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

基本信息

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
项目类别：
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

项目摘要

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，骨骼破坏可能与骨头敏感的过度活性有关破骨细胞（OCS），这不仅是由于太多OC的分化而导致的，而且还来自过度 OC的成熟。虽然当前大多数骨质流失治疗可以通过减少OC数来阻止骨质流失，但如果将来的治疗策略专注于靶向OC成熟而不是早期OC，则可能会更好分化以避免抑制取决于骨形成之间相互作用的耦合骨形成成骨细胞和OC。为了靶向成熟，我们先前确定了免疫球蛋白超家族成员11（IGSF11）作为调节OC分化的新细胞表面受体，而不是新骨形成。为了表征IGSF11信号传导，我们通过质谱法分析了蛋白质磷酸化在IGSF11激活并鉴定出丙酮酸激酶M2（PKM2）之后，催化了最后一步的酶糖酵解，作为下游靶标。这一发现突出了可能比以前已知的更大的 OC分化和炎症性骨质流失期间代谢调节的决定性作用。因此，我们提出以下具体目的：1。检查IGSF11-PKM2信号传导在炎症性骨质流失中的作用。我们将通过使用LPS诱导的炎症性骨质流失的背景下调查OC表达的IGSF11 骨丢失模型。为了测试PKM2依赖性效应的贡献，我们将处理LPS诱导的IGSF11 - / - PKM2的小分子调节剂（TEPP-46，Shikonin）的小鼠。我们的初步数据表明Tepp- 46 PKM2的激活减少了DSS诱导的骨质流失。检查IGSF11表达是否影响结肠炎相关的骨质流失，我们将使用IGSF11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shikonin处理的IGSF11 - / - 小鼠进行DSS诱导的结肠炎实验。这些研究对于建立IGSF11和PKM2对临床相关的贡献至关重要炎症性骨质流失。 2。破骨细胞中IGSF11-PKM2信号传导机制的表征分化。我们在OC分化过程中制定了IGSF11-PKM2函数的四步模型，我们将借助HCD3-IFL（逆转录病毒（RV）构建体直接激活细胞内IGSF11 区分OC。我们将首先调查等级和IGSF11-PKM2之间可能的串扰，我们猜测是由Traf6依赖性K63连接的多泛素化介导的IGSF11支架蛋白PSD- 95。第二，我们旨在鉴定与磷酸化PKM2的IGSF11-PSD-95复合物接近的激酶。第三，我们将使用RV突变体确认各种PKM2修饰的重要性PKM2变构确认和PKM2亚细胞定位到OC分化。最后，PKM2是一个很好的特征糖酵解的酶调节剂，因此我们将使用代谢测定法和抑制剂来确认显着性 PKM2函数与OC分化的这一方面。这些研究对初始研究至关重要推定的IGSF11-PKM2途径及其在OC分化过程中的功能的验证和表征。