Elucidating the JNK2-mediated redox regulation of cartilage matrix remdeling

描述（由申请人提供）：高龄是发展骨关节炎（OA）的单一最大危险因素。越来越多的证据表明，氧化应激是导致衰老和与年龄有关的疾病（如OA）发展的重要因素。在OA患者的软骨中观察到活性氧（ROS）水平升高，可能在刺激关节软骨细胞外基质（ECM）降解中起作用。ROS已被证明是整合素和细胞因子信号通路中的次级信使，刺激软骨细胞产生ECM降解酶，称为基质金属蛋白酶（MMPs）。纤维连接蛋白片段（FN-f）刺激a5¿1整合素可增加软骨细胞内ROS水平，而ROS抑制可阻断FN-f刺激的MMP-13表达。然而，ROS的来源和ROS介导整合素信号通路的机制尚不清楚。因此，本研究的目的是阐明软骨细胞中受刺激的a5¿1整合素通路中氧化还原调节的关键信号事件。我们的一般假设是，整合素途径中氧化还原调节的信号蛋白，包括c-Jun n -末端激酶2 (JNK2)，被FN-f刺激的ROS靶向，并在特定的半胱氨酸残基上氧化，产生半胱氨酸磺酸。利用不同氧化酶和Rho家族蛋白的化学和分子抑制剂，我们将研究ROS的酶促来源以及参与翻译整合素信号以激活软骨细胞中ROS产生的蛋白质。这项工作还将利用质谱、突变蛋白分析和一种特定的硫酸标记试剂，确定在a5¿1整合素途径中由半胱氨酸磺酸形成调节的ROS蛋白靶点。这些研究的结果将有助于解释在骨性关节炎的发展过程中，过多的ROS产生导致软骨稳态失衡和ECM降解增加的机制。骨关节炎是一种与年龄有关的疾病，是美国最常见的关节炎类型，预计到2020年将影响约6000万美国人。本研究将阐明活性氧在促进骨关节炎关节软骨破坏中的作用。这些研究的结果可能有助于确定潜在的治疗靶点，可以减缓或阻止骨关节炎患者的软骨损失。

DESCRIPTION (provided by applicant): Advanced age is the single greatest risk factor for developing osteoarthritis (OA). Accumulating evidence points to oxidative stress, as an important factor contributing to both ageing and the development of age- related diseases like OA. Elevated levels of reactive oxygen species (ROS) have been observed in the cartilage of OA patients and may play a role in the stimulation of articular cartilage extracellular matrix (ECM) degradation. ROS have been shown to serve as secondary messengers in integrin and cytokine signaling pathways which stimulate chondrocyte production of ECM degrading enzymes called matrix metalloproteinases (MMPs). Fibronectin fragment (FN-f) stimulation of the a5¿1 integrin has been shown to increase intracellular levels of ROS in chondrocytes, while ROS inhibition blocked FN-f stimulated MMP-13 expression. However, both the source of ROS and the mechanism by which ROS mediate this integrin signaling pathway remains unclear. Thus, the objective for this proposed research is to elucidate the key redox regulated signaling events in the stimulated a5¿1 integrin pathway in chondrocytes. Our general hypothesis is that redox-regulated signaling proteins within the integrin pathway, including c-Jun N-terminal kinase 2 (JNK2), are targeted by FN-f stimulated ROS and oxidized at specific cysteine residues generating cysteine sulfenic acids. Using chemical and molecular inhibitors of different oxidases and Rho family proteins, we will investigate the enzymatic source of ROS and the proteins involved in translating the integrin signal to activate ROS production in chondrocytes. This proposed work will also determine the protein target(s) of ROS that are regulated by cysteine sulfenic acid formation within the a5¿1 integrin pathway utilizing mass spectrometry, mutational protein analysis, and a specific sulfenic acid-labeling reagent called dimedone. The results of these studies will help to explain the mechanism by which excessive ROS production can lead to an imbalance in cartilage homeostasis and increased ECM degradation in the development of OA. Osteoarthritis, an age-related disease, is the most common type of arthritis in the United States and is predicted to affect approximately 60 million Americans by 2020. The proposed research will elucidate the role of reactive oxygen species in promoting cartilage breakdown in osteoarthritic joints. The results of these studies could help in the identification of potential therapeutic targets that can slow or stop cartilage loss in osteoarthritis patients.