Conversion of 25-(OH)D3to 24,25-(OH)2D3and nuclear binding of 24,25-(OH)2D3in chondrocytes

Conversion of 25-(OH)D3to 24,25-(OH)2D3and stimulation of proteoglycans synthesis in cultured chondrocytes has been previously demonstrated (Corvol et al., 1978). To investigate subcellular localization, labelled vitamin D metabolites, 26,27-3H, were incubated with cultured chondrocytes from prepubertal rabbits for various time periods. Medium and cells were extracted separately with methanol-chloroform (v : v) and analyzed by high pressure liquid chromatography. When labelled 25-(OH)D3was incubated, 8 × 10-9M, most radioactivity was recovered as 24,25-(OH)2D3in the mitochondrial fraction and reached a plateau after 60 min. Same labelled 24,25-(OH)2D3was also found in the nuclei. When labelled 24,25-(OH)2D3was incubated, the radioactivity was mainly retained in the nuclei. Nuclear uptake of this label was inhibited by preincubating the cells with excess non labelled 24,25-(OH)2D3. There was no specific nuclear binding of 25-(OH)D3. In conclusion, in chondrocytes the 24-hydroxylation of vitamin D is within mitochondria as already shown for the kidney. Specific nuclear binding of 24,25-(OH)2D3suggests that this is the active vitamin D metabolite in chondrocytes.