Novel GPCR Kinase Interactions in Macrophages and Role in Arthritis

描述（由申请人提供）：单核细胞/巨噬细胞是包括肌肉骨骼疾病在内的许多疾病炎症过程中的关键细胞类型。破骨细胞（来源于单核细胞/巨噬细胞）具有独特的骨吸收能力，因此在肌肉骨骼系统的稳态中起着核心作用。在类风湿性关节炎等疾病中观察到的炎症性骨溶解（由于炎症引起的骨吸收或溶解）中，破骨细胞侵蚀关节周围骨，导致关节塌陷和毁容。肿瘤坏死因子（TNF）是在这些炎症性疾病的病理生理中起关键作用的主要细胞因子之一，巨噬细胞中过度的TNF信号传导可归因于关节炎等疾病中破骨细胞募集和活性增加。TNF也是作用于单核/巨噬细胞的最有效的促炎细胞因子之一。因此，了解TNF刺激单核/巨噬细胞信号通路的生化机制，以及TNF诱导的信号通路影响巨噬细胞存活的机制，对关节炎的靶向治疗具有重要意义。我们在初步数据中证明，g蛋白偶联受体激酶（GPCR激酶或GRKs） -2和-5在巨噬细胞中TNF受体的信号传导中起着新的关键作用。这表明GRKs可能在关节炎的发病机制中发挥关键作用。GRKs是丝氨酸/苏氨酸蛋白激酶，最初因其磷酸化激动剂占据的g蛋白偶联受体（gpcr）的能力而被发现。GRK2和gr5在TNF信号传导中的作用代表了一种新的信号传导模式，因此了解这些GRKs在巨噬细胞和TNF介导的疾病（如关节炎）中调节TNF信号传导的机制值得进一步研究。在初步的蛋白质组学实验中，我们已经确定死亡相关蛋白激酶（DAPK）是GRK2的一种新的相互作用蛋白。在许多细胞类型中，DAPK是一种关键的信号蛋白，参与调节tnf诱导的生长/凋亡(3)。在这项资助中，我们建议扩大我们关于巨噬细胞中GPCR激酶在TNF信号传导中的新作用的初步发现。本应用的中心假设是GPCR激酶在TNF信号传导和巨噬细胞存活中起关键作用，因此在关节炎的发病机制中起关键作用。为了验证这一假设，我们建议检查以下具体目标：1。确定GRK2与死亡相关蛋白激酶（DAPK）相互作用在tnf诱导的巨噬细胞存活/凋亡中的作用。2. 鉴定和表征巨噬细胞TNF刺激下新的grk2相互作用信号体。3. 确定GRK2和gr5在小鼠关节炎模型中的作用。本文提出的生化、蛋白质组学和体内方法将回答GRKs在巨噬细胞中TNFR信号传导的细胞、生化和病理生理基础中的作用的关键问题，这将为识别炎症性疾病（如关节炎）中TNFR信号传导的分子/治疗靶点奠定基础。相关性：肿瘤坏死因子（TNF）作用于巨噬细胞，并通过激活信号通路在包括关节炎在内的各种疾病的发展中发挥重要作用。本应用程序的主要目的是了解巨噬细胞和关节炎中TNF如何调节这些信号通路。

DESCRIPTION (provided by applicant): Monocytes/macrophages are crucial cell types in inflammatory processes in many diseases including musculoskeletal diseases. Osteoclasts, (cells derived from monocytes/macrophages) have the unique capacity to resorb bone and thus play a central role in the homeostasis of musculoskeletal system. In Inflammatory osteolysis (resorption or dissolution of bone due to inflammation) observed in disorders such as rheumatoid arthritis, osteoclasts erode periarticular bone, leading to joint collapse and disfigurement. Tumor necrosis factor (TNF) is one of the dominant cytokines that play a critical role in the pathophysiology of these inflammatory disease conditions and excessive TNF signaling in macrophages has been attributed to an increased osteoclastic recruitment and activity in diseases such as arthritis. TNF is also one of the most potent pro-inflammatory cytokines that act on monocytes/macrophages. Thus understanding the biochemical mechanisms by which TNF stimulates signaling pathways in monocytes/macrophages and the mechanisms by which TNF-induced signaling affects survival of macrophages is of vital importance for therapeutic targeting in arthritis. We demonstrate in preliminary data that G-protein coupled receptor kinases (GPCR kinases or GRKs) -2 and -5 play novel and crucial roles in the signaling of TNF receptors in macrophages. This suggests that GRKs could potentially play a key role in the pathogenesis of arthritis. GRKs are serine/threonine protein kinases originally discovered for their ability to phosphorylate agonist-occupied G-protein coupled receptors (GPCRs). Role of GRK2 and 5 in TNF signaling represents a novel signaling paradigm and thus understanding the mechanisms by which these GRKs regulate TNF signaling in macrophages and in TNF-mediated diseases such as arthritis deserves further investigation. In preliminary proteomics experiments we have identified death-associated protein kinase (DAPK) as a novel interacting protein of GRK2. DAPK is a critical signaling protein involved in the regulation of TNF-induced growth/apoptosis in many cell types (3). In this grant, we propose to expand our preliminary findings on the novel roles of GPCR kinases in TNF signaling in macrophages. The central hypothesis of this application is that GPCR kinases play a crucial role in TNF signaling and macrophage survival and thus are critically involved in the pathogenesis of arthritis. To test this hypothesis we propose to examine the following specific aims: 1. Determine the role of GRK2 interaction with death-associated protein kinase (DAPK) in TNF-induced macrophage survival/apoptosis. 2. Identify and characterize novel GRK2-interacting signalosomes upon TNF stimulation in macrophages. 3. Determine the role of GRK2 and 5 in a mouse model of arthritis. Biochemical, proteomic and in vivo approaches proposed here will answer critical questions on the role of GRKs in cellular, biochemical and pathophysiological basis of TNFR signaling in macrophages which will form the basis for identifying molecular/therapeutic targets for TNFR signaling in inflammatory disease conditions such as arthritis. RELEVANCE: Tumor necrosis factor (TNF) acts on macrophages and plays an important role in the development of various diseases including arthritis by activating signaling pathways. Major goal of this application is to understand how these signaling pathways are regulated by TNF in macrophages and in arthritis.