SIRT1 Signaling in Injurious Chondrocyte Mechaotransduction

项目总结 关节的高能量创伤会给软骨组织带来机械过载，并导致 软骨细胞的损伤反应，经常导致创伤后骨关节炎(PTOA)。因为软骨 内在修复能力有限，临床上对治疗软骨损伤的新疗法的需求尚未得到满足。 并抑制PTOA的进展。介导细胞损伤反应的机械敏感信号通路 软骨细胞对机械过载的影响还不是很清楚。填补这些知识空白可能会提供 关节损伤后预防或延缓PTOA发展的新治疗靶点。 我们的初步数据表明，Sirtuin1(SIRT1)是一种依赖NAD的蛋白去乙酰基酶，是一种新的- 在软骨细胞对损伤性超负荷的反应中发现了机械敏感信号分子。SIRT1活动 在亚致死冲击超载后的5分钟内，牛软骨移植块中的含量下降，并保持在 被压制了至少24小时。初步的实验结果也提示了可能的调节途径 SIRT1停用。重要的是，SIRT1的药理激活完全挽救了急性损伤 软骨移植中的反应。这项研究的第一个目标是定义上游信号通路， 调节软骨细胞损伤机械反应中的SIRT1失活。这将在#年完成 目的1使用药物抑制剂和CRISPR/Cas9策略。此外，主要的机制是 SIRT1调节细胞过程是蛋白质去乙酰化。因此，第二个目标是分析 用乙酰化组法测定机械负载软骨细胞中SIRT1下游底物，并阐明 这些脱乙酰靶在软骨细胞损伤反应和/或软骨细胞行为中的作用。这 将在目标2中用蛋白质组学方法用机械方法鉴定脱乙酰基底物来实现目标 超载。这些靶点中至少有一个在机械过载和/或软骨损伤反应中的作用 将对健康状况进行评估。最后，SIRT1的调节是否维持了软骨的动态平衡 受伤情况将在目标3中进行评估。 这项拟议的研究有了新的突破，因为它研究了机械诱导的酶 SIRT1的失活，它以前没有被认为是软骨细胞中的机械敏感性，在 软骨的撞击损伤反应。由于sirtuins最初被认为是衰老和 长寿，成功完成拟议的工作可能会在伤害诱导和 与年龄相关的骨关节炎，改变了我们对这种疾病的理解。此外，了解 SIRT1酶失活上游和下游的信号通路将识别新的分子 在软骨细胞的损伤反应中的事件，并可能揭示以前未发现的软骨调节 健康。这些结果有望成为治疗软骨损伤的新方法和新方法的基础 诱导关节软骨修复的治疗靶点。

PROJECT SUMMARY High-energy trauma to an articular joint delivers a mechanical overload to cartilage tissue and causes an injury response in chondrocytes that frequently leads to post-traumatic osteoarthritis (PTOA). Because cartilage has limited intrinsic repair capabilities, there is an unmet clinical need for new therapies to treat cartilage injury and inhibit progression of PTOA. The mechanosensitive signaling pathways that mediate the injury response of chondrocytes to mechanical overloads are not well understood. Filling these gaps in knowledge may provide new therapeutic targets following joint injury that prevent or delay the development of PTOA. Our preliminary data identify Sirtuin1 (SIRT1), an NAD+-dependent protein deacetylase, as a newly- discovered mechanosensitive signaling molecule in chondrocytes’ response to injurious overload. SIRT1 activity decreased in bovine cartilage explants within 5 minutes of a sublethal impact overload, and remained suppressed for at least 24 hours. Preliminary experimental results also suggest the likely pathway that regulates SIRT1 deactivation. Importantly, pharmacological activation of SIRT1 completely rescued the acute injury response in the cartilage explants. The first objective of this study is to define upstream signaling pathways that regulate SIRT1 deactivation in the injurious mechanoresponse of chondrocytes. This will be accomplished in Aim 1 using pharmacological inhibitors and a CRISPR/Cas9 strategy. Additionally, the major mechanism for SIRT1 to regulate cellular processes is to deacetylate proteins. Therefore, a second objective is to analyze the acetylome to determine the downstream substrates of SIRT1 in mechanically loaded chondrocytes, and to clarify the role of these deacetylation targets in the chondrocyte injury response and/or chondrocyte behavior. This objective will be met in Aim 2 with a proteomics approach to identify deacetylation substrates with mechanical overload. The role of at least one of these targets in the injury response to mechanical overload and/or cartilage health will be evaluated. Finally, whether SIRT1 regulation maintains cartilage homeostasis following cartilage injury will be assessed in Aim 3. The proposed study breaks new ground, as it investigates the mechanically induced enzymatic deactivation of SIRT1, which had not previously been recognized as mechanosensitive in chondrocytes, in the impact injury response in cartilage. As sirtuins were initially recognized as pivotal regulators of aging and longevity, successful completion of the proposed work may provide a molecular link between injury-induced and age-related osteoarthritis, transforming our understanding of the disease. Furthermore, understanding the signaling pathways that are upstream and downstream of SIRT1 enzyme deactivation will identify new molecular events in the injury response of chondrocytes and may reveal previously undiscovered regulators of cartilage health. These results are expected to form the basis for new approaches to treat cartilage injury and novel therapeutic targets to induce articular cartilage repair.