Ulcerative colitis is characterized by both an acute and chronic inflammatory infiltrate that elaborates a complex array of inflammatory mediators and cytokines. Although the initiating pathogenic factor in this disease process remain unknown, it is generally agreed that the release of pro-inflammatory mediators is responsible, to a large extent, for the tissue damage in this disease. In the regard, Interleukin-8 (IL- 8) is a pro-inflammatory cytokine that has potent chemoattractive and activating function for neutrophils and is produced by a variety of cell types including various epithelial cells. It has recently been demonstrated that transcriptional activation of multiple cytokine genes, including IL-8, immunoreceptors, and other substances important in the modulation of the inflammatory response are regulated by critical nuclear transcription factors such as nuclear factor Kappa B (NFkB) and nuclear factor IL 6 (NF-IL 6). An emerging paradigm suggests that activation of NFkB and/or NF-IL 6 in concert with other transcription factors allows the expression of inflammatory cytokines by epithelial cells. In preliminary data, I demonstrate that IL-8 mRNA can be induced by IL-1beta in the colon cancer cell line Caco-2. Furthermore, activation of NFkB is shown to play a critical role in the transcriptional regulation of the IL-8 gene in this cell line. These data from the basis for the following related hypotheses: 1) The transcriptional regulation of the IL 8 gene may be used as a model to identify nuclear transcription factors that play a central role in the expression and induction of inflammatory modulators in Caco-2 cells, and 2) Activation of NFkB and other nuclear factors are required for the transcriptional activation of the IL-8 gene in the colonic epithelium in vivo. Therefore, the overall goal of this grant is to study the activation and functional effect of nuclear transcription factors responsible for regulating IL-8 gene transcription in the normal and inflamed colonic epithelium. In light of the central role that NFkB plays in the transcriptional regulation of multiple inflammatory mediators, initial studies will characterize both the structural and functional aspects of this transcription factors in the Caco-2 cell line. The ability of NFkB to transcriptionally activate genes in the context of other transcription factors such as NF-IL 6 will then be examined by characterizing the IL-8 promoter in vitro. Finally, results of the Caco-2 cell culture model will then be validated in a murine model of colonic inflammation using both normal and transgenic mice. Elucidation of factors that play a central role in the transcriptional regulation of inflammatory cytokines in the colonic epithelium may provide insight into the pathogenesis and the perpetuation of the inflammatory response in ulcerative colitis.
