Aminothiazoles Inhibit RANKL-mediated Osteoclastogenesis and PGE₂ Production in RAW 264.7-cells

Objectives: Periodontitis is characterized by chronic inflammation and osteoclast-mediated bone loss regulated by the receptor activator of nuclear factor-kappaB (RANK), the RANK ligand (RANKL) and osteoprotegerin (OPG) system. The anti-inflammatory drugs aminothiazoles, acting as PGE synthase inhibitors, have recently been reported to reduce bone resorption in experimental periodontitis in rats as well as to inhibit prostaglandin E₂ (PGE₂) production in gingival fibroblasts in vitro. The aim of this study was to investigate the effect of aminothiazoles on RANKL-mediated osteoclastogenesis in vitro using RAW 264.7 cells.

Methods: The osteoclast precursors RAW 264.7 cells were treated with RANKL (3 ng/ml) alone or in combination with the aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) or 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) at different doses for 4 days. The differentiated cell layers were either fixed and histologically stained for tartrate resistant acid phosphatase (TRAP) or collected for gene expression analysis of TRAP, Cathepsin K and OPG. The expression of the osteoclast markers TRAP and Cathepsin K as well as the RANKL decoy receptor OPG was analyzed by RT-qPCR. The cell medium was collected for analysis of PGE₂, determined by EIA.

Results: The aminothiazoles TH-848 and TH-644 decreased the formation of multinucleated TRAP-positive osteoclast-like cells in RANKL activated RAW 264.7 cells. The mRNA expression of TRAP and Cathepsin K was down-regulated by the aminothiazoles, in contrast to OPG that was up-regulated. In agreement with mRNA expression of TRAP and Cathepsin K, the aminothiazoles also decreased the production of PGE₂ in RANKL-stimulated RAW 264.7 cells.

Conclusion: Aminothiazoles reduce osteoclastogenesis associated with decreased PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of inflammatory bone diseases including periodontitis.