Regulation of Osteoclast Function : Role of the Cbl-PI3K Complex

描述（申请人提供）：破骨细胞是在正常和病理条件下迁移至潜在骨吸收部位并吸收骨的主要细胞。我们的初步数据表明，在破骨细胞中，cbl基因的缺失会损害破骨细胞在发育过程中的迁移。类似地，Cbl-/-破骨细胞响应趋化刺激而重组肌动蛋白细胞骨架的能力也显著受损。我们还表明，酪氨酸磷酸化依赖性结合的Cbl的PI 3-激酶，破骨细胞功能所需的信号蛋白，是组织整合素激活后的信号事件的关键要求之一。消除Cbl和PI 3 K激酶之间的相互作用显著损害了体内和体外破骨细胞的骨吸收能力，表明Cbl-PI 3 K复合物的形成和该复合物下游的后续信号传导事件对于破骨细胞的正常功能是必需的。虽然在理解Cbl和PI 3 K在破骨细胞生物学中的作用方面已经取得了一些进展，但仍有待回答的问题是：这两种蛋白质之间的相互作用机制是什么，以及将这种复合物与破骨细胞功能联系起来的下游事件是什么？我们已经解决了这些问题，具体目标如下：AIM-1。确定哪种酪氨酸激酶（Src或Syk）对α v β 3激活下游的CblY 731的磷酸化至关重要，以及体外调节CblY 731磷酸化对破骨细胞迁移和骨吸收的影响。AIM-2通过使用两种不同的敲入小鼠模型确定Cbl-PI 3 K复合物对骨骼组织的体内影响，特别是破骨细胞功能，其中Cbl-PI 3 K相互作用被消除或组成性激活。AIM-3检查在调节肌动蛋白细胞骨架中重要的Cbl-PI 3 K相互作用下游的信号事件。拟议的研究将明确地证明在破骨细胞的av？3介导的信号下游的事件的相互依赖性。本提案的具体目标将通过采用各种办法和方法来实现。这包括敲除和敲入小鼠的体内研究、表达野生型和突变蛋白的培养细胞的生物化学分析以及从遗传修饰小鼠分离的细胞的生物化学研究。这些目标的成功完成将证明Cbl在破骨细胞功能中发挥的积极作用，并将增加我们对骨吸收调节的理解，可能确定新的治疗干预措施，以控制与骨质疏松症关节炎，骨转移和牙周病相关的骨丢失。骨质疏松症和其他与骨骼有关的疾病困扰着1.5亿美国人，并构成了不断上升的医疗保健费用的重大负担。这里提出的研究将有助于理解骨吸收背后的基本生物学，并将有助于确定治疗干预的目标，以控制骨质流失。

Description (provided by applicant): Osteoclasts are the principal cells that migrate to the potential bone resorption sites and resorb bone under normal and pathological conditions. Our preliminary data has shown that in osteoclasts, deletion of cbl gene impairs osteoclast migration during development. Similarly, the ability of Cbl-/- osteoclasts to reorganize the actin cytoskeleton in response to chemotactic stimuli is also significantly compromised. We have also shown that tyrosine phosphorylation-dependent binding of Cbl to PI3-Kinase, a signaling protein required for osteoclast function, is one of the key requirements to organize the signaling events upon integrin activation. Abolishing the interaction between Cbl and PI3K kinase significantly compromises the bone resorbing capacity of osteoclasts both in vivo and in vitro, indicating that formation of the Cbl-PI3K complex and the subsequent signaling events downstream of this complex are necessary for proper functioning of osteoclasts. Although some progress has been made in understanding the roles of Cbl and PI3K in osteoclast biology the questions that remain to be answered are: what is the mechanism of interaction between the two proteins and what are the downstream events that link this complex to osteoclast function? We have addressed these issues the following specific aims: AIM-1. Determines which tyrosine kinase (Src or Syk) is critical for phosphorylation of the CblY731 downstream of av¿3 activation and the effect of modulating the phosphorylation of CblY731 on osteoclast migration and bone resorption in vitro. AIM-2. Determines the in vivo impact of Cbl-PI3K complex on skeletal tissue in general and osteoclast function particular by using two different knock-in mouse models in which the Cbl-PI3K interaction is either abolished or constitutively activated. AIM-3. Examines the signaling events downstream of Cbl-PI3K interaction that are important in regulating actin cytoskeleton. The proposed studies will unequivocally demonstrate the interdependence of the events in osteoclast downstream of av¿3-mediated signaling. The specific aims of the proposal will be accomplished through the application of varieties of approaches and methodologies. This includes in vivo studies with knock-out and knock-in mice, biochemical analysis of cultured cells expressing wild type and mutant proteins and biochemical studies of cells isolated form the genetically modified mice. Successful completion of these aims will demonstrate the positive role played by Cbl in osteoclast function and will increase our understanding of the regulation of bone resorption, potentially identifying novel therapeutic intervention to control bone loss related to osteoporosis arthritis, bone metastasis and periodontal disease. Osteoporosis and other bone-related maladies afflict 150 million Americans and constitute a significant burden to ever-rising health care cost. The research proposed here will help in understanding the basic biology behind bone resorption and will help identifying targets for therapeutic interventions to control bone loss.