OSTEOCALCIN PRODUCTION IN PRIMARY OSTEOBLAST CULTURES DERIVED FROM NORMAL AND HYP MICE

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevate d circulating osteocalcin levels, as well as altered regulation of ost eocalcin by 1,25(OH)(2)D-3. Whether this osteocalcin abnormality is in trinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production an d its regulation by 1,25(OH)(2)D-3 in primary cultures of murine osteo blasts and examined osteocalcin and its messenger RNA in response to 1 ,25(OH)(2)D-3 in cultures of Hyp mouse-derived osteoblasts. Cell viabi lity and osteocalcin production are optimal when murine cells are harv ested within 36 h of age. Murine primary osteoblast cultures mineraliz e and produce osteocalcin in a maturation-dependent fashion (as demons trated in other species), and continuous exposure to 1,25(OH)(2)D-3, b eginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. How ever, in contrast to other species, exposure to 1,25(OH)(2)D-3, added later(days 17-25) in culture, does not stimulate osteocalcin but arres ts osteocalcin production at current levels. Ambient media levels of o steocalcin were no different in cultures from Hyp mice and their norma l litter mates, and the down-regulatory response to 1,25(OH)(2)D-3 was comparable in cultures from normal and Hyp mice. Furthermore, express ion of osteocalcin messenger RNA in murine cultures is reduced with ex posure to 1,25(OH)(2)D-3, and there is no difference between normal an d Hyp cultures in this response. Thus, primary murine osteoblasts mani fest a species-specific effect of 1,25(OH)(2)D-3 on osteocalcin produc tion. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)(2)D-3 in Hyp mic e, are not observed in osteoblast cultures derived from the mutant str ain. These data indicate that abnormalities of osteocalcin described i n intact Hyp mice require factors other than those present in cultured cells.