Effect of vitamin D metabolites on the expression of alkaline phosphatase activity by epiphyseal hypertrophic chondrocytes in primary cell culture

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Abstract
The effects of three vitamin D3 metabolites, 25-hydroxyvitamin D3 (25-(OH)D3), 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), and 24R,25-dihydroxyvitamin D3 (24R,25-(OH)2D3) on the activity of alkaline phosphatase (AP), a key enzyme involved in biomineralization, have been studied in primary cultures of chicken epiphyseal growth plate chondrocytes. Dosages of 1α,25-(OH)2D3 (10−12 to 10−7 M) caused a progressive, dosage-and time-dependent decrease in cellular AP levels, IC50 occurring at approximately 10−12 M. In contrast, 24R,25-(OH)2D3 at 10−13 to 10−10 M stimulated cellular AP activity, half-maximal stimulation occurring at about 10−13 M. At higher levels (10−10 to 10−7 M), 24R,25-(OH)2D3 caused progressive reduction in AP activity. Maximal effects of 24R,25-(OH)2D3 were evident 48 h after administration of the metabolite. 25-(OH)D3 initially (24 h) caused a weak, dosage-dependent decrease in cellular AP activity, but after 48–72 h, low levels (10−13 to 10−11 M) caused a dosage-dependent increase in AP activity. Higher levels of 25-(OH)D (> 10−10 M) were clearly inhibitory to AP. These findings reveal that the AP activity of growth plate chondrocytes is exquisitely sensitive to both 1α,25- and 24R,25-(OH)2D3 but the response to each is in opposite directions. The paradoxical response of the cells to 25-(OH)D3 can be explained if the metabolite is slowly metabolized by a 24-hydroxylase to 24R,25-(OH)2D3 leading to stimulation of cellular AP. The inhibitory effects of all metabolites at higher levels (10−8 to 10−7 M) may be explained if both 25-(OH)D3 and 24R,25-(OH)2D3 act as weak agonists for the 1α,25-(OH)2D3 receptor, mimicking its inhibitory action. The data show that vitamin D metabolites exert a direct effect on the growth plate chondrocytes. Studies are in progress to elucidate the mechanism of this AP response.
Funding Information
  • United States Public Health Service, National Institute of Arthritis (AM-18983)