Mechanisms Governing Monocyte Recruitment during Periodontal Inflammation

描述(申请人提供)：炎症是牙周疾病的主要诱因，了解控制炎症反应的分子机制是合理设计抗击疾病的治疗方法的必要先导。我们的总体目标是发现调节单核细胞募集以促进炎症的特定信号通路，从而促进牙周病的发展和进展。趋化因子是单核细胞在体内运输的分子向导，肌动蛋白、细胞骨架和整合素黏附是响应趋化因子信号影响单核细胞迁移的分子机制。在我们的研究中，我们正在研究一种与牙周病相关的趋化因子CX3CL1是如何通过细胞骨架调节因子ROCK来促进单核细胞从循环中招募到牙周组织的。在这项提案中，我们从两个方面探讨了这一信号通路。首先，我们在问CX3CL1促进的黏附是否是由岩石活动的时间调节所介导的。我们的初步数据显示，CX3CL1和ROCK的抑制都能促进单核细胞与内皮细胞黏附分子的黏附。已知趋化因子调节ROCK及其上游Rho GTP酶激活剂，尽管特定的Rho GTP酶对CX3CL1的反应动力学尚未被研究。其目的是验证CX3CL1信号通过不同的Rho GTP酶瞬时抑制ROCK并促进单核细胞与内皮细胞黏附分子黏附的假设。其次，我们正在研究ROCK信号的新机制，通过询问ROCK如何与潜在的下游效应因子Coronin1A相互作用。我们已经通过蛋白质组学的方法确定了Coronin1a是一种可能的岩石相互作用蛋白，最近对Coronin1a在白细胞中的功能的研究揭示了Coronin1a在体内白细胞黏附、扩散和运输中的重要作用。ROCK和Coronin1A的功能重叠，结合我们的蛋白质组学结果，强烈表明这两个蛋白质之间存在以前未知的功能相互作用。这一目标的目的将是确定ROCK是否直接与Coronin1a结合，或者Coronin1a是否为ROCK的底物。我们预计，目标1的结果将提供所需的数据，将CX3CL1、岩石和粘附力之间的关联关系转换为直接的机械关系。同样，虽然ROCK和Coronin1A之间的相互作用是令人信服的，但目标2中的实验是必要的，以提供关键的初步数据来直接测试它们的相互作用。总之，这些AIMS的结果将提供必要的初步数据，以支持成功的R01应用程序，研究CX3CL1通过抑制ROCK和随后激活Coronin1A诱导黏附而触发单核细胞募集的假设。 公共卫生相关性：炎症是牙周疾病的一个重要因素，开发对抗病理性炎症反应的策略是口腔健康研究人员的一个关键目标。我们的研究旨在了解控制牙周炎症的分子机制，为合理开发治疗干预措施提供必要的信息。

DESCRIPTION (provided by applicant): Inflammation is a major contributor to periodontal disease, and understanding the molecular mechanisms that govern the inflammatory response is a necessary precursor to the rational design of therapeutics to combat disease. Our overall objective is to discover specific signaling pathways that regulate monocyte recruitment to promote inflammation, and thereby contribute to the development and progression of periodontal disease. Chemokines are the molecular guides for monocyte trafficking throughout the body & the actin cytoskeleton and integrin adhesion are the molecular machinery that respond to the chemokine signals to effect monocyte migration. In our study, we are investigating how a chemokine associated with periodontal disease, CX3CL1, signals through the cytoskeletal regulator, ROCK, to promote monocyte recruitment from the circulation into periodontal tissue. In this proposal, we are pursuing two aspects of this signaling pathway. First, we are asking if CX3CL1-promoted adhesion is mediated by temporal regulation of ROCK activity. Our preliminary data shows that CX3CL1 and inhibition of ROCK both promote adhesion of monocytes to endothelial adhesion molecules. Chemokines are known to regulate ROCK and its upstream Rho GTPase activators, although the kinetics of specific Rho GTPases in response to CX3CL1 have not been investigated. The purpose of this aim will be to test the hypothesis that CX3CL1 signals through distinct Rho GTPases to transiently inhibit ROCK and promote monocyte adhesion to endothelial adhesion molecules. Second, we are investigating novel mechanisms of ROCK signaling by asking how ROCK interacts with a potential downstream effector, Coronin1A. We have identified Coronin1A as a putative ROCK interacting protein through a proteomics-based approach, and recent studies of Coronin1A function in leukocytes reveal an important role for Coronin1A in leukocyte adhesion, spreading and trafficking in vivo. The functional overlap of ROCK and Coronin1A, in combination with our proteomics results, strongly suggests a previously unrecognized functional interaction between these two proteins. The purpose of this aim will be to determine if ROCK binds to Coronin1A directly, or if Coronin1A is a substrate of ROCK. We anticipate that the results of Aim 1 will provide the data needed to convert our correlative relationship between CX3CL1, ROCK and adhesion into a direct mechanistic relationship. Similarly, while the case for interaction between ROCK and Coronin1A is compelling, the experiments in Aim 2 are necessary to provide the critical preliminary data to directly test their interaction. Together, the results of these aims will provide the necessary preliminary data needed to support a successful R01 application investigating the hypothesis that CX3CL1 triggers monocyte recruitment by inducing adhesion mediated by inhibition of ROCK and subsequent activation of Coronin1A. PUBLIC HEALTH RELEVANCE: Inflammation is a large contributor to periodontal disease, and developing strategies to combat pathological inflammatory reactions is a key goal for oral health investigators. Our studies are designed to understand the molecular mechanisms that govern periodontal inflammation, which will provide necessary information for rational development of therapeutic interventions.