Pro-inflammatory Mediator Crosstalk Induces COX-2 in Myofibroblasts via PKC/PKD

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DESCRIPTION (provided by applicant): Following the completion of my surgical training in General and Colorectal Surgery, I sought an academic position where I would have the best opportunity to achieve my long-term career goals of becoming an independently funded investigator, as well as an outstanding clinician and teacher. My life-long mentor, Dr. Jeffrey Matthews, guided my decisions in terms of choosing an academic environment where these goals would be strongly supported. I met with Dr. Enrique Rozengurt during the recruitment process, and his willingness to serve as my research mentor was a major factor in my decision to join the UCLA Department of Surgery in 2006. With the full support of my department, I have been working with Dr. Rozengurt since my arrival at UCLA. Under his guidance, I have laid the groundwork of a research proposal (this grant) that will serve as the foundation of an independent research program. With the additional guidance and expertise of Dr. Charalabos Pothoulakis, and the full support of my Department of Surgery under the leadership of Dr. Ronald Busuttil, I feel well positioned to succeed in my pursuit of a successful research career. Dr. Rozengurt, Dr. Pothoulakis, and Dr. Busuttil comprise my Advisory Committee, and with their help I have designed a Career Development Plan consisting of graduate level courses, weekly research seminars/lectures, formal laboratory meetings, and participation in national research conferences. My short-term goals are to gain expertise in areas of signal transduction and murine models of inflammation and colitis-associated cancer as I develop my own distinct focus of research. The association between chronic ulcerative colitis and the development of colorectal cancer is a well- established but incompletely understood phenomenon. Though the fundamental mechanism is not known, there has been a growing awareness of the contribution of the underlying intestinal stroma on inflammation-associated cancer. Colonic myofibroblasts are a subpopulation of cells located within the stroma of the GI tract that have been linked to both inflammation and tumorigenesis, and are a major source of cyclo-oxygenase 2 (COX-2), a critical enzyme also involved in these processes. However, the cell signaling pathways regulating COX-2 expression in myofibroblasts have not been well studied. Protein kinase D (PKD), the founding member of a new family of protein kinases that includes PKD2 and PKD3, has been implicated in important cellular functions but the expression, regulation and function of PKD in myofibroblasts, both in response to pro-inflammatory mediators and/or in the production of local regulators, has not been investigated. The central hypothesis of this proposal is that pro-inflammatory mediators activate a novel PKD phosphorylation cascade that plays a critical role in signal transduction pathways regulating colonic myofibroblast function. Using the human colonic myofibroblast cell line 18Co, our Preliminary Data demonstrate a synergistic upregulation of COX-2 protein expression following treatment with the inflammatory mediators TNF-( (TNF) and bradykinin (BK). This effect appears to be mediated by unique cross talk interactions between the distinct signaling pathways downstream of TNF and BK, leading to amplified PKD activation in direct association with a dramatic increase in COX-2 expression. The Specific Aims of this proposal are to 1) fully characterize the synergistic expression of COX-2 in response to pro-inflammatory mediators in colonic myofibroblasts, 2) identify mechanisms of signaling cross talk between pro-inflammatory mediators responsible for the synergy, 3) define the role of PKC/PKD signaling in the expression of COX-2 in myofibroblasts, and 4) substantiate the physiologic significance of these findings using a mouse model of colitis-associated cancer. Experimental results will also be confirmed using myofibroblast cells extracted from human colonic tissue taken at the time of surgery. Potential cross talk between TNF and BK in the synergistic enhancement of COX-2 expression in colonic myofibroblasts, mediated through PKD, may play an important role in colitis-associated cancer. PKD signaling may regulate key biological functions of myofibroblasts in the setting of inflammation.