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.

Neuronal nicotinic alpha7 receptors modulate inflammatory cytokine production in the skin following ultraviolet radiation

“…Further, UVR exposure is a common and wellcharacterized method of inducing inflammation in the skin, and we have employed this method for many years to study this effect Gahring et al, 1984). Keratinocyte expression of nAChR has been demonstrated to have important roles in keratinocyte adhesion and motility (Chernyavsky et al, 2004;Grando et al, 1995;Zia et al, 1997), differentiation (Arredondo et al, 2002;Grando et al, 1996), apoptosis (Arredondo et al, 2003;Nguyen et al, 2001), and certain components of inflammation (Kalayciyan et al, 2007;Kurzen et al, 2007).…”

The Roles of Growth Factors in Keratinocyte Migration

“…102 Activation of the a3 nAChR induces KC migration via PKCd and RhoA, activation of a7 nAChR induces KC migration via PI3K and Rac/Cdc42, and activation of a9 nAChR induces KC migration both in vitro and in vivo by modulating cell-cell and cell-ECM adhesion. 103,104 The a7 nAChR and M1 mAChR enable KCs to sense ACh gradients, which initiates polarization of the cell and expression of a 2 and a 3 integrins at the leading edge of the cell via activation of the Ras/Raf-1/MEK1/ERK pathway, resulting in the stabilization of lamellipodia at the leading edge during the initial steps of AChmediated chemotaxis. 103 Lastly, activation of the mAChR M4 induces KC migration both in vitro and in vivo by upregulating the ''migratory'' integrins a 5 b 1 , a v b 5 , and a v b 6 , and activation of the mAChR M3 inhibits KC migration both in vitro and in vivo by upregulating the ''sedentary'' integrins a 2 b 1 and a 3 b 1 .…”

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

“…ACh and its congeners are chemotactic for KCs, neurons, and other cell types (2)(3)(4)(5)(6)(7)(8). To characterize the physiologic control of keratinocyte migration, we developed an in vitro model of skin epithelialization, termed the agarose gel keratinocyte outgrowth system (AGKOS) (9).…”

“…To characterize the physiologic control of keratinocyte migration, we developed an in vitro model of skin epithelialization, termed the agarose gel keratinocyte outgrowth system (AGKOS) (9). Using AGKOS, we have demonstrated that activation of the keratinocyte nicotinic ACh receptors (nAChRs) comprised by ␣7 subunits exhibit reciprocal effects on cell motility by stimulating directional (chemotaxis) and inhibiting random (chemokinesis) migration and that ACh-gated ion channels containing ␣3 subunit stimulate chemokinesis of KCs (8).…”

“…: 916-734-6057; Fax: 916-734-6793; E-mail: sagrando@ucdavis.edu. 2 The abbreviations used are: KC, keratinocyte; GW5074, 5-iodo-3-[ (3,5- Redistribution of ␣7 immunoreactivity to the leading edge of KCs upon exposure to a cholinergic chemoattractant precedes crescent shape formation and directional migration (8), suggesting that accumulation of ␣7 nAChRs is required for lamellipodium formation. The signaling downstream from ␣7 nAChR can proceed via several pathways.…”