Regulation of prostaglandin E2 synthesis in human gingival fibroblasts

Abstract: Prostaglandins, especially, prostaglandin E2 (PGE2) play an important role in inflammatory diseases such as rheumatoid arthritis and periodontitis. The aim of the present thesis was to study the production and the regulation of PGE2 with special reference to the key enzymes phospholipase A2 (PLA2) and cyclooxygenase (COX) at the rnRNA level in human gingival fibroblasts. Reverse transcription polymerase chain reaction (RT-PCR) and solution hybridization techniques were used to study the expression of these genes. The cytokines interleukin-1 (IL-1), tumor necrosis factor [alpha] (TNF[alpha]), and to a lesser extent, epidermal growth factor (EGF) stimulated PGE2 production in human gingival fibroblasts. The combination of IL-1ß and TNF[alpha] as well as IL-1 and EGF synergistically stimulated the production of prostaglandins (PGE2 and PGI2). IL-1ß as well as TNF[alpha] induced the rnRNA expression of cPLA2 (85 kDa) in gingival fibroblasts whereas the rnRNA expression of sPLA2 (14 kDa) was not detected. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced expression of cPLA2 rnRNA, accompanied by a corresponding increase in PGE2 production. The cytokines IL-1, TNF[alpha] and, to a lesser extent EGF induced COX-2 rnRNA levels. The combination of IL-1ß and TNF[alpha] as well as IL-1ß and EGF synergistically increased the expression of COX-2 rnRNA. These cytokines neither alone nor in combination affected COXI rnRNA levels. The phorbol ester PMA induced COX-2 rnRNA expression as well as potentiated the stimulatory effect of IL- I ß and/or TNF[alpha] on COX-2 rnRNA, accompanied by a corresponding increase in PGE2 production. Moreover, the inhibitors of tyrosine kinases, herbimycin A and PD 153035, reduced COX-2 rnRNA expression induced by cytokines whereas COX-1 rnRNA level was not affected. The anti-inflammatory drugs, dexamethasone (DEX), indomethacin (INDO) and NS-398 abolished the cytokine-induced PGE2 production in gingival fibroblasts. In addition DEX also blocked the cytokine-induced cPLA2 and COX-2 rnRNA expression. In conclusion, this thesis demonstrates that the cytokine-stimulated PGE2 production is mediated by cPLA2 and COX-2 rnRNA expression. In addition, the cytokines act in concert to synergistically stimulate PGE2 partly due to enhanced gene expression of COX-2 and that the enzyme PKC and tyrosine kinases are involved in the regulation of PGE2 synthesis in gingival fibroblasts.