MECHANISMS OF AVB3 INTEGRIN MEDIATED BONE RESORPTION

骨吸收是一个多步骤的过程，由破骨细胞及其前体细胞附着到骨上，促进细胞骨架重组和细胞内信号通路的激活。 事实上，由于机制知之甚少，识别骨基质驻留蛋白的整合素α v β 3在体外和体内对骨吸收至关重要，因此是潜在的抗骨质疏松症靶点。然而，优化这一治疗目标将取决于对整合素分子结构的详细分析，因为它涉及吸收过程的特定组分，目前还没有信息。 α v β 3结构/功能数据的获取最好通过在α v β 3缺失的破骨细胞中表达整合素的组分来实现，所述整合素的组分对再吸收过程中的特定事件的个体影响可以被评估。在这方面，我们制定了一系列工具，我们建议这些工具将使我们能够实现这些目标。 首先，我们产生了破骨细胞紊乱的β 3基因敲除小鼠。其次，我们建立了一种逆转录病毒为基础的技术，重建β 3-/-破骨细胞与β 3 cDNA。 第三，我们利用达特技术将全长蛋白质递送到破骨细胞及其前体的整个群体中。 这些工具提供了独特的机会，以确定β 3整联蛋白亚基的成分，调节真正的骨吸收细胞中破骨细胞生理学的特定方面。 我们专注于β 3整合素亚基的胞质结构域，因为它是已知的，在其他情况下，调节细胞骨架组织和介导基质衍生的信号。 事实上，我们已经完全拯救了β 3-/-整联蛋白破骨细胞表型的逆转录病毒转导的全长人β 3 cDNA定位我们描绘的细胞质成分，这种整联蛋白的管理具体事件的吸收过程。 我们假设β 3整合素胞质结构域的特定组分：1）调节参与再吸收过程的骨细胞内信号分子的活化; 2）组织破骨细胞细胞骨架; 3）调节破骨细胞在体外和体内降解骨的能力。 因此，我们的特定目的是鉴定β 3整联蛋白胞质结构域的特定组分，其：1）调节参与再吸收过程的骨内信号分子的活化; 2）组织破骨细胞细胞骨架; 3）调节破骨细胞在体外和体内降解骨的能力。

Bone resorption is a multistep process initiated by attachment of osteoclasts and their precursors to bone, prompting cytoskeletal reorganization and activation of intracellular signaling pathways. In fact, by mechanisms poorly understood, the integrin alphavbeta3, which recognizes bone matrix-residing proteins, is essential to osteoclastic bone resorption, in vitro and in vivo, and is therefore a potential anti-osteoporosis target. Optimizing this therapeutic goal will depend, however, on detailed analysis of the integrin's molecular structure as it relates to specific components of the resorptive process, information presently not available. Acquisition of alphavbeta3, structure/function data will be best achieved by expressing, in alphavbeta3 null osteoclasts, constituents of the integrin whose individual impact on particular events in the resorptive process, can be evaluated. In this regard, we developed a series of tools which, we propose, will enable us to achieve these goals. First, we generated beta3 knockout mice with deranged osteoclasts. Second, we have established a retroviral-based technique of reconstituting beta3-/-osteoclasts with beta3 cDNAs. Third, we utilized TAT technology to deliver full length proteins, into entire populations of osteoclasts and their precursors. These tools provide the unique opportunity to identify constituents of the beta3 integrin subunit which regulate specific aspects of osteoclast physiology in authentic bone resorptive cells. We focus on the cytoplasmic domain of the beta3 integrin subunit as it is known, in other circumstances, to regulate cytoskeletal organization and mediate matrix derived signals. The fact we have completely rescued the beta3-/-integrin osteoclast phenotype by retroviral transduction of a full length human beta3 cDNA positions us to delineate the cytoplasmic components of this integrin which govern specific events the resorptive process. We hypothesize that specific components of the beta3 integrin cytoplasmic domain: 1) regulate activation of intraosteoclastic signaling molecules involved in the resorptive process; 2) organize the osteoclast cytoskeleton; 3) regulate the capacity of osteoclasts to degrade bone in vitro and in vivo. Thus, our Specific Aims are to identify specific components of the beta3 integrin cytoplasmic domain which: 1) regulate activation of intraosteoclastic signaling molecules involved in the resorptive process; 2) organize the osteoclast cytoskeleton; 3) regulate the capacity of osteoclasts to degrade bone in vitro and in vivo.