Regulation of Keratinocyte Migration by Acetylcholine

目的：整合表皮角质形成细胞（KCs）的结构和功能信息， 上皮形成与胆碱能机制的知识指导KC通过上皮形成步骤。 基本原理：上皮化是一个自我调节的过程，其中细胞活动介导粘附， 迁移是由一个单一的"起搏器"系统控制的，该系统具有自分泌、阿曲他克林和 旁分泌乙酰胆碱（ACh）作为细胞运动的趋化因子。 背景：KCs通过两种不同类型的胆碱能信号同时刺激 途径：乙酰胆碱与G蛋白偶联毒蕈碱受体结合引起的代谢事件 （mAChR），以及由乙酰胆碱门控离子通道（或nAChR）介导的离子事件。这种多样性使得 单一细胞递质ACh对皮肤上皮形成的不同阶段的KCs发挥不同的作用。 工作假设：MI mAChR和a3| α 34和α 7 nAChR协同作用介导 趋化性通过钙粘蛋白磷酸化状态的Ca2+依赖性改变， 整合素M4 mAChR和a3 nAChR通过蛋白上调迁移整合素促进迁移 激酶（PK）CS依赖性途径，而α 9 nAChR促进细胞-细胞和细胞-细胞间的组装/分解。 衬底键合。含α S的<$3 nAChR增加细胞-细胞粘附的强度。M3弹性稳定 细胞-基质附着并由于Rho依赖性事件的失活而终止迁移。 具体目的：1）鉴定ACh受体亚型，其调节KCs的不同细胞活性， 正常上皮形成。2)确定ACh受体偶联的信号转导通路介导 选择性调节包括角质形成细胞迁移功能的细胞活性。 方法：采用分子生物学方法（实时荧光定量PCR;小干扰RNA; ACh受体）， 基因敲除小鼠）、免疫学（免疫组织化学、蛋白质印迹、ELISA、流式细胞术）和 将采用药理学方法阐明上皮形成的胆碱能调节， 角质形成细胞趋化性、趋化作用以及细胞-细胞和细胞-基质附着的测定。 意义：这项研究的结果将为设计能够治疗糖尿病的治疗方案奠定基础。 由不正常的上皮形成引起的皮肤病。 描述：最近的研究表明，局部产生和释放的类胡萝卜素分子， ACh可以启动、引导和维持伤口床中的皮肤细胞运动。这使得乙酰胆碱和药物作用于 乙酰胆碱受体是伤口愈合的潜在有力工具。这项研究将确定潜在的治疗 作用于特定类型ACh受体以激活信号通路的胆碱能药物中的药物 在伤口上皮形成过程中介导角质形成细胞的功能。

Purpose: To integrate structural and functional information about epidermal keratinocytes (KCs) during epithelialization with the knowledge on cholinergic mechanisms guiding KCs through epithelialization steps. Rationale: Epithelialization is a self-regulated process in which cell activities mediating adhesion and migration are controlled, in part, by a single "pace-maker" system featuring autocrine, juxtacrine and paracrine acetylcholine (ACh) as a chemokine for cell motility. Background: KCs are simultaneously stimulated through two distinct types of cholinergic signaling pathways: the metabolic events, elicited by ACh binding to G protein-coupled muscarinic receptors (mAChRs), and the ionic events, mediated by ACh-gated ion channels (or nAChRs). This diversity allows the single cytotransmitter ACh to exert diverse effects on KCs at various stages of skin epithelialization. Working Hypotheses: The MI mAChR and the a3|34 and a7 nAChRs act synergistically in mediating chemotaxis via Ca2+-dependent alterations in the phosphorylation status of cadherins and sedentary integrins. M4 mAChR and a3 nAChR facilitate migration by upregulating migratory integrins through protein kinase (PK) CS-dependent pathway, whereas a9 nAChR prompts assembly/disassemblyof cell-cell and cell- substrate bonds. The aS-containing ¿3 nAChR increases the strength of cell-cell adhesion. M3 elicits stable cell-substrate attachment and terminates migration due to inactivation of the Rho-dependent events. Specific Aims: 1) Identify ACh receptor subtypes regulating distinct cellular activities of KCs required for normal epithelialization. 2) Identify the ACh receptor-coupled signal transduction pathways mediating selective regulation of cellular activities comprising the keratinocyte migratory function. Methodology: A combination of molecular biological (real-time PCR; small interfering RNA; ACh receptor knockout mice), immunological (immunohistochemistry, Western blot, ELISA, flow cytometry), and pharmacological approaches will be employed to elucidate cholinergic regulation of epithelialization, using the assays of keratinocyte chemotaxis, chemokinesis, and cell-cell and cell-substrate attachments. Significance: Results of this study will lay a groundwork for designing therapeutic regimens able to treat the skin disease caused by abnormal epithelialization. DESCRIPTION: Recent research shows that the locally produced and released hormone-like molecule, ACh, may initiate, guide, and sustain skin cell motility in the wound bed. This makes ACh and drugs acting at the ACh receptors potentially powerful tools for wound healing. This study will identify potential therapeutic agents among cholinergic drugs acting upon specific types of ACh receptors to activate signaling pathways mediating keratinocyte functions during wound epithelialization.