Joint trauma and subsequent osteoarthritis (OA) remain significant injuries in racehorses and the aging human population. Arthritis remains second only to tendon and ligament injury as the leading cause of racehorse retirement. In man, it is the leading disability among adults and the incidence is expected to double by the year 2020.  There are no treatments that markedly alter the progression of OA.  Proteoglycan-4 (PRG4), also known as lubricin, has been identified as the primary boundary lubricant in synovial fluid, where it prevents cartilage fibrillation and degradation under high-load by decreasing friction at the articular cartilage gliding surfaces.  In addition, lubricin may protect against the development of OA by decreasing synovial cell adhesion to the articular cartilage surface, inhibiting synoviocyte overgrowth, and inhibiting protein deposition and pannus formation.  We propose that lubricin may be an effective exogenous biologic for articular injection. The aims of the proposed research are to characterize and evaluate PRG4. This molecule has been recognized as part of the joint surface proteoglycan milieu, but changes in lubricin levels in synovitis and OA, the role of concurrent lubricin synthesis from the synovial lining, and the correlation and binding at both cartilage and synovial layers is poorly understood. The potential for PRG4 as an intraarticular supplement by direct injection or gene delivered PRG4 therapy is novel, and may provide a new and more persisting therapeutic alternative to HA and other routine intraarticular medications. Before this can happen, PRG4 needs extensive evaluation through purification, identification of mechanistic partners, definition of the biophysical organization of lubricin and associated molecules on the surface of articular cartilage, and finally relating this spatial and topographic information to the function of lubricin as both a boundary lubricant and chondroprotective agent.  The studies will lead to later in vivo analyses of exogenous PRG4 in an equine carpal chip model of OA. The long-term goal of this proposal is to fully characterize the physicochemical and metabolic behavior of PRG4. We hypothesize that PRG4 enhances normal joint function by binding to cartilage surface partners to enhance high load frictionless motion, improve cartilage surface layer metabolism, and normalize synovial membrane barrier and synthetic functions.  This hypothesis will be tested by several specific aims.  Aim 1. Determine lubricin levels and expression in normal joints and those with synovitis and OA. Aim 2. Investigate how mechanical properties of the lubricin boundary layer emerge from its nanoscale organization and galectin crosslinking.  Aim 3. Delineate the importance of specific molecular domains in the functional assembly of the lubricin boundary layer.  Aim 4. Determine how cells adhere and migrate along or through the lubricin boundary layer. The broad objectives of this proposal are to examine the biophysical organization of lubricin on articular cartilage and to relate this information to lubricin’s role as a chondroprotective agent.  Studies suggest that lubricin protects articular cartilage from fibrillation by functioning as a boundary lubricant and by preventing synovial cell overgrowth and protein deposition.  We seek to understand the underlying mechanisms by which lubricin confers these properties in the synovial environment.  Little is known about the structural organization of the boundary layer or lubricin’s interaction with other molecules, particularly at the nanoscale.  This fundamental knowledge will inform the design of future therapeutic applications of lubricin for OA prevention.