Regulation of Keratinocyte Migration by Acetylcholine

乙酰胆碱对角质形成细胞迁移的调节

• 批准号: 7540218
• 负责人: SERGEI A GRANDO
• 金额: $ 27.02万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7540218
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acetylcholine; Adenylate Cyclase; Adhesions; Agonist; Antisense Oligonucleotides; Beds; Binding; Biochemical; Biological; Biological Assay; Biological Metamorphosis; Cadherins; Cell Maturation; Cell-Cell Adhesion; Cells; Chemotaxis; Choline; Cholinergic Agents; Cholinergic Receptors; Condition; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Data; Development; Enzyme-Linked Immunosorbent Assay; Enzymes; Epidermis; Event; Exhibits; Flow Cytometry; Funding; GTP-Binding Proteins; Gated Ion Channel; Glycoproteins; Guanylate Cyclase; Hormones; Human; Immigration; Immunohistochemistry; Individual; Integrins; Knockout Mice; Knowledge; Lateral; Locomotion; Mediating; Medical; Metabolic; Methodology; Molecular; Mus; Muscarinic Acetylcholine Receptor; Muscarinics; Nicotinic Agonists; Object Attachment; Paracrine Communication; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphorylation; Polymerase Chain Reaction; Process; Protein Kinase; Protein Kinase C; Purpose; Ras/Raf; Regulation; Research; Research Personnel; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Skin; Small Interfering RNA; Staging; System; Therapeutic; Therapeutic Agents; Time; Treatment Protocols; Western Blotting; Work; Wound Healing; autocrine; calmodulin-dependent protein kinase II; cell growth regulation; cell motility; chemokine; cholinergic; design; keratinocyte; migration; paracrine; receptor; receptor coupling; rho; sedentary; skin disorder; tool; wound

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.

目的：整合有关表皮角质形成细胞（KC）的结构和功能信息 上皮化借助有关胆碱能机制的知识，通过上皮化步骤引导KCS。 理由：上皮化是一个自我调节的过程，在这种过程中，细胞活动介导粘合剂和 迁移由一个单一的“ pace-maker”系统控制，该系统具有自分泌，近距离和 旁分泌乙酰胆碱（ACH）作为细胞运动的趋化因子。 背景：通过两种不同类型的胆碱能信号传导刺激KC 途径：代谢事件，通过与G蛋白偶联的毒蕈碱受体结合引起的代谢事件 （MACHR）和离子事件，由ACH门控离子通道（或NACHRS）介导。这种多样性允许 单细胞递质ACH在皮肤上皮化的各个阶段对KCS执行潜水员的影响。 工作假设：MI MACHR和A3 | 34和A7 NACHRS ACT在中介方面协同作用 通过Ca2+依赖性改变钙粘蛋白和久坐的磷酸化状态的趋化性变化 整合素。 M4 MACHR和A3 NACHR喜欢的迁移通过蛋白质上调迁移蛋白 激酶（PK）CS依赖性途径，而A9 NACHR提示细胞和细胞的组装/拆卸 - 底物债券。含有含量的3 nACHR增加了细胞 - 细胞粘附的强度。 M3引起稳定 由于Rho依赖性事件灭活，细胞基底附着并终止迁移。 具体目的：1）确定调节KCS所需的不同细胞活性的ACH受体亚型 正常上皮化。 2）确定介导的ACH受体耦合信号转导途径 填写角质形成细胞迁移功能的细胞活性的选择性调节。 方法论：分子生物学（实时PCR；小干扰RNA； ACH受体）的组合 敲除小鼠），免疫学（免疫组织化学，Western印迹，ELISA，流式细胞仪）和 将采用药理学方法来阐明上皮化的胆碱能调节，并使用 角质形成细胞趋化性，趋化因子以及细胞细胞和细胞基底附着的测定。 意义：这项研究的结果将为设计治疗方案的基础奠定基础。 由异常上皮化引起的皮肤疾病。 描述：最近的研究表明，本地生产和释放的类似骑马的分子， ACH可以在伤口床上启动，指导和维持皮肤细胞运动。这使ACH和毒品作用 ACH受体可能具有伤口愈合的功能强大的工具。这项研究将确定潜在的治疗 作用于特定类型ACH受体的胆碱能药物中的药物激活信号通路 在伤口上皮化过程中介导角质形成细胞的功能。