The Focus: Localized provoked vulvodynia (LPV) is the most common cause of chronic dyspareunia (painful intercourse) in premenopausal women, and it remains a poorly understood disease. Existing therapies do not target the underlying causes, treatment is trial and error, and intervention typically escalates to cutting away the painful tissue surrounding the vaginal opening, the vestibule. This proposal aims to identify new targets to treat LPV pain in alignment with the missions of at least two NIH institutes by aiming to 1) improve women’s health care (NICHD) and 2) identify non-addictive targets for pain therapy (NIDA). The Premise: We discovered a non-classical inflammatory response in the vestibule of LPV patients that is a key contributor to LPV pain. The vestibule of LPV patients is hypersensitive to inflammatory stimuli, causing a response when one would not otherwise occur, which is characterized by high levels of proinflammatory mediators. There is a direct association between proinflammatory mediator levels and pain; fibroblasts taken from sites of exquisite pain produce the highest levels of these mediators, indicating the vestibule could be uniquely vulnerable and could be specifically targeted to resolve pain. We subsequently identified several candidates for therapeutic intervention (e.g. Dectin-1, Nuclear kappa factor B). However, targeting these would not completely alleviate proinflammatory signaling or might compromise host defenses. Our goal is to identify and validate new therapeutic targets for LPV pain therapy. Our supporting data strongly suggest that transient receptor potential vanilloid receptor 4 (TRPV4) and members of its signaling pathway represent promising and innovative therapeutic targets. We will confirm TRPV4’s role in LPV, validate the likely therapeutic targets, and in the process, enhance our mechanistic understanding of vulvodynia. Organizing Hypothesis: We hypothesize that targeting the TRPV4 pathway will reduce pro-nociceptive signaling in human fibroblasts and tissue and impart analgesia in mice Specific Aim 1: Elucidate the role of site-specific TRPV4 signaling differences to identify new therapeutic targets for LPV. Specific Aim 2: Explore the relationship between inflammation, TRPV4, and alterations in lipid profiles in LPV patients. Specific Aim 3: Validate TRPV4 and other identified targets using 3D tissue culture and an in vivo LPV model. Impact on the field: We plan to accomplish three goals: 1) identify and validate new targets for desperately needed non-invasive and efficacious vulvodynia therapies, 2) improve understanding of the vulvodynia mechanism, and 3) identify mechanisms likely conserved in other pain conditions by focusing on a ubiquitous signaling pathway (TRPV4) suspected to play a role in pain syndromes, targeting of which would be unlikely to result in adverse sequelae, including drug dependence disorders.