This proposal describes a 5 year training program for the development of an academic career in Cardiovascular Transcriptional Biology. The principal investigator has scientific background with expertise in cellular immunology. He will now embark upon a program which is particularly designed to expand his scientific experience, such that he develops the skills required for an independent research career. This program will provide in-depth knowledge and experience in molecular biology, in particular transcriptional regulation, and in transgenic animal studies. Dr. Mukesh K. Jain, MD, will mentor the principal investigator's scientific development. Dr. Jain has a very active laboratory and is the Director of Cardiovascular Research Institute, Case Western Reserve University, Cleveland OH. He has trained several research fellows. The candidate's training will be enhanced by the collaboration with Prof. Diane Mathis, PhD - an expert in the field of arthritis and inflammation research. The project is designed to determine the effect of the transcription factor KLF2 on monocyte activation and function. Preliminary data demonstrate that this factor is expressed in monocytes, and reduced upon cytokine activation or differentiation. Furthermore, forced overexpression of KLF2 in monocytes inhibits the LPS-mediated induction of proinflammatory factors, cytokines and chemokines and reduced phagocytosis. Conversely, short interfering RNA-mediated reduction in KLF2 increased inflammatory gene expression. Mechanistically, KLF2 inhibits the transcriptional activity of both NF-kB and activator protein-1 in part by means of recruitment of transcriptional coactivator p300/CBP-associated factor, PCAF. These observations implicate KLF2 as a novel negative regulator of monocytic activation. The proposed experiments in this application will build on these initial observations and explore them in depth - both mechanistically and in models of arthritis in vivo. Studies will include a detailed analysis of effect on target genes with KLF2 overexpression (by adenoviral overexpression and transgenic models) and siRNA-mediated 'knockdown'approaches. KLF2 transgenic mice will be studied to assess the effects of this factor on the development of arthritis in vivo. Proposal was made to generate acute/ chronic arthritis in KLF2 transgenic mice and will be determined their biological consequences by evaluating arthritis development, macrophage recruitment, immunohistochemistry and gene expression during arthritis development. The monocyte is a key cell type in the regulation of the body's inflammatory response. The applicant's identification of the factor KLF2 as a key regulator of monocyte activation may provide the basis for novel therapies directed at limiting inflammation for a broad spectrum of disease states including arthritis.
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