IgSF11 Signaling Controls Osteoclast Maturation and Pathogenic Bone Loss

IgSF11 信号传导控制破骨细胞成熟和致病性骨质流失

• 批准号: 10337682
• 负责人: YONGWON CHOI
• 金额: $ 35.75万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10337682
• 关键词: Affect; Biological; Biological Assay; Cell Surface Receptors; Cells; Colitis; Complex; Coupled; Data; Disease; Enzymes; Future; Genetic Transcription; Glycolysis; Homeostasis; Human; Immunoglobulins; Inflammation; Inflammatory; JAK2 gene; Link; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Mind; Modeling; Modification; Molecular; Mus; Myelogenous; Nature; Osteoblasts; Osteoclasts; Osteogenesis; Pathogenicity; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotransferases; Polyubiquitination; Process; Proteins; Pyruvate Kinase; Regulation; Role; SRC gene; Scaffolding Protein; Signal Pathway; Signal Transduction; TRAF6 gene; Testing; Therapeutic; Validation; base; bone; bone loss; bone resorbing activity; bone strength; clinically relevant; dextran sulfate sodium induced colitis; experimental study; inflammatory bone loss; inhibitor; member; mutant; novel; prevent; small molecule; src-Family Kinases

Under inflammatory conditions, bone destruction can be linked to excessive activity of bone-resorbing osteoclasts (OCs), which results not only from the differentiation of too many OCs, but also from over- maturation of OCs. While most current bone loss treatments prevent bone loss by reducing OC numbers, it may be better if future therapeutic strategies focus on targeting OC maturation rather than early OC differentiation to avoid inhibiting coupled bone formation that depends on interactions between bone-forming osteoblasts and OCs. In an effort to target maturation, we previously identified immunoglobulin superfamily member 11 (IgSF11) as a novel cell surface receptor that regulates OC differentiation but not new bone formation. To characterize IgSF11 signaling, we analyzed, by mass spectrometry, proteins phosphorylated after IgSF11 activation and identified Pyruvate kinase M2 (PKM2), the enzyme that catalyzes the last step of glycolysis, as a downstream target. This finding highlights a potentially greater than previously known determinative role for metabolic regulation during OC differentiation and inflammatory bone loss. We therefore propose the following specific aims: 1. Examine the role of IgSF11-PKM2 signaling in inflammatory bone loss. We will investigate OC-expressed IgSF11 in the context of inflammatory bone loss by using an LPS-induced model of bone loss. To test the contribution of PKM2-dependent effects, we will treat LPS-induced IgSF11-/- mice with small molecule modulators (TEPP-46, shikonin) of PKM2. Our preliminary data suggests that TEPP- 46 activation of PKM2 reduces DSS-induced bone loss. To examine whether IgSF11 expression affects colitis- associated bone loss, we will perform DSS-induced colitis experiments using IgSF11-deficient mice. We will also perform DSS-induced colitis experiments using IgSF11-/- mice treated with TEPP-46 or shikonin. These studies will be critical to establishing the intersection of IgSF11 and PKM2 contributions to clinically relevant inflammatory bone loss. 2. Characterization of IgSF11-PKM2 signaling mechanisms in osteoclast differentiation. We have formulated a four-step model of IgSF11-PKM2 function during OC differentiation, which we will test with the aid of hCD3-iFL, a retroviral (RV) construct to directly activate intracellular IgSF11 in differentiating OCs. We will first investigate possible crosstalk between RANK and IgSF11-PKM2, which we speculate is mediated by TRAF6-dependent K63-linked polyubiquitination of the IgSF11 scaffold protein PSD- 95. Second, we aim to identify kinases proximal to the IgSF11-PSD-95 complex that phosphorylate PKM2. Third, we will use RV mutants to confirm the importance of various PKM2 modifications, PKM2 allosteric confirmation, and PKM2 subcellular localization to OC differentiation. Finally, PKM2 is a well-characterized enzymatic regulator of glycolysis, so we will employ metabolic assays and inhibitors to confirm the significance of this aspect of PKM2 function to OC differentiation. These studies will be critically important to initial validation and characterization of a putative IgSF11-PKM2 pathway and its function during OC differentiation.

在炎症条件下，骨破坏可能与骨吸收的过度活动有关 破骨细胞（OCs），这不仅是由于太多的OCs分化，而且也是由于过度， OCs的成熟。虽然目前大多数骨丢失治疗通过减少OC数量来防止骨丢失，但 如果未来的治疗策略集中于靶向OC成熟而不是早期OC， 分化，以避免抑制依赖于骨形成之间的相互作用的偶联骨形成。 成骨细胞和OC。为了靶向成熟，我们先前鉴定了免疫球蛋白超家族， 成员11（IgSF 11）作为一种新的细胞表面受体，调节OC分化，但不调节新骨 阵为了表征IgSF 11信号传导，我们通过质谱分析了磷酸化的蛋白质， 在IgSF 11活化后，鉴定了丙酮酸激酶M2（PKM 2），该酶催化了 糖酵解作为下游靶点。这一发现强调了一个潜在的比以前已知的更大的 在OC分化和炎性骨丢失过程中代谢调节的决定性作用。因此我们 提出以下具体目标：1.检查IgSF 11-PKM 2信号在炎性骨丢失中的作用。 我们将通过使用LPS诱导的免疫荧光技术研究炎性骨丢失背景下OC表达的IgSF 11。 骨质流失模型为了测试PKM 2依赖性作用的贡献，我们将LPS诱导的IgSF 11-/- 用PKM 2的小分子调节剂（TEPP-46，紫草素）的小鼠。我们的初步数据表明，TEPP- PKM 2的活化减少DSS诱导的骨丢失。为了检查IgSF 11表达是否影响结肠炎- 因此，我们将使用IgSF 11缺陷型小鼠进行DSS诱导的结肠炎实验。我们将 还使用用TEPP-46或紫草素处理的IgSF 11-/-小鼠进行DSS诱导的结肠炎实验。这些 研究对于建立IgSF 11和PKM 2对临床相关疾病的交叉贡献至关重要。 炎性骨质流失2.破骨细胞IgSF 11-PKM 2信号转导机制的研究 分化我们已经建立了OC分化过程中IgSF 11-PKM 2功能的四步模型， 我们将在hCD 3-iFL的帮助下进行测试，hCD 3-iFL是一种逆转录病毒（RV）构建体，可直接激活细胞内IgSF 11， 区分OC。我们将首先研究RANK和IgSF 11-PKM 2之间可能的串扰， 推测是由TRAF 6依赖性K63连接的IgSF 11支架蛋白PSD的多聚泛素化介导的。 95.其次，我们的目标是确定激酶的IgSF 11-PSD-95复合物磷酸化PKM 2的近端。 第三，我们将使用RV突变体来证实各种PKM 2修饰的重要性，PKM 2变构， 确认，和PKM 2亚细胞定位到OC分化。最后，PKM 2是一种特征良好的 糖酵解的酶调节剂，因此我们将采用代谢试验和抑制剂来证实其意义 PKM 2在OC分化中的功能。这些研究对最初的研究至关重要。 验证和表征推定的IgSF 11-PKM 2途径及其在OC分化过程中的功能。