Periodontitis is a bacteria-driven inflammatory bone loss disease affecting 47% of adults in the United States. Oral pathogens induce the generation of inflammatory cytokines (including IL-1β, IL-6, TNF-α, and RANKL), which attract monocytes to gingival tissues. Under RANKL stimulation, these mononuclear cells can further differentiate and fuse to form multinucleated osteoclasts, resulting in alveolar bone loss. Meanwhile, inflammatory cytokines associated with periodontitis inhibit osteoblast differentiation and bone regeneration. With current limited treatment modalities, periodontitis is still the major cause of alveolar bone loss and tooth loss in adults. Our long-term goal is to develop effective new therapies to treat the disease. We were the first to demonstrate that sphingosine-1-phosphate receptor 2 (S1PR2, a G-protein-coupled receptor) plays a key role in regulating the inflammatory bone loss response in periodontitis. Knockdown of S1PR2 by a S1PR2-directed shRNA or inhibition of S1PR2 by its specific inhibitor (JTE013) reduced PI3K, NF-κB, and MAPKs protein kinases induced by the oral pathogen Aggregatibacter actinomycetemcomitans (Aa), and decreased IL-1β, IL- 6, TNF-α levels induced by Aa. Moreover, treatment with the S1PR2 shRNA or JTE013 decreased cells adhesion units (podosome components) induced by RANKL, inhibited osteoclastogenesis and bone resorption induced by RANKL. Oral topical administration of JTE013 alleviated inflammatory bone loss in C57BL/6 mice with periodontitis induced by ligature placement. However, there is a knowledge gap on how S1PR2 regulates osteoblastogenesis and bone regeneration. Thus, the goal of this application is to determine the role of S1PR2 in controlling osteoblastogenesis and bone regeneration. Our preliminary data show that inhibition of S1PR2 by JTE013 in bone marrow-derived stromal cells (BMSCs) cultured in osteogenic media increases osteoblastogenesis compared with vehicle treatment. Treatment with JTE013 increased the mRNA levels of osteogenic genes, including alkaline phosphatase, Runt-related transcription factor 2 (Runx2), osteocalcin, osterix, and bone morphogenetic protein (BMP) 2 compared with vehicle treatment. Additionally, treatment with JTE013 increased the levels of osteogenic proteins, including BMP2, BMP7, BMP receptor II, BMP receptor 1A, and p-Smad 1/5/9 protein compared with control vehicle treatment. Thus, we hypothesize that inhibition of S1PR2 in pre-osteoblast cells promotes osteoblastogenesis and bone regeneration. Our specific aims will determine 1) if shRNA knockdown or inhibition of S1PR2 in vitro will increase osteoblastogenesis via BMPs/Smad signaling pathway and/or other signaling pathways; and 2) whether pharmacological inhibition of S1PR2 by JTE013 in mice with periodontitis promotes alveolar bone regeneration following inflammatory bone loss. This application defines novel signaling pathways regulated by S1PR2 in promoting osteoblastogenesis. It will lay the foun...