Prostaglandin E synthases in periodontitis-affected gingival tissue and in gingival fibroblasts

Abstract: Periodontitis is a chronic inflammatory disease resulting in the destruction of the tissue and alveolar bone supporting the teeth and leading ultimately to tooth loss. Prostaglandin E2 (PGE2) is an important inflammatory mediator in the pathogenesis of periodontitis. The biosynthesis of PGE2 is catalysed by three groups of enzymes acting sequentially: phospholipase A2 (PLA2), cyclooxygenases (COX-1 and COX-2) and prostaglandin E (PGE) synthases, which catalyse the final step of PGE2 synthesis. Three PGE synthase isoforms have been identified: i) the inducible microsomal membrane-associated and glutathione-dependent PGE synthase, mPGES-1, ii) the constitutively expressed cytosolic PGE synthase, cPGES, and iii) the glutathione-independent, membrane-associated mPGES-2. The aim of this thesis was to investigate the expression of PGE synthases in gingival tissue from periodontitis patients, as well as to study their expression and regulation in relation to PGE2 production in gingival fibroblasts. In periodontitis-affected gingival tissue, we demonstrated in vivo protein expression of mPGES-1, mPGES-2 and cPGES, as well as COX-2 in fibroblasts, endothelial cells, smooth muscle cells, epithelial cells and immune cells. We further showed that, in cell cultures of gingival fibroblasts and smooth muscle cells, the inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β), or co-culture with lymphocytes, markedly induced mPGES-1 and COX-2 expression, accompanied by an increase in PGE2 production. In cultured endothelial cells, only TNFα was found to increase PGE2 production, via enhanced COX-2 expression. In mast cell cultures, basal levels of PGE2 were detected, but no increase was observed in response to TNFα or IL-1β. To elucidate the impact of mPGES-1 inhibition on mPGES-2 and cPGES expression, as well as on PGE2 production we used knock-down of mPGES-1 expression by small interfering RNA (siRNA). The cytokine-induced protein expression of mPGES-1 was reduced by up to 79% by siRNA silencing, without affecting mPGES-2 or cPGES expression. Moreover, mPGES-1 siRNA did not affect the cytokine-stimulated PGE2 production, whereas levels of the downstream prostaglandin F2α (PGF2α) were enhanced. Using inhibitors and activators of various signalling pathways, we demonstrated that cytokine-induced mPGES-1 expression in gingival fibroblasts did not involve protein kinase C, p38 mitogen-activated protein kinase or tyrosine kinase pathways, in contrast to COX-2 expression. We further observed a possible positive feedback loop in which PGE2 and PGF2α increased the expression of mPGES-1. Furthermore, cytokine-induced mPGES-1 expression and PGE2 production were reduced after the inhibition of the upstream enzyme PLA2 and increased after the addition of arachidonic acid, the product of PLA2. The proposed anti-inflammatory prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), reduced mPGES-1 expression but not COX-2 expression or PGE2 production. To further explore the pathways involved in increased PGE2 synthesis in TNFα- stimulated gingival fibroblasts, a global gene expression profile was established using a microarray platform. Enrichment analysis of the gene expression data led to further investigation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signalling pathways, revealing that these pathways are involved in the signal transduction of TNFα- induced mPGES-1 and COX-2 expression. In conclusion, all three PGE synthases are expressed in gingival tissue from patients with periodontitis. The isoenzyme mPGES-1 is the main PGE synthase involved in cytokine-induced PGE2 production in gingival fibroblasts. The cytokine-increased expression of mPGES-1 involves the signal pathways JNK and NF-κB. Furthermore, the prostaglandins PGE2 and PGF2α increase mPGES-1 expression, which may create a positive feedback loop. Collectively, these results suggest that inflammation-induced production of PGE2 by gingival fibroblasts, mediated by the increased expression of mPGES-1 and COX-2, may contribute to chronic inflammation in periodontitis. The results provide new insights into the expression and regulation of mPGES-1 in gingival fibroblasts and gingival tissue.