FIGURE 3.

Loss or gain of Smpd3/nSMase2 function promotes or suppresses BMP-2-induced chondrogenic maturation, respectively. A, ATDC5 chondrocytes were transfected with control siRNA (siCont) or Smpd3 siRNA (siSmpd3) for 16 h and then treated with or without BMP-2 (300 ng/ml) for 6 days. Quantitative RT-PCR analysis was performed for Smpd3, Col2a1, and Col10a1. B, ATDC5 cells were treated with BMP-2 (300 ng/ml) in combination with GW4869 at a concentration of 0.1 or 1 μm for 4 days to analyze expression of Col2a1 by quantitative RT-PCR. C, ATDC5 cells or primary chondrocytes were stimulated with BMP-2 (300 ng/ml) in combination with GW4869 (1 μm) and C₂-ceramide (10 μm) for 17 days. Cells were subjected to Alcian blue staining. Scale bar, 200 μm. A parallel experiment was done with ATDC5 with a stimulation time of 7 days, and immunoblotting was performed for aggrecan and tubulin. D, ATDC5 cells were stimulated with BMP-2 (300 ng/ml) in combination with C₂-ceramide at 10 μm for 14 days. E, ATDC5 chondrocytes were infected with adenovirus (Ax) carrying LacZ or Smpd3 for 2 h, and further cultured with or without BMP-2 (300 ng/ml) for 7 days. Expression of Smpd3, Col2a1, and Col10a1 was evaluated by quantitative RT-PCR. F, mouse primary chondrocytes were infected with adenovirus carrying lacZ or Smpd3 for 2 h and further cultured with or without BMP-2 (300 ng/ml) for 6 days. Expression of Smpd3, Col2a1, and Col10a1 was evaluated by quantitative RT-PCR. *, p < 0.05; **, p < 0.01.