Receptor-mediated tobacco toxicity: regulation of gene expression through alpha3beta2 nicotinic receptor in oral epithelial cells.

Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-alpha3 small interfering RNA or treated with the alpha3beta2-preferring antagonist alpha-conotoxin MII. Functional inactivation of alpha3-mediated signaling in alpha3-/- mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal alpha3+/+ and alpha3-/- littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified alpha3beta2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by alpha3beta2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-kappaB, and STAT-1. The expression of the nAChR subunits alpha5 and beta2 and the muscarinic receptor subtypes M(2) and M(3) was also altered. This novel mechanism offers innovative solutions to ameliorate the tobacco-related cell damage and intercede in disease pathways, and may shed light on general mechanisms regulating and driving tobacco-related morbidity in human cells.

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

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.