TGF beta 1 regulates 25-hydroxyvitamin D-3, 1 alpha- and 24-hydroxylase activity in cultured growth plate chondrocytes in a maturation-dependent manner

Chondrocytes metabolize 25-(OH)D-3 to the two active dihydroxylated forms o f the secosteroid, 1,25-(OH)(2)D-3 and 24,25-(OH)(2)D-3. The aim of the pre sent study was to examine the activity of the enzymes responsible for this metabolism, 1 alpha-hydroxylase and 24R-hydroxylase, and their regulation b y TCF beta 1. Basal 1 alpha- and 24R-hydroxylase activities were measured i n homogenates of confluent, fourth passage rat costochondral resting zone a nd 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. Homog enates were incubated for 90 minutes and 1 alpha- and 24R-hydroxylase activ ities determined by measuring the conversion of [H-3]-25-(OH)D-3 to [H-3]-1 ,25-(OH)(2)D-3 and [H-3]-24,25-(OH)(2)D-3 using an HPLC with an inline radi oisotope detector. Resting zone cells were also treated with various concen trations of recombinant human TGF beta 1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determ ined. In addition, [H-3]-1,25-(OH)(2)D-3 and [H-3]-24,25-(OH)(2)D-3 release d into the conditioned media by resting zone chondrocyte cultures in respon se to TGF beta 1 were measured. In culture, all three cell types were found to contain 1 alpha- and 24R-hyd roxylase activities. Basal 1 alpha-hydroxylase specific activity was signif icantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hy droxylase. Treatment of resting zone cells with TGF beta 1 increased 24R-hy droxylase mRNA levels in a dose-dependent manner. TGF beta 1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased la-hydroxylase activit y by 20-30%. Similar changes were observed with MCT cells, but not growth z one cells. Production of [H-3]-24,25- (OH)(2)D-3 by resting zone cells incr eased with TCF beta 1 treatment, while [H-3]-1,25-(OH)(2)D-3 production dec reased. The effect was time- and dose-dependent, correlating with hydroxyla se activity and 24-hydroxylase gene expression. These results demonstrate t hat growth plate chondrocytes contain the necessary enzymes to produce 1,25 -(OH)(2)D-3 and 24,25-(OH)(2)D-3 from 25-(OH)D-3. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regu lated by TGF beta 1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vita min D metabolite production.