FUNCTION AND REGULATION OF OSTEONECTIN IN BONE

描述(改编自申请者摘要)：骨联素或SPARC (酸性分泌蛋白，富含半胱氨酸)是含量最丰富的 骨骼中的非胶原性基质蛋白。它由成骨细胞合成， 结合钙离子和I型胶原，并抑制体外矿化。 在非骨骼细胞中的研究表明，骨连蛋白调节 血管生成、金属蛋白酶表达、细胞增殖、细胞形态 以及细胞与基质的相互作用。虽然骨连蛋白在骨骼中含量丰富，但其 功能仍不清楚。本提案的具体目标1是界定 利用SPARC基因敲除小鼠研究骨连接蛋白在骨中的功能。这个 野生型和基因敲除小鼠的骨骼将通过组织形态计量学进行分析 傅里叶变换红外显微光谱分析。功能和 骨细胞的反应性将在体外用器官和 细胞培养分析。在特定的目的2中，骨连接蛋白和 它在MC3T3成骨细胞中的片段将被用来进一步定义 骨连接蛋白在骨中的作用。细胞增殖试验和Northern 金属蛋白酶和基质RNA的印迹分析将被用来评估 成骨细胞基因表达的变化。有研究表明，骨连蛋白 在发育、伤口愈合和基质重塑中起着重要作用， 成纤维细胞生长因子(FGFs)参与了这一调节 骨骼发育和骨折修复。初步数据显示， 碱性成纤维细胞生长因子下调成骨细胞骨连接蛋白的表达 转录后机制。这项建议的具体目标3是 探讨碱性成纤维细胞生长因子破坏骨连接蛋白基因表达的机制 成骨细胞。骨连接蛋白转录本中介导变化的区域 RNA的稳定性将通过缺失/替换突变和 核糖核酸酶保护试验。骨连接蛋白RNA结合蛋白将是 通过RNA迁移率变化分析、UV交联研究和 蛋白质印迹分析。如有必要，酵母三杂交系统可用于检测 将利用RNA-蛋白质相互作用来克隆骨连蛋白RNA结合 蛋白质。申请人建议，这些研究将提供 骨连接蛋白在骨中的作用及其关键信息 受一种关键的形态因子BGF的调节。

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.