1α,25-(OH)₂D₃ regulates 25-hydroxyvitamin D₃ 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C

Rat costochondral chondrocytes possess 25-hydroxyvitamin D₃ 1α- and 24R-hydroxylase activities and metabolize 25-(OH)D₃ to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ in a cell maturation-specific and time-dependent manner. This study examined the hypothesis that 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1α,25-(OH)₂D₃ exerts its effects via mechanisms involving protein kinase C (PKC) mediated pathways. Rat costochondral growth zone chondrocytes were treated with 10^-9-10^-7 M 1α,25-(OH)₂D₃ or 24R,25-(OH)₂D₃ for 24 hours, and 1α- and 24R-hydroxylase activities in cell homogenates determined by measuring the conversion of [³H]-25-(OH)D₃ to [³H]-1,25-(OH)₂D₃ and [³H]-24,25-(OH)₂D₃. Metabolite production by intact cells was determined at 6 and 24 hours. Involvement of PKC was assessed using chelerythrine, and the requirement for protein synthesis was assessed using cycloheximide. In addition, the effect of 10^-10-10^-8 M 1α,25-(OH)₂D₃ on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1α,25-(OH)₂D₃ (1,25-mVDR), which exerts its effects via PKC, was assessed by blocking the 1,25-mVDR with an antibody (Ab99) generated against the 1,25-mVDR in chick enterocyte membranes. Specificity of the 1,25-(OH)₂D₃-dependent effect on 24,25-(OH)₂D₃ production was determined by comparing the response to 1α,25-(OH)₂D₃ to the response to 1 β,25-(OH)₂D₃. 1α,25-(OH)₂D₃ increased 24R-hydroxylase specific activity in a dose-dependent manner; 24,25-(OH)₂D₃ production by intact cells was also increased. The effect of 1α,25-(OH)₂D₃ on 24,25-(OH)₂D₃ production was stereospecific. Only 1 1α,25-(OH)₂D₃ caused an increase; 1β,25-(OH)₂D₃ was without effect. 24R,25-(OH)₂D₃ had no effect on 24R-hydroxylase activity at 24 hours, 1α-hydroxylase activity was unaffected by either metabolite at 24 hours, 1α,25-(OH)₂D₃ affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1α,25-(OH)₂D₃ caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1α,25-(OH)₂D₃-dependent stimulation of 24R-hydroxylase. These results suggest an interrelationship between the 1,25-mVDR and gene expression via the nuclear VDR (nVDR) and/or a PKC-mediated mechanism in modulating 24R-hydroxylase in growth zone chondrocytes.