The Role of Osteoactivin in Osteoblast Development and Function

骨激活素在成骨细胞发育和功能中的作用

• 批准号: 7737516
• 负责人: FAYEZ F SAFADI
• 金额: $ 33.75万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7737516
• 关键词: Affect; Age; Alkaline Phosphatase; Alleles; BMP2 gene; Cell Death; Cell physiology; Cell surface; Cytoskeletal Modeling; Data; Development; Diagnosis; Disease; Down-Regulation; Elements; Evaluation; Exhibits; Focal Adhesions; Fracture; Funding; Generations; Genes; Genetic Transcription; Growth Factor; Hand; Health Care Costs; Healthcare; Homeodomain Proteins; Hospitalization; In Vitro; Incidence; Insulin-Like Growth Factor I; Integrins; Investigation; Knockout Mice; Mediating; Mediator of activation protein; Messenger RNA; Molecular; Mus; Mutant Strains Mice; Mutation; Nodule; Nonsense Codon; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Osteoporosis; PTK2 gene; Patients; Pattern; Phenotype; Physicians; Process; Production; Protein Binding; Protein Isoforms; Proteins; Recombinants; Regulation; Role; Secondary to; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; Stress; Stromal Cells; Structure-Activity Relationship; Study Section; TNFSF11 gene; Testing; Toxic effect; Transcriptional Regulation; Transgenic Organisms; Visit; Work; bone; bone cell; bone growth factor; bone loss; bone mass; clinically significant; gain of function; in vivo; interest; loss of function; mineralization; mouse model; mutant; novel; novel therapeutics; osteoactivin; osteoblast differentiation; promoter; public health relevance; research study; response; skeletal

DESCRIPTION (provided by applicant): From a study in which we discovered osteoactivin (OA) in bone, we demonstrated that OA mRNA and protein are expressed in osteoblasts and its expression exhibited a temporal pattern being at highest levels during the later stages of matrix maturation and mineralization. Furthermore, the protein is synthesized, processed, glycosylated and secreted by osteoblasts. Using gain-of-function and loss-of-function approaches in osteoblasts, we found that down-regulation of OA decreased osteoblast differentiation and function and over-expression increased osteoblast differentiation and function in vitro. We also demonstrated that the secreted form of OA regulates osteoblast differentiation and function. Treatment with recombinant OA promotes bone formation in vivo. The importance of OA in osteogenesis was confirmed in mice with the null allele for OA and in mice with a natural mutation in the OA gene caused a premature stop codon that results in the generation of a truncated OA protein. Both of these mice exhibit a skeletal phenotype associated with decreased bone mass. During the previous funding period and since the last submission, we established colonies of OA KO and OA mut. mice, and also generated transgenic (Tg) mice that over-express OA in bone. Preliminary data from OA KO, OA mut. and Tg mice support the hypothesis that OA is a novel bone anabolic factor which is synthesized and secreted by osteoblasts, and acts either as an ECM-associated signaling molecule or downstream of BMP2 to regulate osteoblast differentiation and function. In addition to its effects on osteoblasts, we present data showing that OA affects osteoclast differentiation, and we hypothesize that these abnormalities are secondary to altered production of osteoclastogenic factors (e.g. RANKL) by stromal cells/osteoblasts in the bone microenvironment. Studies proposed in aim 1 will evaluate the effects of OA deficiency (OA KO), truncated OA (OA mut.) or OA over-expression (Tg) on bone in vivo, and assess the differentiation and function of bone cells (osteoblasts and osteoclasts) derived from these mice in primary cultures. The presence of various domains in OA might reflect different functions, and evaluation the structure/function relationship and role of the various domains of OA on normal osteoblast differentiation will be investigated in aim 2 of this application. During the previous funding period, we also showed that the secreted isoform of OA can function as an ECM-associated (matricellular) protein and demonstrated that osteoblasts attach to OA via the av¿1 integrin, resulting in the formation of focal adhesions, cytoskeletal reorganization and the activation of FAK. Studies proposed in aim 3 will test the hypothesis that OA acts as a matricellular protein that binds to specific cell surface integrins on osteoblasts to initiate integrin-activated signaling, cytoskeletal reorganization, and regulate cell function. We recently demonstrated that BMP2 regulates OA expression and that OA is a downstream mediator of BMP2-induced osteoblast function, a response that is mediated by the Smad signaling pathway. We present preliminary data that BMP2 stimulates the recruitment of Smad1, Dlx5 and CBP to the OA promoter and this effect is dependent on the stage of osteoblast differentiation. Studies proposed in aim 4 will investigate the mechanism whereby OA acts as a downstream mediator of BMP2-induced osteoblast differentiation and function, and will evaluate the effects of BMP2 in stimulating the recruitment of Smad1, homeodomain proteins, and CBP co-activators to the OA promoter for transcriptional regulation during osteoblast differentiation. Proposed experiments are expected to generate novel information regarding the effects of OA deficiency or over-expression on bone formation/remodeling in vivo, its mechanisms of action in osteoblasts, and the molecular requirements for OA induction by BMP2 in osteoblasts. PUBLIC HEALTH RELEVANCE: Osteoporosis is a major health care problem since approximately 10 million people over the age of 50 have been diagnosed with the disease and 33.6 million more are estimated to have low bone mass (osteopenia). Low bone mass is accompanied by an increased incidence of fracture, and it is estimated that the direct health care costs in the Untied States from fractures related to osteopenia (hospitalizations, ER visits, physician visits, etc.) ranges from $12-$18 billion annually. Osteoactivin is a novel growth factor in bone and the proposed studies will generate new information regarding its effects and mechanisms of action on bone cell development and function. Once we understand its full effects on bone and how it works to promote bone formation, this information will be helpful in developing new therapeutic strategies to selectively enhance bone formation in patients with clinically significant bone loss.

描述（由适用提供）：根据我们在骨骼中发现骨活蛋白（OA）的研究，我们证明了OA mRNA和蛋白质在成骨细胞中表达，其表达表现出在基质成熟和矿化的后期表现出最高水平的临时模式。此外，该蛋白是由成骨细胞合成，加工，糖基化和分泌的。使用功能收益和成骨细胞的功能丧失方法，我们发现OA的下调减少了成骨细胞分化，功能和过表达增加了成骨细胞的分化和体外功能。我们还证明了OA的分泌形式调节成骨细胞的分化和功能。重组OA治疗可在体内促进骨形成。在无效等位基因的小鼠中证实了OA在成骨中的重要性对于OA和OA基因中自然突变的小鼠导致过早的终止密码子，从而导致截断的OA蛋白产生。这两只小鼠都暴露了与骨骼减少有关的骨骼表型。在上一个资金期间，自上次提交以来，我们建立了OA KO和OA MUT的殖民地。小鼠，还产生过表达OA的转基因（TG）小鼠。 OA KO，OA MUT的初步数据。 TG小鼠支持以下假设：OA是一种新型的骨合成代谢因子，由成骨细胞合成和分泌，并且可以用作与ECM相关的信号分子或BMP2的下游来调节成骨细胞的分化和功能。除了对成骨细胞的影响外，我们还提供数据表明OA会影响破骨细胞的分化，并且我们假设这些异常是由于基质细胞/骨微细胞在骨微观环境中通过基质细胞/成骨细胞的成骨细胞生成因子（例如RANKL）的产生而发生的。 AIM 1提出的研究将评估OA缺乏症（OA KO），截短的OA（OA Mut。）或OA过表达（TG）对体内骨骼的影响，并评估骨细胞（成骨细胞和骨细胞）的分化和功能。 OA中各个域的存在可能反映出不同的功能，评估OA的各个域在正常成骨细胞分化中的结构/功能关系以及作用将在本应用的AIM 2中研究。在上一个融资期间，我们还表明，OA的分泌同工型可以作为与ECM相关的（矩阵）蛋白的作用，并证明成骨细胞通过AV¿1整合素附着在OA上，从而形成局灶性粘合剂，细胞骨架重新组织和FAK的活化。 AIM 3中提出的研究将检验以下假设：OA充当基质蛋白，该基质蛋白与成骨细胞上特定细胞表面整合素结合以启动整合素激活的信号传导，细胞骨架重组和调节细胞功能。我们最近证明了BMP2调节OA表达，OA是BMP2诱导的成骨细胞功能的下游介质，该响应由SMAD信号通路介导。我们介绍了BMP2刺激SMAD1，DLX5和CBP向OA启动子募集的初步数据，并且这种效应取决于成骨细胞分化的阶段。 AIM 4提出的研究将研究OA充当BMP2诱导的成骨细胞分化和功能的下游介体，并将评估BMP2在刺激SMAD1，同源蛋白蛋白和CBP共同激活剂对OA启动者对OA启动者的募集的募集中的影响。预计提出的实验将产生有关OA缺乏症或过表达对体内骨形成/重塑的影响的新信息，其成骨细胞中的作用机制以及BMP2在成骨细胞中诱导OA诱导的分子要求。公共卫生相关性：骨质疏松症是一个主要的医疗保健问题，因为大约50岁以上的人被诊断出患有该疾病，据估计还有3360万人患有低骨量（骨质减少）。低骨质量是通过增加的骨折事件来实现的，据估计，与骨质减少症有关的骨折（住院，急诊就诊，实体访问等）的直接医疗保健成本范围为12至180亿美元。骨活性蛋白是骨骼中的新生长因子，所提出的研究将产生有关其作用对骨细胞发育和功能的作用和机制的新信息。一旦我们了解了其对骨骼的全部影响及其如何促进骨形成的作用，该信息将有助于开发新的治疗策略，以选择性地增强临床上骨质损失显着的患者的骨骼形成。