PROJECT SUMMARY Osteoarthritis (OA) is a total joint disease characterized by articular cartilage degradation, synovial inflammation, meniscus degeneration, subchondral bone sclerosis, osteophyte formation, and joint pain. It afflicts nearly 1/4 of the US population resulting in healthcare expenditures exceeding $185 billion annually. Despite the severity and impact of OA on individuals and our health care system, only recently have there been advances in understanding the molecular, cellular and tissue events underlying OA development. It is well established that inappropriate expression and activation of catabolic enzymes underlies the joint cartilage destruction observed in OA, however the precise molecular mechanisms responsible for promoting joint cartilage catabolism is not well understood, nor is there a defined understanding of the molecular mediators of OA-associated pain. Recently an up-regulation in the NOTCH signaling pathway has been documented in human and murine post-traumatic OA (PTOA), suggesting a connection between aberrant NOTCH signaling and OA. Using cartilage-specific loss-of-function and gain-of-function mouse genetic approaches, we demonstrated that physiological NOTCH signaling is required for long-term maintenance of the joint cartilages, however enhanced NOTCH signaling leads to a progressive OA pathology and chronic pain. We further utilized RNA-sequencing, immunohistochemistry (IHC), and biochemical approaches to identify several NOTCH targets potentially responsible for NOTCH-mediated joint cartilage catabolism and OA-associated pain; factors that are also dysregulated during OA progression in both mice and humans. Our published and preliminary data identified IL-6/JAK/STAT and nerve growth factor (NGF) signaling as highly relevant NOTCH targets that may significantly contribute to joint cartilage catabolism and pathological pain observed in NOTCH-induced OA and PTOA. Therefore, we will test the overarching hypothesis that pathologic NOTCH signaling activation promotes IL-6/JAK/STAT and NGF signaling to induce joint cartilage catabolism and pain in OA. A variety of in vivo and in vitro approaches will identify the NOTCH pathway and critical downstream effectors (IL-6/JAK/STAT and NGF) as important regulators of OA-associated cartilage catabolism and pain, while simultaneously testing NOTCH neutralizing antibodies as a translational disease modifying osteoarthritis drug (DMOAD) therapy.