Figure 8.

S1P increased RANKL expression by inducing PGE₂ secretion through COX2 regulation. (A) Calvarial osteoblasts were treated for 24 h with S1P and VtD₃ (10⁻⁸ M). PGE₂ levels in the culture supernatants were determined by EIA. ^*P<0.01; ^**P<0.005 versus control. (B) Osteoblasts were treated with S1P (1 μM) and/or VtD₃ (10⁻⁸ M) for 24 h. COX2 and mPGES1 protein levels were determined. (C) Osteoblasts were incubated for 24 h with the indicated CM. COX2 and mPGES1 proteins were detected. (D, E) Osteoblasts were stimulated with S1P (1 μM) and/or VtD₃ (10⁻⁸ M) for 10 min. The COX2 and mPGES1 activities were determined as described in Materials and methods. (F) Osteoblasts were treated with S1P (1 μM), VtD₃ (10⁻⁸ M), and NS398 (1 μM). RANKL and OPG mRNA levels were examined by RT–PCR. (G) Cocultures of BMMs and osteoblasts were carried out in the presence of VtD₃ (10⁻⁹ M), S1P (1 μM), and NS398. TRAP⁺ MNCs were scored.