Receptor-mediated tobacco toxicity: Regulation of gene expression through α3β2 nicotinic receptor in oral epithelial cells

Juan Arredondo, Alexander I. Chernyavsky, Lisa M. Marubio, Arthur L. Beaudet, David L. Jolkovsky, Kent E Pinkerton, Sergei A. Grando

Research output: Contribution to journalArticle

70 Scopus citations

Abstract

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-α3 small interfering RNA or treated with, the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3-/- 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 α3+/+ and α3-/- 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 α3β2 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 α3β2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-κB, and STAT-1. The expression of the nAChR subunits α5 and β2 and the muscarinic receptor subtypes M2 and M3 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.

Original languageEnglish (US)
Pages (from-to)597-613
Number of pages17
JournalAmerican Journal of Pathology
Volume166
Issue number2
StatePublished - Feb 2005

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

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    Arredondo, J., Chernyavsky, A. I., Marubio, L. M., Beaudet, A. L., Jolkovsky, D. L., Pinkerton, K. E., & Grando, S. A. (2005). Receptor-mediated tobacco toxicity: Regulation of gene expression through α3β2 nicotinic receptor in oral epithelial cells. American Journal of Pathology, 166(2), 597-613.