EPITHELIAL ACETYLCHOLINE IN ORAL BIOLOGY AND PATHOLOGY

描述（由申请人提供）：要求提供资金，以支持我们正在进行的研究，以确定乙酰胆碱（ACh），其药理同源物和烟草产品对口腔角质形成细胞（OKC）介导作用的分子机制。角质形成细胞出生和死亡的连续循环是一个自我维持的过程，部分由局部激素ACh通过信号通路控制，该信号通路将每种类型的ACh受体与特定细胞功能的调节偶联。游离细胞递质ACh以生理相关浓度存在于上消化道上皮中。OKC表达ACh合成和降解酶，以及烟碱和毒蕈碱类ACh受体。在对被称为SLURP（分泌型哺乳动物Ly-6/尿激酶纤溶酶原激活物受体相关蛋白）-1和-2的胆碱能蛋白的研究中，已经发现了通过烟碱型ACh受体（nAChR）进行细胞调节的新范例。初步结果表明，SLURP-1和-2调节角质形成细胞增殖，凋亡和分化。最重要的是，SLURPs和专职烟碱拮抗剂可以部分消除烟碱衍生的亚硝胺4-（甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮（NNK）和N '-亚硝基去甲烟碱（NNN）引起永生化OKC转化的能力。我们将测试以下工作假设：1）NNK的病理生物学作用主要通过α 7和/或α 9 nAChR（s）介导，而NNN的病理生物学作用主要通过α 3-产生的nAChR（s）介导; 2）SLURP蛋白可以在体内和体外防止口腔细胞的亚硝胺依赖性转化，并消除角质形成细胞周期、生长和分化中的烟草/尼古丁依赖性改变; SLURP-1主要与NNK竞争结合同五聚体nAChR（s），SLURP-2主要与NNN竞争结合异五聚体nAChR（s），两种SLURP均干扰亚硝胺诱导的nAChR信号转导。具体目的是确定：1）角质形成细胞nAChR在介导烟草亚硝胺的病理生物学效应中的作用; 2）SLURP-1和-2在OKC免受烟草毒性的生理保护中的作用; 3）受体介导的信号传导机制介导SLURP-1和-2对OKC的作用。该应用的主要意义在于其目的在于阐明烟碱乙酰胆碱受体如何介导烟草衍生的亚硝胺的病理生物学效应以及SLURP如何防止亚硝胺的毒性作用。整合SLURPs和上皮乙酰胆碱轴的结构和功能信息将有助于更好地了解上消化道上皮的正常发育和功能。了解SLURP与亚硝胺对口腔角质形成细胞作用的药理学将有助于制定有效的预防计划，其中烟草产品的有害影响是预期的，甚至是消除的，通过特定烟碱乙酰胆碱受体的药理学配体作为解毒剂。

DESCRIPTION (provided by applicant): 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.