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044 PROTEINASE-ACTIVATED RECEPTORS INCREASE INTERLEUKIN-8 EXPRESSION IN COLONIC EPITHELIAL CELLS H Wang, F Moreau, WK MacNaughton Proteinase-activated receptors (PARs) are G protein-coupled receptors that are activated by serine proteinases such as thrombin, trypsin and tryptase, and which play important roles in the processes of inflammation and tumorigenesis. It has previously been shown that activation of PARs stimulates interleukin-8 (IL-8) expression in airway epithelial cells. IL-8 is a potent chemoattractant for neutrophils and is also involved in angiogenesis. We tested whether PARs stimulate IL-8 expression in colonic epithelial cells and sought to determine the mechanism whereby this occurred. HT29 colonic epithelial cells were exposed to the PAR1-activating peptide (AP), TFLLR-NH2 (50µM), or the PAR2-AP, SLIGRL-NH2 (50µM). The expression of IL-8 mRNA was determined by semi-quantitative RT-PCR, and IL-8 protein in the cell culture medium was measured by ELISA. Both the PAR1-AP and PAR2-AP increased the mRNA level of IL-8 by 2 hours and the release of IL-8 protein by 18 hours. An IL-8 promoter luciferase reporter (bp 135 to +46) assay revealed that PAR1- and PAR2-activating peptides caused a 3-fold increase in wild type reporter activity. Further experiments were conducted in which HT29 cells were transfected with IL-8 promoter constructs containing mutations of either nuclear factor-kappaB (NF-kappaB) or activator protein-1 (AP-1) activation sites. Both mutations completely blocked PAR1- and PAR2-induced IL-8 promoter activation in the luciferase assay. Further experiments, in which HT29 cells were transfected with a loss of function mutation of the CCAAT/Enhancer-binding Protein (C/EBP) site, showed a 50% reduction in PAR-induced IL-8 promoter luciferase activity. These data indicated that the binding of NF-kappaB and AP-1 elements are required for PAR1- and PAR2-induced transcriptional activation of IL-8. Activation of the C/EBP site on the IL-8 promoter plays a partial role.
Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta
In conclusion, activation of both PAR1 and PAR2 stimulates the transcriptional expression and release of IL-8 through NF-kappaB- and AP-1 transcription pathways in HT-29 cells. Therefore, this study provides evidence suggestive of a new role for serine proteinases, acting through PAR1 and PAR2, in colonic inflammation and tumorigenesis.
H Wang is supported by a CAG-CCFC Fellowship