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.
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.
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