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 ex...