ACh receptor; PKA, protein kinase A; PKG, protein kinase G; ROK, Rho-associated protein kinase; siRNA, small interfering RNA; WT, wild-type.

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