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First published online 19 October 2004
doi: 10.1242/jcs.01492
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Research Article |
1 Department of Dermatology, University of California Davis, 4860 Y Street, Sacramento, CA 95817, USA
2 Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
* Author for correspondence (e-mail: sagrando{at}ucdavis.edu)
Accepted 16 August 2004
Nicotinergic agents can act as both chemokines and chemoattractants for cell migration. Epidermal keratinocytes both synthesize acetylcholine and use it as a paracrine and autocrine regulator of cell motility. To gain a mechanistic insight into nicotinergic control of keratinocyte motility, we determined types of nicotinic acetylcholine receptors and signaling pathways regulating keratinocyte chemokinesis and chemotaxis, using respective modifications of the agarose gel keratinocyte outgrowth assay. Random migration of keratinocytes was significantly (P<0.05) inhibited by hemicholinum-3, a metabolic inhibitor of acetylcholine synthesis, as well as by the 
-conotoxins MII and AuIB, preferentially blocking 3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the 3ß2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of 3ß2 included activation of the protein-kinase-C isoform 
and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of 7 nicotinic acetylcholine receptor. The 7-preferring antagonist -bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the 7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-7 antisense oligonucleotides and 7-knockout mice. The signaling pathway mediating 7-dependent keratinocyte chemotaxis included intracellular calcium, activation of calcium/calmodulin-dependent protein-kinase II, conventional isoforms of protein-kinase C, phosphatidylinositol-3-kinase and engagement of Rac/Cdc42. Redistribution of 7 immunoreactivity to the leading edge of keratinocytes upon exposure to a chemoattractant preceded crescent shape formation and directional migration. Application of high-resolution deconvolution microscopy demonstrated that, on the cell membrane of keratinocytes, the nicotinic acetylcholine receptor subunits localize with the integrin ß1. The obtained results demonstrate for the first time that 3 and 7 nicotinic acetylcholine receptors regulate keratinocyte chemokinesis and chemotaxis, respectively, and identify signaling pathways mediating these functions, which has clinical implications for wound healing and control of cancer metastases.
Key words: Keratinocytes, Chemokinesis, Chemotaxis, Acetylcholine, Nicotinic receptors 3 and 7, Knockout mice, Antisense oligonucleotides
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