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: 8073624
• 负责人: FAYEZ F SAFADI
• 金额: $ 13.13万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8073624
• 关键词: Affect; Age; Alkaline Phosphatase; Alleles; BMP2 gene; Beryllium; Cell Death; Cell physiology; Cell surface; Cytoskeletal Modeling; Data; Development; Diagnosis; Disease; Down-Regulation; Evaluation; Exhibits; Extracellular Matrix; Focal Adhesions; Fracture; Funding; Generations; Genes; Genetic Transcription; Growth Factor; Hand; Health; 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; Osteopenia; 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 Mice; 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; 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（由申请人提供）：通过一项在骨骼中发现骨激活素（OA）的研究，我们证明了 OA mRNA 和蛋白质在成骨细胞中表达，并且其表达呈现出在基质成熟后期处于最高水平的时间模式此外，该蛋白质由成骨细胞合成、加工、糖基化和分泌，利用成骨细胞的功能获得和功能丧失方法，我们发现 OA 的下调。成骨细胞分化和功能以及过度表达增加了体外成骨细胞分化和功能，我们还证明了OA的秘密减少形式调节成骨细胞分化和功能，从而促进了体内骨形成。OA在成骨中的重要性得到了证实。在具有 OA 无效等位基因的小鼠和具有 OA 基因自然突变的小鼠中，导致过早终止密码子，导致产生截短的 OA 蛋白。与骨量减少相关的骨骼表型在上一次资助期间和自上次提交以来，我们建立了 OA KO 和 OA mut 小鼠的群体，并且还产生了在骨骼中过度表达 OA 的转基因 (Tg) 小鼠。 OA KO、OA mut. 和 Tg 小鼠支持这样的假设：OA 是一种由成骨细胞合成和分泌的新型骨合成代谢因子，并且充当 ECM 相关信号分子或下游。 BMP2 调节成骨细胞分化和功能 除了对成骨细胞的影响外，我们还提供了数据显示 OA 影响破骨细胞分化，并且我们发现这些异常是继发于基质细胞/成骨细胞破骨细胞生成因子（例如 RANKL）产生的改变。目标 1 中提出的研究将评估 OA 缺乏 (OA KO)、截短 OA (OA mut.) 或 OA 过度表达的影响。 （Tg）在体内骨骼上，并评估原代培养物中来自这些小鼠的骨细胞（成骨细胞和破骨细胞）的分化和功能。OA中不同结构域的存在可能反映不同的功能，并评估其结构/功能。 OA 各个结构域对正常成骨细胞分化的关系和作用将在本申请的目标 2 中进行研究。在之前的资助期间，我们还表明 OA 的分泌亚型可以作为 ECM 相关（基质细胞）蛋白发挥作用。成骨细胞通过 av 附着在 OA 上1 整合素，导致粘着斑形成、细胞骨架重组和 FAK 激活。目标 3 中提出的研究将检验 OA 作为基质细胞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信号传导的假设。我们最近证明 BMP2 调节 OA 表达，并且 OA 是 BMP2 诱导的下游介质。成骨细胞功能，这是由 Smad 信号通路介导的反应，我们提供的初步数据表明，BMP2 刺激 Smad1、Dlx5 和 CBP 向 OA 启动子的募集，并且这种作用取决于目标 4 中提出的成骨细胞分化的阶段。将研究 OA 作为 BMP2 诱导的成骨细胞分化和功能的下游介质的机制，并将评估 BMP2 在刺激 Smad1、同源域蛋白和OA 启动子的 CBP 共激活剂在成骨细胞分化过程中进行转录调节，预计将产生关于 OA 缺乏或过度表达对体内骨形成/重塑的影响、其在成骨细胞中的作用机制以及骨形成的新信息。成骨细胞中 BMP2 诱导 OA 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 1000 万 50 岁以上的人已被诊断出骨质疏松症。据估计，还有 3360 万人患有这种疾病，骨量低（骨质减少）伴随着骨折发生率的增加，据估计，美国因骨折而产生的直接医疗费用与骨质减少有关。骨激活素是一种新型的骨骼生长因子，拟议的研究将产生有关其作用和作用机制的新信息。一旦我们了解了它对骨骼的全面影响以及它如何促进骨形成，这些信息将有助于开发新的治疗策略，以选择性地增强临床上显着骨质流失患者的骨形成。