Abstract Hip osteoarthritis (OA) affects one in four people by the age of 85, and it is linked to abnormal hip morphology including Cam-type femoroacetabular impingement (FAI). Hence, symptomatic FAI represents an ideal condition to identify key regulators in hip OA progression. To this end, we performed RNA sequencing (RNA- seq) comparing FAI and hip OA cartilage, revealing that WNT16 expression is negatively correlated with OA severity. We also showed that WNT16 is decreased during cartilage cell (i.e., chondrocyte) hypertrophy (a hallmark of OA) and that WNT16 is up-regulated in chondrocytes under mechanical loading. As Transient Receptor Potential Vanilloid 4 (TRPV4), a Ca2+-permeable ion channel, is an essential mechanosensor in chondrocytes, our results suggest a novel link between TRPV4, WNT16, and chondrocyte mechanobiology. Our preliminary data predict that WNT16 in chondrocytes is potentially regulated by TRPV4-mediated signaling pathways, and WNT16 inhibits chondrocyte hypertrophy via G protein-associated signal transduction. Thus, to elucidate the functional role of WNT16 in hip OA pathogenesis and develop novel therapies, we propose the following aims: Specific Aim 1: To determine the genetic and epigenetic mechanism(s) of mechanoinduction of WNT16 in chondrocytes. Agarose constructs encapsulating human stem cell-derived chondrocytes and hip primary chondrocytes will be subjected to loading in combination of TRPV4 activator/inhibitor, and their effects on WNT16 will be assessed. We will use novel next-generation sequencing to identify genetic and epigenetic factors that regulate WNT16 expression in chondrocytes. Specific Aim 2: To determine the effects of WNT16 gain- and loss-of-function on chondrocyte specification, hypertrophy, and hip OA development. We will investigate if cartilage-specific Wnt16 knockout mice exhibit enhanced hip OA compared to control mice. We will measure OA severity, synovitis, and bone remodeling. We will also quantify behavioral changes and pain responses, as well as determine the association of these measurements with hip OA severity. We will use human hip primary chondrocytes with WNT16 modulation to investigate its effects on hypertrophy. Specific Aim 3: To elucidate altered chondrocyte cell-cell crosstalk in hip healthy/FAI/OA cartilage and to determine if WNT16 mRNA delivery can mitigate hip OA by restoring normal WNT signaling among chondrocyte crosstalk. We will fully characterize distinct chondrocyte phenotypes in human healthy, FAI, and OA hip cartilage by integrating scRNA/Spatial-seq datasets. We will determine if WNT16 mRNA delivery using nanoparticles to hip FAI/OA cartilage explants is sufficient to mitigate hip OA progression. Impact: A mechanistic understanding of the role of WNT16 in hip cartilage homeostasis and FAI/OA will provide insights into the development of therapeutic interventions for hip OA by targeting WNT16 signaling.