FUNCTION AND REGULATION OF OSTEONECTIN IN BONE

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DESCRIPTION (Adapted from the Applicant's Abstract): Osteonectin or SPARC (secreted protein acidic and rich in cysteine) is one of the most abundant non-collagenous matrix proteins in bone. It is synthesized by osteoblasts, binds Ca2+ and type I collagen, and inhibits mineralization in vitro. Studies in non-skeletal cells indicate that osteonectin regulates angiogenesis, metalloproteinase expression, cell proliferation, cell shape and cell-matrix interactions. Although osteonectin is abundant in bone, its function remains unknown. Specific Aim 1 of this proposal is to define the function of osteonectin in bone utilizing a SPARC knock-out mouse. The bones of wild-type and knock-out mice will be analyzed by histomorphometry and Fourier transform infrared microspectroscopy. The function and responsiveness of bone cells will be evaluated, in vitro, using organ and cell culture assays. In Specific Aim 2, over-expression of osteonectin and its fragments in MC3T3 osteoblasts will be used to further define the function of osteonectin in bone. Cell proliferation assays and Northern blot analysis of metalloproteinase and matrix RNAs will be used to assess changes in osteoblast gene expression. It is suggested that osteonectin plays an important role in development, wound healing and matrix remodeling, and fibroblast growth factors (FGFs) have been implicated in the regulation of skeletal development and fracture repair. Preliminary data indicate that basic FGF (bFGF) down-regulates osteonectin expression in osteoblasts by a post-transcriptional mechanism. Specific Aim 3 of this proposal is to determine the mechanisms by which bFGF destabilizes osteonectin mRNA in osteoblasts. Regions of the osteonectin transcript that mediate changes in RNA stability will be determined by deletion/substitution mutagenesis and RNase protection assay. Osteonectin RNA binding proteins will be characterized by RNA mobility shift assay, UV crosslinking studies and Western blot analyses. If necessary, a yeast 3-hybrid system for detecting RNA-protein interactions will be used to clone osteonectin RNA binding proteins. It is suggested by the applicant that these studies will provide critical information on the function of osteonectin in bone, and its regulation by a key morphogen, bGF.