FUNCTION AND REGULATION OF OSTEONECTIN IN BONE

描述（改编自申请人摘要）：骨粘连蛋白或骨钙素 （分泌的蛋白质酸性和富含半胱氨酸）是最丰富的 骨中的非胶原基质蛋白。 它由成骨细胞合成， 结合Ca ~（2+）和I型胶原，并在体外抑制矿化。 在非骨骼细胞中的研究表明，骨连接素调节 血管生成，金属蛋白酶表达，细胞增殖，细胞形态 和细胞-基质相互作用。 虽然骨粘连蛋白在骨中丰富，但其 功能仍然未知。 本提案的具体目标1是确定 利用β-内酰胺酶基因敲除小鼠研究骨粘连蛋白在骨中的功能。 的 将通过组织形态计量学分析野生型和基因敲除小鼠的骨 和傅里叶变换红外显微光谱。 的功能和 骨细胞的反应性将在体外使用器官和 细胞培养测定。 在特异性目标2中，骨连接素和 其在MC 3 T3成骨细胞中的片段将被用于进一步定义 骨粘连蛋白在骨中的作用。 细胞增殖测定和北方 金属蛋白酶和基质RNA的印迹分析将用于评估 成骨细胞基因表达的变化。 提示骨粘连蛋白 在发育、伤口愈合和基质重塑中起重要作用， 和成纤维细胞生长因子（FGF）参与调节 骨骼发育和骨折修复。 初步数据表明 碱性成纤维细胞生长因子（bFGF）下调成骨细胞中骨连接蛋白的表达， 转录后机制 该提案的具体目标3是 确定bFGF使骨粘连蛋白mRNA不稳定的机制， 成骨细胞 骨粘连蛋白转录物中介导骨粘连蛋白表达变化的区域 RNA稳定性将通过缺失/取代诱变来确定， RNA酶保护测定。 骨连接蛋白RNA结合蛋白将是 通过RNA迁移率变动分析、UV交联研究和 Western印迹分析。 如果需要，可以使用酵母3-杂交系统来检测 RNA-蛋白质相互作用将用于克隆骨连接素RNA结合 proteins. 申请人建议，这些研究将提供 关于骨粘连蛋白在骨中的功能的重要信息， 由一个关键的形态发生蛋白bGF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.