Regulation of Keratinocyte Migration by Acetylcholine

目的：了解表皮角质形成细胞(KC)如何控制自身的迁移功能，并在未来的临床研究中利用这一生理机制影响胆碱能药物的创面愈合。背景：KC类似于神经元，形成局部通讯网络，其中乙酰胆碱(ACh)通过激活不同类型的ACh受体(AChRs)来调节KC的重要功能，包括迁移。原理：角质形成细胞迁移是一个自我调节的过程，在这个过程中，介导迁移的细胞活动部分地由一个单独的“起搏器”系统控制，该系统以自分泌、旁分泌和旁分泌ACh作为细胞运动的趋化因子。不同类型的角质形成细胞烟碱型和毒酪碱型AChRs(nAChRs和mAChRs)分别介导了对介导迁移的细胞活动的不同控制。主要目的：了解每种角质形成细胞AChR类型在伤口再上皮化中的作用。工作假设：1)ACh对KC的趋化作用是通过激活α3nAChR介导的。2)M4mAChR促进KC的迁移，α7nAChR抑制迁移，α9nAChR控制爬行KC局部粘连的组装和拆解。具体目的：1)在使用受体选择性胆碱能激动剂和拮抗剂的实验中，鉴定介导KC向ACh梯度趋化的角质形成细胞AChRs，这些受体选择性胆碱能激动剂和拮抗剂将分别诱导或阻断从Alpha3、Alpha7、Alpha9 nAChR或M4 mAChR基因敲除小鼠培养的人KC和小鼠KC的定向迁移。2)确定每种AChR类型对介导KC在特定细胞外基质蛋白上迁移的基本细胞内事件的胆碱能控制的贡献。野生型和缺乏AChR的KC将用于通过nAChR和mAChR类型偶联的离子和代谢事件的药理学和分子生物学分析。所有实验都将在皮肤再上皮化的体外模型中进行，该模型允许对药物化合物进行随机研究，以及浓度梯度和直流电场诱导的KC定向迁移。我们将展示AChR特异性的胆碱能对爬行的KC的膜电位、细胞内钙、氢和cAMP水平的影响，以及肌动蛋白细丝聚合、局灶性粘连的组装/分解以及整合素受体的表达。意义：我们将展示nAChRs和mAChRs如何不同地调节KC在伤口愈合中的功能和迁移，这将为无法愈合的皮肤伤口的治疗提供新的解决方案。

Purpose: To understand how epidermal keratinocytes (KC) control their own migratory function and exploit this physiologic mechanism to affect wound closure with cholinergic drugs in future clinical studies. Background: KC, similarly to neurons, form local communication networks wherein acetylcholine (ACh) regulates vital functions of KC, including migration, by activating different types of ACh receptors (AChRs). Rationale: Keratinocyte migration is a self-regulated process in which cell activities mediating migration are controlled, in part, by a single "pace-maker" system featuring autocrine, juxtacrine and paracrine ACh as a chemokine for cell movement. The differential control of cellular activities mediating migration are mediated by different types of keratinocyte nicotinic and muscarinic AChRs (nAChRs and mAChRs), respectively. Major goal: To understand contribution of each keratinocyte AChR type to wound re-epithelialization. Working Hypotheses: 1) The chemotactic effect of ACh on KC is mediated by activation of alpha3 nAChR. 2) The m4 mAChR stimulates migration, alpha7 nAChR inhibits it, and alpha9 nAChR controls assembly/disassembly of focal adhesions in crawling KC. Specific Aims: 1) To identify keratinocyte AChRs that mediate chemotaxis of KC toward ACh gradient in experiments with receptor-selective cholinergic agonists and antagonists that will either elicit or block, respectively, directional migration of human KC and murine KC grown from the alpha3, alpha7, alpha9 nAChR or m4 mAChR knockout mice. 2) To identify contribution of each AChR type to cholinergic control of the basic intracellular events mediating migration of KC over specific extracellular matrix proteins. The wild type, and AChR-deficient KC will be used in pharmacologic and molecular biological assays of ionic and metabolic events coupled by nAChR and mAChR types. All experiments will be performed in an in vitro model of skin re- epithelialization that allows studies of pharmacologic compounds on random, and concentration gradient-and DC electric field- elicited directional migration of KC. We will demonstrate AChR- specific cholinergic effects on membrane potential, intracellular Ca2+, hydrogen, and cAMP levels, as well as actin filament polymerization, assembly/disassembly of focal adhesions, and integrin receptor expression in crawling KC. Significance: We will show how nAChRs and mAChRs differentially modulate the function and migration of KC in wound healing which will offer novel solutions for management of skin wounds that fail to heal.