Rat growth plate chondrocytes express low levels of 25-hydroxy-1α-hydroxylase

Abstract

Long standing disturbances of Vitamin D-metabolism as well as null-mutant animals for 25-hydroxy-1α-hydroxylase results in disorganised growth plates. Cultured chondrocytes were shown to be target for the hydroxylated Vitamin D-metabolites 1α,25(OH) 2D 3 and 24,25(OH) 2D 3. Because studies on production of these metabolites were inconclusive in in vitro systems, the expression of the Vitamin D-system was examined in rat growth plate chondrocytes in vitro as well as ex vivo. Gene expression for 25-hydroxy-1α-hydroxylase, 25-hydroxy-24-hydroxylase as well as Vitamin D-receptor and collagen II and X were analysed on mRNA level by RT-PCR and quantitative real-time PCR, on protein level by western blotting and by immunohistochemistry in isolated growth plate chondrocytes or intact growth plates. Compared to UMR or CaCo 2 cells and renal homogenates cultured growth plate chondrocytes expressed low levels of 25-hydroxy-1α-hydroxylase mRNA and 25-hydroxy-24-hydroxylase mRNA. The expression of both was modulated by 25(OH)D 3, but 1α,25(OH) 2D 3 affected only 25-hydroxy-24-hydroxylase. These data were confirmed by Western blotting. Immunohistochemistry demonstrated predominant staining for 25-hydroxy-1α-hydroxylase in chondrocyte nodules and cells embedded in matrix in vitro. Ex vivo, 25-hydroxy-1α-hydroxylase was detected predominantly in late proliferative and hypertrophic zone of the growth plate. In conclusion, growth plate chondrocytes express the key components for a paracrine/autocrine Vitamin D-system.