Role of sphingosine-1-phosphate receptor 2 in osteoblastogenesis and bone regeneration in periodontitis

牙周炎是一种细菌驱动的炎症性骨丢失疾病，在美国有47%的成年人受到影响。 口腔病原体诱导产生炎性细胞因子(包括IL-1β、IL-6、肿瘤坏死因子-α和RANKL)， 将单核细胞吸引到牙龈组织中。在RANKL刺激下，这些单个核细胞可以进一步 分化融合形成多核破骨细胞，导致牙槽骨丢失。同时， 与牙周炎相关的炎性细胞因子抑制成骨细胞分化和骨再生。 在目前有限的治疗方式下，牙周炎仍然是牙槽骨丢失和牙齿的主要原因。 成人的损失。我们的长期目标是开发有效的新疗法来治疗这种疾病。我们是第一个 证明神经鞘氨醇-1-磷酸受体2(S1PR2，一种G蛋白偶联受体)起关键作用 在调节牙周炎的炎症性骨丢失反应方面。由S1PR2导向的S1PR2基因敲除 ShRNA或其特异性抑制剂(JTE013)抑制S1PR2使PI3K、NF-κB和MAPKs蛋白减少 口腔病原菌伴生放线杆菌(Aggregatibacter Actinmycetcomitans，AA)诱导的β、IL-1、IL-4、IL-6、IL-4、IL-4和IL-4的表达 6、AA诱导的肿瘤坏死因子-α水平。此外，用S1PR2 shRNA或JTE013处理细胞减少 RANKL诱导的黏附单位(足体成分)抑制破骨细胞生成和骨吸收 由RANKL诱导。口服JTE013减轻C57BL/6小鼠炎性骨丢失 因扎扎术引起的牙周炎。然而，关于S1PR2是如何监管的，还存在一个知识缺口 成骨细胞生成和骨再生。因此，本应用程序的目标是确定S1PR2的角色 在控制成骨细胞形成和骨再生方面的作用。我们的初步数据显示，通过抑制S1PR2 成骨介质中培养的骨髓基质细胞中JTE013表达增加 成骨细胞移植与赋形剂治疗的比较。经JTE013处理后，大鼠肾小管上皮细胞基因表达水平升高。 成骨基因包括碱性磷酸酶、Runx2相关转录因子、骨钙素、 Osterix、骨形态发生蛋白(BMP)2与赋形剂治疗进行比较。此外，还可以使用 JTE013增加成骨蛋白水平，包括BMP2、BMP7、BMP受体II、BMP受体 1a和p-Smad1/5/9蛋白，与对照载体处理比较。因此，我们假设抑制作用 成骨前细胞中S1PR2的表达促进成骨细胞的形成和骨再生。我们的具体目标 将确定1)体外下调shRNA或抑制S1PR2是否会通过以下途径促进成骨细胞的形成 BMPS/Smad信号通路和/或其他信号通路；2)药物抑制 JTE013诱导牙周炎小鼠S1PR2促进炎症骨后牙槽骨再生 损失。这一应用定义了由S1PR2调控的促进成骨细胞形成的新的信号通路。 这将为开发一种新的牙周炎治疗方法奠定基础 炎症性骨丢失，同时促进骨再生。

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 foundation to develop a novel therapeutic approach for patients with periodontitis to alleviate inflammatory bone loss, while promoting bone regeneration.