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Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Departments of Pediatrics (T.O.C., K.C.M., B.E.), Orthopaedics and Rehabilitation (M.A., D.B., C.M.G.), and Surgery (T.L.M., M.C.), Yale University School of Medicine, New Haven, Connecticut 06520-8064

Address all correspondence and requests for reprints to: Thomas O. Carpenter, M.D., Department of Pediatrics, Yale University School of Medicine, P.O. Box 208064, New Haven, Connecticut 06520-8064. E-mail: carpenteto{at}maspo3.mas.yale.edu

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)₂D₃. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)₂D₃ in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)₂D₃ in cultures of Hyp mouse-derived osteoblasts.

Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)₂D₃, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)₂D₃, added later (days 17–25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)₂D₃ was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)₂D₃, and there is no difference between normal and Hyp cultures in this response.

Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)₂D₃ on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)₂D₃ in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

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