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，骨破坏可能与骨吸收过度活动有关破骨细胞（OC），这不仅是由于过多的 OC 分化造成的，而且也是由于过度的破骨细胞分化所致。 OC 的成熟。虽然目前大多数骨质流失治疗方法都是通过减少 OC 数量来预防骨质流失，但如果未来的治疗策略侧重于 OC 成熟而不是早期 OC 可能会更好分化以避免抑制依赖于成骨细胞之间相互作用的耦合骨形成成骨细胞和 OC。为了以成熟为目标，我们之前鉴定了免疫球蛋白超家族成员 11 (IgSF11) 作为一种新型细胞表面受体，调节 OC 分化，但不调节新骨形成。为了表征 IgSF11 信号传导，我们通过质谱分析了磷酸化的蛋白质 IgSF11 激活后并鉴定出丙酮酸激酶 M2 (PKM2)，该酶催化最后一步糖酵解，作为下游目标。这一发现凸显了潜在的比之前已知的更大的 OC 分化和炎症性骨丢失过程中代谢调节的决定性作用。我们因此提出以下具体目标： 1. 检查 IgSF11-PKM2 信号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来研究 OC 表达的 IgSF11 骨质流失模型。为了测试 PKM2 依赖性效应的贡献，我们将治疗 LPS 诱导的 IgSF11-/- 具有 PKM2 小分子调节剂（TEPP-46、紫草素）的小鼠。我们的初步数据表明 TEPP- 46 PKM2 激活可减少 DSS 引起的骨质流失。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 缺陷小鼠进行 DSS 诱导的结肠炎实验。我们将还使用经 TEPP-46 或紫草素处理的 IgSF11-/- 小鼠进行 DSS 诱导的结肠炎实验。这些研究对于确定 IgSF11 和 PKM2 对临床相关贡献的交叉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差异化。我们制定了 OC 分化过程中 IgSF11-PKM2 功能的四步模型，我们将借助 hCD3-iFL 进行测试，hCD3-iFL 是一种逆转录病毒 (RV) 构建体，可直接激活细胞内 IgSF11 区分 OC。我们将首先调查 RANK 和 IgSF11-PKM2 之间可能存在的串扰，我们将其推测是由 IgSF11 支架蛋白 PSD- 的 TRAF6 依赖性 K63 连接的多泛素化介导的 95. 其次，我们的目标是鉴定靠近 IgSF11-PSD-95 复合物、磷酸化 PKM2 的激酶。第三，我们将使用RV突变体来确认各种PKM2修饰、PKM2变构的重要性确认，以及 PKM2 亚细胞定位至 OC 分化。最后，PKM2 是一个特性良好的糖酵解的酶调节剂，因此我们将采用代谢测定和抑制剂来确认其重要性 PKM2 在这方面对 OC 分化发挥作用。这些研究对于初步假定的 IgSF11-PKM2 通路及其在 OC 分化过程中的功能的验证和表征。