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Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
Published on: February 10, 2012
A receptor-mediated mechanism of nicotine toxicity in oral keratinocytes
J Arredondo1, V T Nguyen, A I Chernyavsky
1Department of Dermatology, School of Medicine, University of California, Davis 95817, USA.
Abstract:
Smoking and smokeless tobacco cause morbidity that originates from the epithelium lining of the skin and upper digestive tract. Oral keratinocytes (OKC) express nicotinic acetylcholine receptors (nAChRs) that bind nicotine (Nic). We studied the mechanism of the receptor-mediated toxicity of tobacco products on OKC. Preincubation of normal human OKC with Nic altered the ligand-binding kinetics of their nAChRs, suggesting that the nAChRs underwent structural changes. This hypothesis was confirmed by the finding that exposure of OKC to Nic causes transcriptional and translational changes. Through RT-PCR and immunoblotting, we found a 1.5- to 2.9-fold increase in the mRNA and protein levels of alpha3, alpha5, alpha7, beta2, and beta4 nAChR subunits. Exposure of OKC to Nic also changed the mRNA and protein levels of the cell cycle and cell differentiation markers Ki-67, PCNA, p21, cyclin D1, p53, filaggrin, loricrin, and cytokeratins 1 and 10. The nicotinic antagonist mecamylamine prevented these changes, which indicates that the Nic-induced changes in the expression of both the nAChR and the cell cycle and cell differentiation genes resulted from pharmacologic stimulation of nAChRs with Nic. To establish the relevance of these findings to the pathobiologic effects of tobacco products in vivo, we studied the above parameters in the oral tissue of rats and mice after their exposure for 3 weeks to environmental cigarette smoke or drinking water containing equivalent concentrations of Nic that are pathophysiologically relevant. The changes of the nAChRs and the cell cycle and cell differentiation genes were similar to those found in vitro. The results of indirect immunofluorescence assay of tissue specimens validated these findings. Thus, some pathobiologic effects of tobacco products in oral tissues may stem from Nic-induced alterations of the structure and function of keratinocyte nAChRs responsible for the physiologic regulation of the cell cycle by the cytotransmitter acetylcholine.
Insights
Nicotine in tobacco alters oral keratinocyte nicotinic acetylcholine receptors (nAChRs), affecting cell cycle and differentiation. These changes, observed in vitro and in vivo, contribute to tobacco-related oral tissue damage.
Area of Science:
- Cell Biology
- Toxicology
- Dermatology
Background:
- Tobacco use, including smoking and smokeless forms, is a significant cause of oral tissue morbidity.
- Oral keratinocytes (OKC) possess nicotinic acetylcholine receptors (nAChRs) that interact with nicotine (Nic).
Purpose of the Study:
- To investigate the mechanism of nicotine-induced toxicity on oral keratinocytes.
- To elucidate how nicotine affects nAChR structure, function, and gene expression in OKC.
- To determine the in vivo relevance of these findings in oral tissues.
Main Methods:
- Exposure of human OKC and rodent oral tissues to nicotine or environmental cigarette smoke.
- Analysis of nAChR subunit expression using RT-PCR and immunoblotting.
- Assessment of cell cycle and differentiation markers (e.g., Ki-67, p53, filaggrin) via molecular and immunofluorescence techniques.
- Pharmacological blockade of nAChRs using mecamylamine.
Main Results:
- Nicotine exposure altered nAChR ligand-binding kinetics and increased mRNA/protein levels of specific nAChR subunits (alpha3, alpha5, alpha7, beta2, beta4).
- Nicotine modulated the expression of cell cycle regulators (Ki-67, PCNA, p21, cyclin D1, p53) and differentiation markers (filaggrin, loricrin, cytokeratins).
- These nicotine-induced changes were prevented by the nAChR antagonist mecamylamine, confirming receptor mediation.
- Similar alterations in nAChRs and related genes were observed in rodent oral tissues exposed to cigarette smoke or nicotine.
Conclusions:
- Nicotine-induced structural and functional changes in keratinocyte nAChRs contribute to tobacco-related pathobiological effects in oral tissues.
- These alterations impact the regulation of cell cycle and differentiation pathways.
- The findings highlight a key mechanism underlying oral tissue damage from tobacco products.
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