Funding is requested to support our ongoing studies toward identification of molecular mechanisms mediating effects of acetylcholine (ACh), its pharmacologic congeners and tobacco products on oral keratinocytes (OKC). The continuous cycle of keratinocyte birth and death is a self-sustained process controlled, in part, by the local hormone ACh through the signaling pathways that couple each type of ACh receptors to regulation of a particular cell function. Free cytotransmitter ACh is present in physiologically-relevant concentrations in the epithelium lining the upper digestive tract. OKC express both the ACh synthesizing and degrading enzymes, and both nicotinic and muscarinic classes of ACh receptors. A novel paradigm of cell regulation via nicotinic ACh receptors (nAChRs) has been discovered in studies of the cholinergic proteins termed SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)-1 and -2. Preliminary results indicate that SLURP-1 and -2 regulate keratinocyte proliferation, apoptosis and differentiation. Most importantly, SLURPs and professional nicotinic antagonists can abolish, in part, the abilities of the nicotine- derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) to cause transformation of immortalized OKC. We will test the following working hypotheses: 1) the pathobiologic effect of NNK is mediated predominantly via a7 and/or a9 nAChR(s), and that of NNN-via a3- made nAChR(s);2) SLURP proteins can prevent nitrosamine-dependent transformation of oral cells both in vivo and in vitro, and abolish tobacco/nicotine-dependent alterations in the keratinocyte cell cycle, growth and differentiation;and 3) SLURP-1 competes mainly with NNK for binding to the homopentameric nAChR(s) and SLURP-2-with NNN at the binding site of heteropentameric nAChR(s), and both SLURPs interfere with the nitrosamine-induced nAChR signaling.
The Specific Aims will be to determine: 1) the role of keratinocyte nAChRs in mediating the pathobiologic effects of tobacco nitrosamines;2) the roles for SLURP-1 and -2 in the physiologic protection of OKC from tobacco toxicity;and 3) the receptor-mediated signaling mechanisms mediating SLURP-1 and -2 actions on OKC. The primary significance of the application lies in its goal to elucidate how nicotinic acetylcholine receptors mediate pathobiologic effects of tobacco-derived nitrosamines and how SLURP can prevent the toxic effects of nitrosamines. Integrating structural and functional information about SLURPs and the epithelial acetylcholine axis will facilitate a better understanding of normal development and function of the epithelium lining the upper digestive tract. Learning the pharmacology of the SLURP vs. nitrosamine action on oral keratinocytes will help develop an effective prevention programs wherein hazardous effects of tobacco products are anticipated, or even abolished, by a pharmacologic ligand of a specific nicotinic acetylcholine receptors acting as an antidote.
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