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

1-磷酸鞘氨醇受体2在牙周炎成骨细胞生成和骨再生中的作用

• 批准号: 10445306
• 负责人: Hong Yu
• 金额: $ 18.88万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-06 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10445306
• 关键词: Actinobacillus actinomycetemcomitans; Adult; Affect; Alkaline Phosphatase; Alveolar Bone Loss; BMP2 gene; BMP7 gene; BMPR2 gene; Bacteria; Bone Marrow; Bone Morphogenetic Proteins; Bone Regeneration; Bone Resorption; Bone Tissue; C57BL/6 Mouse; Cell Adhesion; Cell fusion; Cells; Chronic; Couples; Data; Dimethyl Sulfoxide; Disease; Excision; Food; Foundations; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Generations; Genes; Gingiva; Goals; In Vitro; Infection; Inflammatory; Interleukin-1 beta; Interleukin-6; Knowledge; Ligature; Lipopolysaccharides; MAP Kinase Gene; Mammalian Cell; Maxilla; Membrane; Messenger RNA; Microbial Biofilms; Modality; Mononuclear; Mus; Natural regeneration; Oral; Osteoblasts; Osteocalcin; Osteoclasts; Patients; Periodontitis; Pharmacology; Play; Powder dose form; Process; Production; Protein Family; Protein Kinase; Proteins; Role; Signal Pathway; Signal Transduction; Silk; Smad Proteins; Sphingosine-1-Phosphate Receptor; Stromal Cells; TNF gene; TNFSF11 gene; Testing; Tissues; Tooth Loss; Tooth structure; Topical application; United States; alveolar bone; bone; bone morphogenetic protein receptors; cytokine; dysbiosis; inflammatory bone loss; inhibitor; knock-down; macrophage; mesenchymal stromal cell; monocyte; novel; novel therapeutic intervention; novel therapeutics; oral pathogen; osteoblast differentiation; osteoblast proliferation; osteoclastogenesis; osteogenic; osteogenic protein; receptor; recruit; response; small hairpin RNA; transcription factor

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.

牙周炎是一种由细菌引起的炎症性骨质流失疾病，影响着美国 47% 的成年人。 口腔病原体诱导炎症细胞因子的产生（包括 IL-1β、IL-6、TNF-α 和 RANKL）， 在 RANKL 刺激下，这些单核细胞可以进一步吸引单核细胞到牙龈组织。 分化并融合形成多核破骨细胞，导致牙槽骨丢失。 与牙周炎相关的炎症细胞因子抑制成骨细胞分化和骨再生。 由于目前治疗方式有限，牙周炎仍然是牙槽骨流失和牙齿缺失的主要原因。 我们的长期目标是开发有效的新疗法来治疗这种疾病。 证明 1-磷酸鞘氨醇受体 2（S1PR2，一种 G 蛋白偶联受体）发挥关键作用 通过 S1PR2 介导的敲低 S1PR2 来调节牙周炎中的炎症性骨质流失反应。 shRNA 或通过其特异性抑制剂 (JTE013) 抑制 S1PR2 可减少 PI3K、NF-κB 和 MAPK 蛋白 由口腔病原体放线菌聚集杆菌 (Aa) 诱导的激酶，并减少 IL-1β、IL- 如图6所示，Aa诱导的TNF-α水平此外，用S1PR2 shRNA或JTE013处理降低了细胞。 RANKL 诱导的粘附单位（足小体成分），抑制破骨细胞生成和骨吸收 RANKL 口服局部给药 JTE013 可减轻 C57BL/6 小鼠的炎症性骨质流失。 然而，对于 S1PR2 如何调节仍存在知识空白。 因此，本申请的目标是确定 S1PR2 的作用。 我们的初步数据表明，通过抑制 S1PR2。 成骨培养基中培养的骨髓基质细胞 (BMSC) 中的 JTE013 增加 与载体治疗相比，JTE013 治疗增加了成骨细胞的 mRNA 水平。 成骨基因，包括碱性磷酸酶、Runt相关转录因子2（Runx2）、骨钙素、 osterix 和骨形态发生蛋白 (BMP) 2 与媒介物治疗相比。 JTE013增加成骨蛋白的水平，包括BMP2、BMP7、BMP受体II、BMP受体 1A 和 p-Smad 1/5/9 蛋白与对照载体处理相比因此，我们捕获了这种抑制。 前成骨细胞中的 S1PR2 促进成骨细胞生成和骨再生。 将确定 1) 体外 shRNA 敲低或抑制 S1PR2 是否会通过以下方式增加成骨细胞生成 BMPs/Smad信号通路和/或其他信号通路；以及2)是否有药理抑制作用； JTE013 的 S1PR2 在牙周炎小鼠体内促进炎症骨后牙槽骨的再生 该申请定义了 S1PR2 在促进成骨细胞生成中调节的新信号通路。 这将为开发一种新的治疗方法来缓解牙周炎患者的症状奠定基础。 炎症性骨质流失，同时促进骨再生。