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Human and Murine Osteocalcin Gene Expression: Conserved Tissue Restricted Expression and Divergent Responses to 1,25-Dihydroxyvitamin D₃ in Vivo

Bone and Mineral Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales, Australia

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)₂D₃ treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)₂D₃ injection (23.6 ± 3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2 ± 0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)₂D₃ treatment in animals on a low calcium diet (25.2 ± 7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)₂D₃ production (7.5 ± 0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)₂D₃ injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)₂D₃. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)₂D₃ is not. The mouse osteocalcin genes do not respond to 1,25-(OH)₂D₃ treatment, but the human osteocalcin-directed transgene is markedly up-regulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)₂D₃ are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.

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