THE ROLE OF CTGF IN OSTEOBLAST DEVELOPMENT AND FUNCTION

CTGF 在成骨细胞发育和功能中的作用

• 批准号: 7077813
• 负责人: STEVEN N. POPOFF
• 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: 2002-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7077813
• 关键词: autocrine; biological signal transduction; bone morphogenetic proteins; cell adhesion; cell differentiation; connective tissue; connective tissue growth factor; extracellular matrix; gene expression; gene targeting; genetically modified animals; growth factor receptors; in situ hybridization; integrins; laboratory mouse; laboratory rat; messenger RNA; northern blottings; osteoblasts; osteocalcin; osteogenesis; osteoporosis; phenotype; protein binding; protein structure function; tissue /cell culture; transforming growth factors

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated protein that regulates diverse cellular functions including proliferation, adhesion, migration, differentiation, matrix production and survival. In a study examining differential gene expression in bone from normal and osteopetrotic rats, we discovered that CTGF mRNA was expressed in normal bone. Subsequent studies demonstrated that CTGF is produced and secreted by osteoblasts. Recombinant rat CTGF (rCTGF) was generated and used to examine its effects on osteoblasts in culture. In addition to its effect as a mitogen, rCTGF stimulated functions associated with osteoblast differentiation including alkaline phosphatase activity, osteocalcin gene expression and mineralization. Using a local injection model to test its anabolic effect in vivo, rCTGF induced a potent osteogenic response. The finding that CTGF knockout mice have a skeletal phenotype including severe osteopenia highlights the important role that this growth factor plays in osteoblast development and bone formation. We hypothesize that CTGF is an anabolic bone growth factor. We propose that CTGF 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 in an autocrine fashion. Experiments proposed in this application will focus on the association between CTGF and bone with the following aims. Experiments in aim 1 will provide a more comprehensive evaluation of the effects of rCTGF on osteoblast development and function in vitro and bone formation in vivo. Studies in aim 2 will characterize rCTGF binding to osteoblasts and determine whether integrins serve as receptors for CTGF on osteoblasts. The morphological, cytoskeletal and signaling responses of osteoblasts adhered to immobilized rCTGF will also be examined. In aim 3, studies will examine whether specific effects of TGF-beta1 and BMP-2 on osteoblast proliferation/differentiation are CTGF-dependent, and determine whether selected regulatory elements of the CTGF promoter are necessary for transcriptional activation by TGF-beta1 and/or BMP- 2. Experiments in the first part of aim 4 will characterize the skeletal phenotype in CTGF null (-/-) mice and examine the ability of -/- osteoblasts to differentiate in vitro. In the final part of this aim we propose to generate transgenic mice expressing CTGF 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 its mechanisms 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.

结缔组织生长因子（CTGF）是一种分泌的细胞外基质相关蛋白，可调节多种细胞功能，包括增殖，粘附，迁移，分化，基质产生和存活率。 在一项研究中，研究了正常和骨质膜大鼠骨骼中差异基因表达的研究，我们发现CTGF mRNA在正常骨中表达。 随后的研究表明，CTGF是由成骨细胞产生和分泌的。 生成重组大鼠CTGF（RCTGF），并用于检查其对培养成骨细胞的影响。 除了其作为有丝分裂原的作用外，RCTGF还刺激了与成骨细胞分化有关的功能，包括碱性磷酸酶活性，骨钙素基因表达和矿化。 RCTGF使用局部注射模型在体内测试其合成代谢作用，诱导了有效的成骨反应。 CTGF敲除小鼠具有骨骼表型的发现，包括严重的骨质减少症，突出了该生长因子在成骨细胞发育和骨形成中起着的重要作用。 我们假设CTGF是合成代谢的骨生长因子。 我们建议CTGF由成骨细胞合成和分泌，在该成骨细胞中充当ECM相关的信号分子或可溶性因子，以促进成骨细胞分化和自身分泌方式。 本应用程序中提出的实验将集中于CTGF和BONE之间的关联与以下目的。 AIM 1中的实验将对RCTGF在体外体外和骨形成成骨细胞发育和功能的影响提供更全面的评估。 AIM 2中的研究将表征RCTGF与成骨细胞结合，并确定整联蛋白是否用作成骨细胞上CTGF的受体。 还将检查符合固定RCTGF的成骨细胞的形态，细胞骨架和信号反应。 在AIM 3中，研究将检查TGF-BETA1和BMP-2对成骨细胞增殖/差异化的特定影响是否依赖于CTGF，并确定CTGF启动子的选定调节元素是否需要TGF-BETA1和/或BMP-2的TGF-BETA1和/或BMP-2。在第一个AIL AIL的第一个组合中的tgf-Beta1和/或BMP-2。 （ - / - ）小鼠并检查 - / - 成骨细胞在体外区分的能力。 在此目的的最后部分，我们建议生成在骨钙蛋白启动子控制下表达CTGF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。 骨骼中新型合成代谢剂及其作用机理的鉴定最终将导致新的治疗策略的发展，以选择性地刺激与骨质流失和断裂修复有关的疾病中的成骨。