Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

Abstract

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 α-conotoxins MII and AuIB, preferentially blocking α3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the α3β2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of α3β2 included activation of the protein-kinase-C isoform δ and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of α7 nicotinic acetylcholine receptor. The α7-preferring antagonist α-bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the α7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-α7 antisense oligonucleotides and α7-knockout mice. The signaling pathway mediating α7-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 α7 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 β1. The obtained results demonstrate for the first time that α3 and α7 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.