Figure 7.

S1P potentiates osteoclast differentiation in coculture of BMMs and osteoblasts. (A) Coculture cells were treated with VtD₃ and S1P (1 μM) for 6 days and TRAP⁺ MNCs generated were scored. ^**P<0.005 compared with S1P-untreated controls. (B) Cocultures were treated with PGE₂ and S1P (1 μM) for 6 days. TRAP⁺ MNCs were counted. ^**P<0.005 versus S1P-untreated controls. NS, not significant. (C) Cocultures were carried out in the presence of PGE₂ (10⁻⁸ M), VtD₃ (10⁻¹⁰ M), and S1P (1 μM). TRAP⁺ MNC formation was assessed. ^**P<0.005 versus S1P-untreated controls. (D) Primary osteoblasts were treated with S1P and VtD₃ (10⁻⁸ M) for 24 h. RANKL and OPG mRNA levels were analyzed by RT–PCR. (E) Osteoblasts were incubated for 24 h with indicated CM and RANKL mRNA levels were examined by RT–PCR. (F) T cells were activated with ionomycin and PMA and treated with S1P for 24 h. RANKL mRNA levels were determined by RT–PCR. (G) T cells were activated and treated with SPHK1-CM for 24 h. RT–PCR was performed to detect RANKL mRNA. (H) Coculture was carried out in the presence of FTY-720, VtD₃ (10⁻⁹ M), and S1P (1 μM). TRAP⁺ MNCs were scored. (I) Osteoblasts were incubated with VtD₃ (10⁻⁹ M), S1P, and FTY-720 for 24 h. RANKL mRNA was analyzed by RT–PCR.