Bone sialoprotein (BSP) is a noncollagenous extracellular matrix component of the bone that is intimately associated with initial matrix mineralization and bone remodeling. The Bsp gene has been an important tool used by the project laboratory to understand mechanisms of tissue-specific gene expression in bone. BSP has also been useful for understanding mechanisms of biomineralization. During the previous funding period, we isolated a 2.5 kb murine Bsp gene promoter containing sufficient information to direct osteoblast-selective expression both in cell culture and transgenic mice. Within this promoter, we identified a homeodomain protein-binding site that is required for the osteoblast-selective expression of Bsp in cell culture. The role of this and related sites in the in vivo expression of Bsp is not known. The transcriptional activity of this element can be stimulated by homeodomain proteins associated with bone such as Dlx-5, but we do not know the identity of the protein/s regulating with this site in vivo. Considerable in vitro evidence supports the hypothesis that BSP is a nucleator of hydroxyapatite crystals. We showed that it can also stimulate mineralization when overexpressed in non-mineralizing preosteoblast cell lines. Matrix Gla protein (MGP), in contrast, is a potent inhibitor of mineralization. PTH and PTHrP, both potent inhibitors of osteoblast-mediated biomineralization, inhibit BSP synthesis while rapidly inducing MGP, suggesting that these molecules have opposing actions and are reciprocally regulated. Building on these important findings, this competitive renewal will achieve the following specific aims:
Aim 1. Define the in vivo significance of a homeodomain protein-binding site in the murine BSP promoter.
Aim 2. Identify the nuclear protein(s) that interact with and regulate the proximal homeodomain site in osteoblast.
Aim 3. Define the molecular basis for the regulation of MGP by PTH and PTHrP; assess the ability of BSP and MGP to antagonize each other in cell culture and in vivo. Studies supported by this project will describe a novel mechanism for controlling gene expression in bone and explain important actions of PTH/PTHrP on the regulation of mineralization. Such knowledge is essential for designing new strategies for bone regeneration to treat trauma and pathological conditions such as osteoporosis and periodontal disease.
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