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J. Biol. Chem., Vol. 280, Issue 47, 39220-39228, November 25, 2005

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The Ras/Raf-1/MEK1/ERK Signaling Pathway Coupled to Integrin Expression Mediates Cholinergic Regulation of Keratinocyte Directional Migration^*

Alexander I. Chernyavsky, Juan Arredondo, Evert Karlsson, Ignaz Wessler¶, and Sergei A. Grando1

From the Department of Dermatology, University of California, School of Medicine, Davis, California 95616, the Section of Experimental Geriatrics, NEUROTEC, Karolinska Institute, 141 86 Huddinge, Sweden, and the ^¶Institute of Pathology, University Hospital, Johannes Gutenberg University of Mainz, D-55101 Mainz, Germany

The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell re-orientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by high-performance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of 7 nicotinic and M₁ muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from 7 and M₁ through the MEK1/ERK was an up-regulated expression of ₂ and ₃ integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, 7 works together with M₁ to orient a keratinocyte toward direction of its future migration. Both 7 and M₁ apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge.

Received for publication, April 21, 2005 , and in revised form, August 8, 2005.

^* This work was supported by the National Institutes of Health Grants GM62136 and DE14173 and by a research grant from the Flight Attendant Medical Research Institute to (to S. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Dermatology, University of California, Davis, UC Davis Medical Center, 4860 Y St., 3400, Sacramento, CA 95817. Tel.: 916-734-6057; Fax: 916-734-6793; E-mail: sagrando{at}ucdavis.edu.

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