OSTEOACTIVIN IN OSTEOBLAST DEVELOPMENT AND FUNCTION

骨活性素在成骨细胞发育和功能中的作用

• 批准号: 7106431
• 负责人: FAYEZ F SAFADI
• 金额: $ 28.51万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106431
• 关键词: alkaline phosphatase; antisense nucleic acid; bone development; cell differentiation; cell proliferation; complementary DNA; gene expression; genetically modified animals; growth factor; integrins; laboratory mouse; laboratory rat; osteoblasts; osteocalcin; protein binding; recombinant proteins; tissue /cell culture; transfection; transforming growth factors

DESCRIPTION (provided by applicant): In a study examining differential gene expression in bone from normal and osteopetrotic (op) rats, we discovered a novel cDNA, termed osteoactivin (OA) that was over-expressed in op when compared to normal bone. Subsequent in situ hybridization and immunohistochemistry studies demonstrated that OA mRNA and protein are expressed by osteoblasts. In primary osteoblast cultures, OA mRNA levels exhibited a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization. Furthermore, the protein is synthesized by osteoblasts and secreted into the medium. Using an OA-anti-sense oligonucleotide, we were able to block OA expression. Down-regulation of OA expression inhibited osteoblast differentiation and function including decreased alkaline phosphatase activity, osteocalcin production and matrix mineralization. A CMV-rOA construct was generated and used to examine the effect of OA over-expression on osteoblast development and function in osteoblast cultures. OA over-expression increased nodule formation, alkaline phosphatase activity, osteocalcin production and matrix mineralization. Using a local injection model to test the anabolic effect of a small peptide of OA (OA-P) in vivo, OA-P induced a potent osteogenic response. We hypothesize that OA is an anabolic bone growth factor. We propose that OA is synthesized and secreted by osteoblasts, where it acts as an ECM-associated signaling molecule or a soluble factor to promote osteoblast differentiation and function Experiments proposed in this application will focus on the association between OA and bone with the following aims. Experiments in aim I will provide a more comprehensive evaluation on the effects of down-regulation of OA expression using an OA-anti-sense oligo transfection approach. OA over-expression will be achieved using a CMV-rOA construct and its effects on osteoblast development and function will be evaluated. Studies in aim 2 will examine the effects of recombinant OA (rOA) or OA-P on osteoblast development and function in vitro and bone formation in vivo. Studies in aim 3 will characterize rOA binding to osteoblasts and determine whether integrins serve as receptors for OA on osteoblasts. In aim 4, studies will examine transcriptional regulation of OA by three osteotropic factors, 1,25(OH)2D3, BMP-2 and TGF-beta1. Additional experiments will determine whether specific effects of BMP-2 on osteoblast differentiation are OA-dependent. Experiments in the first part of aim 5 will focus on the generation of OA null (-/-) mice and characterization of their skeletal phenotype. We will also examine the ability of OA -/- osteoblasts to differentiate in vitro. In the final part of this aim, we propose to generate transgenic mice expressing OA under control of the osteocalcin promoter and evaluate whether osteoblast-specific expression has an anabolic effect on bone. The identification of a novel anabolic agent in bone and elucidation of its mechanism(s) of action will eventually lead to the development of new therapeutic strategies to selectively stimulate osteogenesis in diseases associated with bone loss and in fracture repair.

描述（由申请人提供）：在一项检查正常和骨石症 (op) 大鼠骨骼中差异基因表达的研究中，我们发现了一种新的 cDNA，称为骨激活素 (OA)，与正常骨骼相比，它在 op 中过度表达。随后的原位杂交和免疫组织化学研究表明，OA mRNA 和蛋白质由成骨细胞表达。在原代成骨细胞培养物中，OA mRNA 水平表现出一种时间表达模式，在基质成熟和矿化的后期阶段表达最高水平。此外，该蛋白质由成骨细胞合成并分泌到培养基中。使用 OA 反义寡核苷酸，我们能够阻断 OA 表达。 OA表达下调抑制成骨细胞分化和功能，包括碱性磷酸酶活性、骨钙素产生和基质矿化降低。生成了 CMV-rOA 构建体并用于检查 OA 过表达对成骨细胞培养物中成骨细胞发育和功能的影响。 OA 过度表达增加结节形成、碱性磷酸酶活性、骨钙素产生和基质矿化。使用局部注射模型测试 OA 小肽 (OA-P) 体内的合成代谢作用，OA-P 诱导了有效的成骨反应。我们假设 OA 是一种合成代谢骨生长因子。我们提出OA是由成骨细胞合成和分泌的，它作为ECM相关信号分子或可溶性因子来促进成骨细胞分化和功能。本申请中提出的实验将集中于OA与骨之间的关联，其目标如下。目的 I 的实验将使用 OA 反义寡核苷酸转染方法对 OA 表达下调的效果进行更全面的评估。将使用 CMV-rOA 构建体实现 OA 过度表达，并将评估其对成骨细胞发育和功能的影响。目标 2 的研究将检查重组 OA (rOA) 或 OA-P 对体外成骨细胞发育和功能以及体内骨形成的影响。目标 3 的研究将表征 rOA 与成骨细胞的结合，并确定整合素是否作为成骨细胞上 OA 的受体。在目标 4 中，研究将检查三种成骨因子 1,25(OH)2D3、BMP-2 和 TGF-β1 对 OA 的转录调节。其他实验将确定 BMP-2 对成骨细胞分化的具体影响是否依赖于 OA。目标 5 第一部分的实验将重点关注 OA null (-/-) 小鼠的产生及其骨骼表型的表征。我们还将检查 OA -/- 成骨细胞在体外分化的能力。在该目标的最后部分，我们建议在骨钙素启动子的控制下产生表达 OA 的转基因小鼠，并评估成骨细胞特异性表达是否对骨具有合成代谢作用。骨中新型合成代谢剂的鉴定及其作用机制的阐明最终将导致新的治疗策略的开发，以选择性地刺激与骨质流失相关的疾病和骨折修复中的成骨。