Elucidating the JNK2-mediated redox regulation of cartilage matrix remdeling

描述(由申请人提供)：高龄是发生骨关节炎(OA)的最大危险因素。越来越多的证据表明，氧化应激是导致衰老和与年龄相关的疾病(如骨性关节炎)发展的重要因素。在骨性关节炎患者的软骨中观察到活性氧物种(ROS)水平的升高，这可能在促进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第二信使，刺激软骨细胞产生称为基质金属蛋白酶(MMPs)的ECM降解酶。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，而抑制ROS可阻断Fn-f刺激的基质金属蛋白酶-13的表达。然而，ROS的来源以及ROS介导这一整合素信号通路的机制仍不清楚。因此，这项研究的目的是阐明软骨细胞中受刺激的a5？1整合素途径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氨基末端激酶2(JNK2)，被Fn-f刺激的ROS靶向，并被氧化到产生半胱氨酸磺酸的特定半胱氨酸残基上。使用不同的氧化酶和Rho家族蛋白的化学和分子抑制剂，我们将研究ROS的酶来源以及参与翻译整合素信号以激活软骨细胞ROS产生的蛋白质。这项拟议的工作还将利用质谱学、突变蛋白质分析和一种名为二美酮的特定磺酸标记试剂来确定ROS的蛋白质靶标(S)，这些蛋白质靶标受半胱氨酸在a5？1整合素途径中形成的调节。这些研究结果将有助于解释在骨性关节炎的发生发展过程中，过量的ROS产生导致软骨内环境平衡失衡和ECM降解增加的机制。骨关节炎是一种与年龄相关的疾病，是美国最常见的关节炎类型，预计到2020年将影响大约6000万美国人。这项拟议的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可以减缓或阻止骨关节炎患者软骨丢失的潜在治疗靶点。

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.