Effects of inflammatory factors on cyclooxygenase expression in human gingival fibroblasts

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We have previously demonstrated that pro-inflammatory cytokine, IL-1β induced prostaglandin E_2 release in human gingival fibroblasts. In this research project, we investigated the regulation of cyclooxygenase-2 expression induced by IL- 1β in human gingival fibroblasts. Cyclooxygenase-2 is a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. The immediate early gene cyclooxygenase-2 encodes an inducible prostaglandin synthase enzyme, which is implicated in inflammatory and proliferative disease. Cyclooxygenase-2 is highly induced during cell activation by various factors, including mitogens, hormones, and cytokines. Nuclear factor kappaB (NFκB) is a major activator of inflammatory transcription factor, appears to play a primary role in the regulation of cyclooxygenase-2 expression. Since pro-inflammatory cytokine IL-1β has been shown to induce prostaglandin E_2 release in human gingival fibroblasts. We analyzed the effect of on expression of cyclooxygenase-2 a … More nd activation of NFκB in human gingival fibroblasts. Northern hybridization analysis revealed that IL-1β increased the expression of cyclooxygenase-2 mRNA.The effects of was abrogated by herbimycin A, a protein tyrosine kinase inhibitor, and enhanced by orthovanadate, a protein tyrosine phosphatase inhibitor. IL-1β-induced prostaglandin E_2 release was blocked by the tyrosine kinase inhibitor and increased by the protein tyrosine phosphatase inhibitor. The results of transient transfection assays using chimeric constructs of the human cyclo-oxygenase-2 promoter (nt-1432〜+59) ligated to a luciferase reporter gene indicated that stimulated the transcriptional activity 〜 1.5-fold. Gel mobility shift assay with radiolabelled cyclooxygenase-2 NFκB oligonucleotide (nst -223 to -214) revealed an increase in the binding of nuclear proteins from IL-1β-stimulated human gingival fibroblasts. This increase of DNA-protein complex formation induced by IL-1β was blocked by herbimycin A and another tyrosine kinase inhibitor, genisteine.These results suggests that NFκB is an important transcription factor for IL-1β-induced cyclooxygenase gene expression, and involved in inducing cyclooxygenase-2 gene transcription through tyrosine phosphorylation in human gingival fibroblasts. Less