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HISTONE ACETYLTRANSFERASE IS INVOLVED IN PROTEINASE-ACTIVATED RECEPTOR (PAR)-INDUCED INTERLEUKIN-8 EXPRESSION AND NF-kappaB ACTIVATION IN COLONIC EPITHELIAL CELLS
H Wang, F Moreau, WK MacNaughton
Inflammation Research Network, University of Calgary, Calgary, Alberta
BACKGROUND: 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 inflammation and tumorigenesis. IL-8 is a potent chemoattractant for neutrophils and is also involved in angiogenesis. We determined (a) whether activation of PARs stimulates IL-8 expression in colonic epithelial cells and (b) the cellular mechanism whereby this occurs.
METHODS & RESULTS: HT-29 colonic epithelial cells were exposed to the PAR1-activating peptide (AP), TFLLR-NH2 (50 µM), or the PAR2-AP, SLIGRL-NH2 (50 µM). The expression of IL-8 mRNA was determined by semi-quantitative RT-PCR, and IL-8 protein secretion into the culture medium was measured by ELISA. Both the PAR1-AP and PAR2-AP increased the mRNA level of IL-8 by 2 hr and the release of IL-8 protein by 18 hr. An IL-8 promoter luciferase reporter (bp –135 to +46) assay revealed that PAR1-AP and PAR2-AP caused a 3-fold increase in wild type reporter activity. Elevation of NF-kappaB-driven luciferase activity by PAR activation implied that NF-kappaB mediated PAR-induced IL-8 upregulation. Moreover, mutation of the NF-kappaB binding site completely blocked PAR1- and PAR2-induced IL-8 promoter activation in the luciferase assay. Unlike classical NF-kappaB activation pathways, there was no change in IkappaB phosphorylation, degradation and disassociation from p65 after PAR activation. Further studies showed that PAR activation increased the phosphorylation of p65 and also the binding of p65 and p300, a well-known histone acetyltransferase (HAT).
CONCLUSIONS: Activation of PAR1 and PAR2 stimulates the transcriptional expression and release of IL-8 through a HAT-mediated NF-kappaB transcription pathway in HT-29 cells. Thus, 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