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 alpha-conotoxins MII and AuIB, preferentially blocking alpha3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the alpha3beta2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of alpha3beta2 included activation of the protein-kinase-C isoform delta and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of alpha7 nicotinic acetylcholine receptor. The alpha7-preferring antagonist alpha-bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the alpha7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-alpha7 antisense oligonucleotides and alpha7-knockout mice. The signaling pathway mediating alpha7-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 alpha7 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 beta1. The obtained results demonstrate for the first time that alpha3 and alpha7 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.
Highlights
Lateral migration of eukaryotic cells is central to many important biological processes, such as embryogenesis, angiogenesis, metastasis, inflammation and wound healing
We investigated effects of functional inhibition of different KC nAChR types by pharmacological antagonists, antisense oligonucleotides (AsOs) and null mutation of the nAChR subunit gene in receptor knockout (KO) mice on random and directional migration using the chemokinesis and chemotaxis modifications of the agarose gel KC outgrowth system (AGKOS) assay, respectively
Significantly differ from those of KCs transfected with AsOsβ2 alone (P>0.05) (Fig. 2D). These results indicated that α3 nAChR and α7 nAChR play opposing roles in nicotinergic control of KC chemokinesis
Summary
Lateral migration of eukaryotic cells is central to many important biological processes, such as embryogenesis, angiogenesis, metastasis, inflammation and wound healing (for reviews, see Firtel and Chung, 2000; Lauffenburger and Horwitz, 1996). Various kinds of chemical stimuli can modulate random cell migration (chemokinesis), some of which have been shown to stimulate directional migration (chemotaxis) (Lauffenburger and Horwitz, 1996; Mitchison and Cramer, 1996). Many chemoattractants are ligands for G protein-coupled receptors (for a review, see Song and Zhong, 2000). It has become evident that many of the intracellular biochemical events mediating chemotaxis can occur in the absence of functional G-proteins, suggesting a role for Gprotein-independent signaling mechanisms (Devreotes and Janetopoulos, 2003). Activation of ligand-gated ion channels has been shown to affect both chemokinesis and chemotaxis. An agonist of acetylcholine (ACh)-gated ion channels or nicotinic ACh
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