Tyrosine Kinase Signaling for Cartilage Maintenance and Osteoarthritis

 描述（由申请人提供）：该R21项目的长期目标是通过对酪氨酸激酶在软骨稳态中的作用的基本了解，开发一种新的骨关节炎（OA）治疗策略。已知酪氨酸激酶如Src和FAK参与关节组织的机械转导，并且据报道其药理学抑制有益于软骨维持。然而，现有的数据表明，Src的活性取决于亚细胞位置，它可以通过机械负荷上调或下调。此外，初步研究表明，Pyk 2（另一种结构与FAK相似的酪氨酸激酶）的部分沉默可消除TNFα诱导的胞质而非膜结合Src活化。由于Pyk 2的整体敲除改变了骨重建，因此软骨和软骨下骨中Pyk 2介导的反应可能会改变它们在骨/软骨界面的相互作用和OA的进展。在这个项目中解决的问题是酪氨酸激酶（Pyk 2，Src和FAK）的网络在软骨维护中的作用。我们的工作假设是Src/FAK在不同的亚细胞位置激活（例如，细胞质和质膜）充当负荷驱动的软骨保护和再生的介体，并且Pyk 2与Src/FAK相互作用以用于软骨基质的合成代谢和分解代谢调节。测试 我们的假设，我们提出了两个具体的目标，使用正常和OA软骨外植体（离体），和野生型和Pyk 2敲除小鼠。目标1：确定软骨/软骨下骨外植体中的酪氨酸激酶信号传导目的2：评估Pyk 2在OA膝关节中的作用在所提出的离体软骨外植体系统中，将通过定制的加载装置在有和没有OA诱导的情况下施加机械载荷。为了在亚细胞水平上确定激酶活性，我们将采用位于特定亚细胞位置的基于荧光共振能量转移（FRET）的生物传感器。将离体测量值与体内机械载荷数据进行比较。我们希望，我们的理解的Pyk 2/Src/FAK在软骨稳态的调节机制将澄清软骨力学转导的优点和缺点，并有助于确定一个分子靶点，用于治疗退行性关节疾病，如OA。

 DESCRIPTION (provided by applicant): The long-term objective of this R21 project is to develop a novel therapeutic strategy for osteoarthritis (OA) through a basic understanding of the role of tyrosine kinases in cartilage homeostasis. Tyrosine kinases such as Src and FAK are known to be involved in mechanotransduction of joint tissues, and their pharmacological inhibition is reported to be beneficial to cartilage maintenance. However, available data suggest that Src activity varies depending on subcellular locations and it can be up- or down-regulated by mechanical loading. Furthermore, preliminary studies indicate that partial silencing of Pyk2, another tyrosine kinase whose structure is similar to FAK, abolishes TNFα-induced cytoplasmic, but not membrane-bound, Src activation. Since global knockout of Pyk2 alters bone remodeling, Pyk2-mediated responses in cartilage and subchondral bone may alter their interactions at a bone/cartilage interface and the progression of OA. The question addressed in this project is the role of a network of tyrosine kinases (Pyk2, Src and FAK) in cartilage maintenance. Our working hypothesis is that Src/FAK activation at distinct subcellular locations (e.g., cytoplasm and plasma membrane) acts as a mediator of load-driven chondro-protection and regeneration, and that Pyk2 interacts with Src/FAK for anabolic and catabolic regulation of cartilage matrix. To test our hypotheses, we propose two specific aims using normal and OA cartilage explants (ex vivo), and wild type and Pyk2 knockout mice. Aim 1: Determine tyrosine kinase signaling in cartilage/subchondral bone explants Aim 2: Evaluate the role of Pyk2 in OA knee joints In the proposed ex vivo cartilage explant system, mechanical loading will be applied by a custom-made loading apparatus with and without OA induction. To determine kinase activities at the subcellular level, we will employ fluorescence resonance energy transfer (FRET)-based biosensors that are located at specific subcellular locations. Ex vivo measurements will be compared with in vivo mechanical loading data. We expect that advancing our understanding of the regulatory mechanism of Pyk2/Src/FAK in cartilage homeostasis will clarify merits and demerits of mechanotransduction of cartilage, and contribute to identifying a molecular target for treatment of degenerative joint diseases such as OA.