MECHANISMS OF AVB3 INTEGRIN MEDIATED BONE RESORPTION

骨吸收是一个多步骤的过程，通过破骨细胞及其前体附着到骨上，促使细胞骨架重组和激活细胞内信号通路。事实上，整合素Alphavbeta3识别骨基质驻留蛋白，在体外和体内对破骨细胞性骨吸收是必不可少的，其机制尚不清楚，因此是一个潜在的抗骨质疏松靶点。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，因为它与吸收过程的特定成分有关，目前尚无信息。在获得alphavbeta3之后，结构/功能数据将通过在alphavbeta3中空的破骨细胞中表达整合素的成分来最好地实现，可以评估其对吸收过程中特定事件的单独影响。在这方面，我们开发了一系列工具，我们建议，这些工具将使我们能够实现这些目标。首先，我们用错乱的破骨细胞培育了Beta3基因敲除小鼠。第二，我们建立了一种基于逆转录病毒的技术，用β3基因重组破骨细胞。第三，我们利用TAT技术将全长蛋白质输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，以确定在真实的骨吸收细胞中调节破骨细胞生理的特定方面的β3整合素亚单位的成分。我们专注于β3整合素亚单位的细胞质区域，因为在其他情况下，它调节细胞骨架组织和中介基质衍生的信号。事实上，我们已经通过逆转录病毒转导全长的人β3-/-整合素来完全挽救了破骨细胞的表型，这一事实使我们能够描绘出这种整合素的细胞质成分，它控制着特定的事件-吸收过程。我们假设β3整合素胞浆结构域的特定成分：1)调节参与骨吸收过程的破骨细胞内信号分子的激活；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，我们的具体目标是确定β3整合素胞浆结构域的特定成分：1)调节参与吸收过程的破骨细胞内信号分子的激活；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.