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: 6778165
• 负责人: FAYEZ F SAFADI
• 金额: $ 31.73万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6778165
• 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）：在一项研究中，研究了正常和骨质术（OP）大鼠骨骼中差异基因表达的研究，我们发现了一种新型cDNA，称为骨活蛋白（OA），与正常骨相比，OP中的OP表达过表达。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。在原发性成骨细胞培养物中，OA mRNA水平表现出一种时间的表达方式，在基质成熟和矿化的后期阶段最高水平表达。此外，该蛋白质由成骨细胞合成并分泌到培养基中。使用OA-ANTI-sense寡核苷酸，我们能够阻止OA表达。 OA表达的下调抑制了成骨细胞分化和功能，包括碱性磷酸酶活性降低，骨钙素的产生和基质矿化。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过表达增加了结节形成，碱性磷酸酶活性，骨钙素的产生和基质矿化。使用局部注射模型测试体内OA（OA-P）较小肽的合成代谢作用，OA-P诱导了有效的成骨反应。我们假设OA是合成代谢的骨生长因子。我们建议OA是由成骨细胞合成和分泌的，在此应用中，它充当了与ECM相关的信号分子或可溶性因子，以促进成骨细胞分化和本应用中提出的功能实验，将重点放在OA和骨骼之间的关联上，以下目的。 AIM我将使用OA-Anti-Sensens固定寡核转染方法对OA表达下调的影响进行更全面的评估。将使用CMV-ROA构建体来实现OA过表达，并评估其对成骨细胞开发和功能的影响。 AIM 2中的研究将检查重组OA（ROA）或OA-P对体内体外和骨形成成骨细胞发育和功能的影响。 AIM 3中的研究将表征ROA与成骨细胞的结合，并确定整联蛋白是否用作成骨细胞上OA的受体。在AIM 4中，研究将通过三个骨骼因子（OH）2d3，BMP-2和TGF-BETA1检查OA对OA的转录调节。其他实验将确定BMP-2对成骨细胞分化的特定影响是否依赖于OA。 AIM 5的第一部分的实验将集中于OA NULL（ - / - ）小鼠的产生以及其骨骼表型的表征。我们还将检查OA - / - 成骨细胞在体外区分的能力。在此目标的最后一部分中，我们建议生成在骨钙素启动子控制下表达OA的转基因小鼠，并评估成骨细胞特异性表达是否对骨骼具有合成代谢作用。在骨骼中鉴定出一种新型的合成代谢剂和阐明其作用机制的鉴定最终将导致发展新的治疗策略，以选择性地刺激与骨质流失和断裂修复相关的疾病中的骨气化。