Novel GPCR Kinase Interactions in Macrophages and Role in Arthritis

描述(申请人提供)：在包括肌肉骨骼疾病在内的许多疾病中，单核/巨噬细胞是炎症过程中的关键细胞类型。破骨细胞(来源于单核/巨噬细胞的细胞)具有独特的吸收骨的能力，因此在肌肉骨骼系统的动态平衡中发挥着核心作用。在类风湿性关节炎等疾病中观察到的炎性骨溶解(由于炎症导致骨吸收或溶解)，破骨细胞侵蚀关节周围骨，导致关节塌陷和毁容。肿瘤坏死因子是在这些炎症性疾病的病理生理过程中起关键作用的主要细胞因子之一，巨噬细胞中过多的肿瘤坏死因子信号被归因于在关节炎等疾病中破骨细胞募集和活性的增加。肿瘤坏死因子也是作用于单核/巨噬细胞的最有效的促炎细胞因子之一。因此，了解肿瘤坏死因子刺激单核/巨噬细胞信号通路的生化机制，以及肿瘤坏死因子诱导的信号转导影响巨噬细胞存活的机制，对于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(GPCRKs)-2和-5在巨噬细胞的肿瘤坏死因子受体信号转导中发挥着新的和关键的作用。这表明GRKs可能在关节炎的发病机制中发挥关键作用。GRK是一种丝氨酸/苏氨酸蛋白激酶，最初被发现是因为它们能够磷酸化激动剂占据的G蛋白偶联受体(GPCRs)。GRK2和GRK5在肿瘤坏死因子信号转导中的作用代表了一种新的信号模式，因此了解这些GRK2和5调节巨噬细胞中的肿瘤坏死因子信号的机制以及在肿瘤坏死因子介导的疾病如关节炎中的作用值得进一步研究。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。DAPK是一种关键的信号蛋白，参与调节肿瘤坏死因子诱导的多种细胞类型的生长/凋亡。在这项授权中，我们建议扩大我们关于GPCRK在巨噬细胞中的肿瘤坏死因子信号转导中的新作用的初步发现。这一应用的中心假设是GPCRK在肿瘤坏死因子信号和巨噬细胞存活中发挥关键作用，因此在关节炎的发病机制中起关键作用。为了验证这一假说，我们建议检验以下特定目标：1.确定GRK2与死亡相关蛋白激酶(DAPK)的相互作用在肿瘤坏死因子诱导的巨噬细胞存活/凋亡中的作用。2.鉴定和鉴定肿瘤坏死因子刺激下巨噬细胞中与GRK2相互作用的新信号体。3.确定GRK2和GRK5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有关GRKs在巨噬细胞中TNFR信号的细胞、生化和病理生理学基础中的作用的关键问题，这将成为确定炎症性疾病(如关节炎)中TNFR信号的分子/治疗靶点的基础。研究背景：肿瘤坏死因子作用于巨噬细胞，通过激活信号通路，在包括关节炎在内的多种疾病的发生发展中发挥重要作用。这项应用的主要目标是了解这些信号通路是如何由巨噬细胞和关节炎中的肿瘤坏死因子调节的。

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.