Expression of prostaglandin E synthase-1 in periodontitis : in vivo and in vitro studies

Abstract: Periodontitis is a chronic inflammatory disease characterized by destruction of the supporting structures of the teeth, including gingival tissues and alveolar bone. In advanced cases of periodontitis the ultimate clinical outcome is tooth loss. The inflammatory mediator prostaglandin E2 (PGE2) plays a central role in the pathogenesis of periodontitis and elevated levels of PGE2 have been observed in gingival crevicular fluid and gingival tissues from patients with periodontitis. The biosynthesis of PGE2 involves three groups of enzymes acting sequentially; phospholipase A2, cyclooxygenases (COX-1 and COX-2) and prostaglandin E synthases (PGES). The PGES enzymes, catalyzing the terminal step in PGE2 biosynthesis, exists in three distinct isoforms; the inducible microsomal membrane- associated and glutathione dependent PGES (mPGES-1); the constitutively expressed cytosolic PGES (cPGES) and the glutathione-independent, membrane-associated PGES (mPGES-2). The aim of this thesis was to investigate the expression of PGES, especially mPGES-1 in gingival tissues and gingival fibroblasts. Furthermore, we also aimed to identify and study the effect of novel mPGES-1 inhibitors on PGE2 synthesis in gingival fibroblasts, experimental periodontitis in rats and osteoclastogenesis using RAW 264.7 cells stimulated with receptor activator of NF-κB ligand (RANKL). In gingival tissues collected from patients with periodontitis, we demonstrated protein expression of mPGES-1, mPGES-2, cPGES and the upstream enzyme COX-2 in fibroblasts, endothelial cells, smooth muscle cells, epithelial cells and immune cells. In cell cultures of human gingival fibroblasts, the mRNA and protein expression of mPGES-1 as well as COX-2 accompanied by subsequent PGE2 production was increased by pro- inflammatory cytokines; tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β). To further investigate additional cell types contributing to elevated levels of PGE2, we used cultures of human airway smooth muscle (HASM) cells, human umbilical vein endothelial cells (HUVECs) and mast cells as a model system. Our results showed that mPGES-1 and COX-2 expression, as well as PGE2 production, was increased by the cytokines, IL-1β and TNFα in HASM cells. In HUVECs, only TNFα increased PGE2 production via an up- regulated expression of COX-2. In mast cells, the expression of PGES and COX-2 was not affected by cytokines and PGE2 production was only observed at basal level. Collectively, these findings suggest that gingival fibroblasts together with smooth muscle cells may contribute to elevated levels of PGE2 in inflamed gingiva. To target mPGES-1 in the context of periodontitis, novel mPGES-1 inhibitors, aminothiazoles, were identified. Studies on PGE2 synthesis in gingival fibroblasts revealed that cytokine-induced PGE2 production was inhibited by the aminothiazoles TH-848 and TH-644. IL-1β-induced mPGES-1 mRNA expression was not affected by aminothiazoles, whereas protein expression was slightly decreased by TH-848 but not by TH-644. In addition, IL-1β-induced expression of COX-2 was not affected by aminothiazoles either at the mRNA or protein level. Similarly, other isoenzymes of PGES, mPGES-2 and cPGES were not affected either by cytokines nor aminothiazoles in gingival fibroblasts. In an in vitro assay for mPGES-1 enzyme activity, TH-848 and TH-644 inhibited mPGES-1 activity without affecting COX-2 activity. In ligature-induced experimental periodontitis in rats, topical treatment with the aminothiazole TH-848 reduced alveolar bone resorption compared to vehicle-treated controls. Furthermore, in vitro studies of aminothiazoles on osteoclastogenesis demonstrated a decreased number of osteoclasts in parallel with decreased PGE2 production in RANKL-stimulated RAW 264.7 cells. In conclusion, all three PGE synthases were expressed in gingival tissue from patients with periodontitis. The isoenzyme mPGES-1 regulated the cytokine-induced PGE2 production in gingival fibroblasts and smooth muscle cells. The novel mPGES-1 inhibitors aminothiazoles inhibited PGE2 production in gingival fibroblasts, reduced alveolar bone resorption in experimental periodontitis and decreased osteoclastogenesis in RANKL- activated RAW 264.7 cells. This suggested that mPGES-1 is an important regulatory enzyme in inflammation-induced PGE2 production and that inhibition of mPGES-1 might be an attractive treatment target for chronic inflammatory bone destruction, such as periodontitis.