Signal pathways JNK and NF-κB, identified by global gene expression profiling, are involved in regulation of TNFα-induced mPGES-1 and COX-2 expression in gingival fibroblasts

Background: Prostaglandin E-2 (PGE(2)) is involved in several chronic inflammatory diseases including periodontitis, which causes loss of the gingival tissue and alveolar bone supporting the teeth. We have previously shown that tumor necrosis factor a (TNF alpha) induces PGE(2) synthesis in gingival fibroblasts. In this study we aimed to investigate the global gene expression profile of TNF alpha-stimulated primary human gingival fibroblasts, focusing on signal pathways related to the PGE(2)-synthesizing enzymes prostaglandin E synthases (PGES), as well as the upstream enzyme cyclooxygenase-2 (COX-2) and PGE(2) production. Results: Microarray and western blot analyses showed that the mRNA and protein expression of the inflammatory induced microsomal prostaglandin E synthase-1 (mPGES-1) was up-regulated by the cytokine TNF alpha, accompanied by enhanced expression of COX-2 and increased production of PGE(2). In contrast, the expression of the isoenzymes microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNF alpha treatment. Using oligonucleotide microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, differentially expressed genes in response to TNF alpha treatment were identified. Enrichment analysis of microarray data indicated two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappa B (NF-kappa B). To evaluate their involvement in the regulation of mPGES-1 and COX-2 expression, we used specific inhibitors as well as phosphorylation analysis. Phosphorylation analysis of JNK (T183/Y185) and NF-kappa B p65 (S536) showed increased phosphorylation in response to TNF alpha treatment, which was decreased by specific inhibitors of JNK (SP600125) and NF-kappa B (Bay 11-7082, Ro 106-9920). Inhibitors of JNK and NF-kappa B also decreased the TNF alpha-stimulated up-regulation of mPGES-1 and COX-2 as well as PGE(2) production. Conclusion: In the global gene expression profile, the enrichment analysis of microarray data identified the two signal transduction pathways JNK and NF-kappa B as positively regulated by the cytokine TNF alpha. Inhibition of these TNF alpha-activated signal pathways reduced the expression of mPGES-1 and COX-2 as well as their end product PGE(2) in gingival fibroblasts. The involvement of the signal pathways JNK and NF-kappa B in the regulation of PGE(2) induced by TNF alpha may suggest these two pathways as possible attractive targets in the chronic inflammatory disease periodontitis.