Regulation of growth plate chondrocytes by 1,25-dihydroxyvitamin D 3 requires caveolae and caveolin-1

Abstract

We examined the role of caveolae and caveolin-1 in the mechanism of 1α,25(OH)2D3 action in growth plate chondrocytes. We found that caveolae are required for rapid 1α,25(OH)2D 3-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1-/- mice. Introduction: 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1α,25(OH)2D3-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1α,25(OH)2D3 action and play an important role in regulating chondrocyte biology and growth plate physiology. Materials and Methods: Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1α,25(OH)2D3 receptors. Caveolae cholesterol was depleted with β-cyclodextrin (CD) and effects of 1α,25(OH)2D3 on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1-/- and C57BL/6 wildtype mice were also treated with 1α,25(OH)2D3. Epiphyses and costochondral junctions of 8-week-old male Cav-1-/- and wildtype mice (N = 8) were compared by histomorphometry and μCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons. Results: Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1α,25(OH)2D3-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1α,25(OH)2D3 effects on all parameters tested. The Cav-1-/- cells did not respond to 1α,25(OH)2D3, although 1α,25(OH) 2D3 increased PKC, alkaline phosphatase, and [ 35S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and μCT showed that Cav-1-/- growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis. Conclusions: The membrane-mediated effects of 1α,25(OH) 2D3 require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology. © 2006 American Society for Bone and Mineral Research.