Tumor necrosis factor α (TNF-α)-induced prostaglanding E2 release is mediated by the activation of cyclooxygenase-2 (COX-2) transcription via NFκB in human gingival fibroblasts

Nuclear factor kappaB (NFkappaB) is a transcription factor and plays a key role in the expression of several genes involved in the inflammatory process. Cyclooxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid synthetic pathway. COX-2 is a highly inducible enzyme by proinflammatory cytokines, of which gene expression is regulated by NFkappaB. TNF-alpha is a pro-inflammatory cytokine. In this paper, we investigated the involvement of NFkappaB on TNF-alpha-mediated prostaglandin E-2 (PGE(2)) release and COX-2 gene expression in human gingival fibroblasts (HGF). TNF-alpha- induced PGE(2) release and COX-2 mRNA accumulation in a time- and concentration-dependent manner in HGF. The results of transient transfection assays using a chimeric construct of the human COX-2 promoter (nts -1432 approximate to +59) ligated to a luciferase reporter gene indicated that TNF-alpha stimulated the transcriptional activity approximate to 1.4-fold. Gel mobility shift assays with a radiolabelled COX-2-NFkappaB oligonucleotide (nts -223 to -214) revealed an increase in the binding of nuclear proteins from TNF-alpha-stimulated HGF. The COX-2-NFkappaB DNA-protein complex disappeared after treatment with pyrrolidine dithiocarbamate (PDTC; an antioxidant) or herbimycin A (a tyrosine kinase inhibitor). PDTC and herbimycin A attenuated TNF-alpha-stimulated PGE(2) release. These results suggest that NFkappaB transcription factor is a key regulator of COX-2 expression in TNF-alpha-induced PGE(2) production, which is mediated through a tyrosine kinase pathway in HGF.