Effects of inflammatory factors on cyclooxygenase expression in human gingival fibroblasts

我们之前已经证明，促炎细胞因子IL-1β诱导人牙龈成纤维细胞释放前列腺素E_2。本研究旨在探讨IL- 1β对人牙龈成纤维细胞环氧化酶-2表达的调控作用。环氧合酶-2是花生四烯酸转化为前列腺素的限速酶。直接早期基因环氧化酶-2编码一种可诱导的前列腺素合成酶，它与炎症和增生性疾病有关。环氧化酶-2在细胞活化过程中受到多种因素的高度诱导，包括有丝分裂原、激素和细胞因子。核因子κ b （NFκB）是炎症转录因子的主要激活因子，在环氧化酶-2的表达调控中起主要作用。由于促炎细胞因子IL-1β已被证明可诱导人牙龈成纤维细胞释放前列腺素E_2。我们分析了其对人牙龈成纤维细胞环氧化酶- 2a - More表达和nf - κ b活化的影响。Northern杂交分析显示，IL-1β增加了环氧化酶-2 mRNA的表达。蛋白酪氨酸激酶抑制剂herbyycin A可消除其作用，蛋白酪氨酸磷酸酶抑制剂原钒酸盐可增强其作用。il -1β诱导的前列腺素E_2释放被酪氨酸激酶抑制剂阻断，蛋白酪氨酸磷酸酶抑制剂增加。结合荧光素酶报告基因的人环氧化酶-2启动子（nt-1432 ~ +59）嵌合构建物瞬时转染实验结果表明，其转录活性可提高1.5倍。用放射性标记的环氧化酶-2 NFκB寡核苷酸（nst -223 ~ -214）进行凝胶迁移试验显示，il -1β刺激的人牙龈成纤维细胞的核蛋白结合增加。这种由IL-1β诱导的dna -蛋白复合物形成的增加被herbimycin A和另一种酪氨酸激酶抑制剂genisteine阻断。提示NFκB是il -1β诱导环氧合酶基因表达的重要转录因子，并通过酪氨酸磷酸化参与人牙龈成纤维细胞中环氧合酶-2基因转录的诱导。少

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