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向潜在的骨吸收部位和骨吸收迁移的主要细胞。我们的初步数据表明，在破骨细胞中，CBL基因的缺失会损害发育过程中破骨细胞的迁移。同样，Cbl-/-破骨细胞重组肌动蛋白细胞骨架的能力也显著受损。我们还表明，依赖酪氨酸磷酸化的Cb1与PI3-Kinase的结合是整合素激活时组织信号事件的关键要求之一。PI3-Kinase是破骨细胞功能所需的信号蛋白。取消Cb1和PI3K激酶之间的相互作用显著影响体内和体外破骨细胞的骨吸收能力，表明Cb1-PI3K复合体的形成和该复合体下游的后续信号事件是破骨细胞正常功能所必需的。尽管在了解Cb1和PI3K在破骨细胞生物学中的作用方面已经取得了一些进展，但仍然需要回答的问题是：这两个蛋白之间的相互作用机制是什么，以及将这个复合体与破骨细胞功能联系起来的下游事件是什么？我们通过以下具体目标解决了这些问题：AIM-1。确定哪个酪氨酸激酶(Src或Syk)对于av？3激活下游的CblY731的磷酸化至关重要，以及在体外调节CblY731的磷酸化对破骨细胞迁移和骨吸收的影响。AIM-2。通过使用Cbl-PI3K相互作用被取消或结构性激活的两种不同的敲入小鼠模型，确定Cbl-PI3K复合体对一般骨骼组织和特定破骨细胞功能的体内影响。AIM-3。研究了Cb1-PI3K相互作用下游的信号事件，这些事件在调节肌动蛋白细胞骨架方面是重要的。拟议的研究将明确地证明av？3介导的信号转导下游破骨细胞中的事件之间的相互依赖。该提案的具体目标将通过采用各种方针和方法来实现。这包括对敲除和敲入小鼠的体内研究，对表达野生型和突变蛋白的培养细胞的生化分析，以及对从转基因小鼠分离的细胞的生化研究。这些目标的成功完成将证明Cbl在破骨细胞功能中所起的积极作用，并将增加我们对骨吸收调节的理解，潜在地寻找新的治疗干预措施来控制与骨质疏松性关节炎、骨转移和牙周病相关的骨丢失。骨质疏松症和其他与骨骼相关的疾病困扰着1.5亿美国人，并对不断上涨的医疗费用构成了重大负担。本文提出的研究将有助于了解骨吸收背后的基本生物学，并将有助于确定治疗干预的靶点，以控制骨丢失。

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.