TGFβ1 regulates 25-hydroxyvitamin D₃ 1α- and 24-hydroxylase activity in cultured growth plate chondrocytes in a maturation-dependent manner

Chondrocytes metabolize 25-(OH)D₃ to the two active dihydroxylated forms of the secosteroid, 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. The aim of the present study was to examine the activity of the enzymes responsible for this metabolism, 1α-hydroxylase and 24R-hydroxylase, and their regulation by TGFβ1. Basal 1α- and 24R-hydroxylase activities were measured in homogenates of confluent, fourth passage rat costochondral resting zone and growth zone chondrocytes and mouse cortico-tubular cells (MCT) were used as a positive control. The cells were harvested and homogenized in buffer optimized to maintain the activity and stability of the hydroxylases. Homogenates were incubated for 90 minutes and 1α- and 24R-hydroxylase activities determined by measuring the conversion of [³H]-25-(OH)D₃ to [³H]-1,25-(OH)₂D₃ and [³H]-24,25-(OH)₂D₃ using an HPLC with an inline radioisotope detector. Resting zone cells were also treated with various concentrations of recombinant human TGFβ1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determined. In addition, [³H]-1,25-(OH)₂D₃ and [³H]-24,25-(OH)₂D₃ released into the conditioned media by resting zone chondrocyte cultures in response to TGFβ1 were measured. In culture, all three cell types were found to contain 1α- and 24R-hydroxylase activities. Basal 1α-hydroxylase specific activity was significantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hydroxylase. Treatment of resting zone cells with TGFβ1 increased 24R-hydroxylase mRNA levels in a dose-dependent manner. TGFβ1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased 1α-hydroxylase activity by 20-30%. Similar changes were observed with MCT cells, but not growth zone cells. Production of [³H]-24,25-(OH)₂D₃ by resting zone cells increased with TGFβ1 treatment, while [³H]-1,25- (OH)₂D₃ production decreased. The effect was time- and dose-dependent, correlating with hydroxylase activity and 24-hydroxylase gene expression. These results demonstrate that growth plate chondrocytes contain the necessary enzymes to produce 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ from 25- (OH)D₃. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regulated by TGFβ1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vitamin D metabolite production.