Fig 3.

1,25-(OH)₂D₃ activates MMP13 through the mitogen-activated protein kinase p38 pathway. (A) Fold change in RNA levels of MMP13. ATDC5 chondrocytes were cultured in DMEM and treated with 10 nM of 1,25-(OH)₂D₃ for 24 hr. SB203580 (10 μM) and SP600125 (20 μM) were added to the culture medium 2 hr before 1,25-(OH)₂D₃ treatment. RNA expression level of MMP13 was determined by quantitative real-time RT-PCR. Level of RNA from control group was normalized to a value of 1. (B) Inhibition by the p38 inhibitor SB203580 of 1,25-(OH)₂D₃-induced increase in MMP13 protein and phosphorylation of p38 in ATDC5 chondrocytes. Confluent chondrocytes were cultured and incubated with or without SB203580 for 2 hr before addition of 10 nM 1,25-(OH)₂D₃ for 24 hr. Total cell lysates were prepared and analyzed by Western blot using antibodies that recognize MMP13, phosphor-p38 and β-actin.