Protocadherin 7 and Osteoclast Maturation

原钙粘蛋白 7 和破骨细胞成熟

基本信息

批准号：
10206010
负责人：
YONGWON CHOI
金额：
$ 34.68万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10206010
关键词：
Actins Address Adhesions Affect Antibodies Biological Assay Biological Markers Biological Process Biology Bone Marrow Bone Marrow Cells CD 200 Cadherins Calcium Cell Adhesion Cell Adhesion Molecules Cell Communication Cell Maturation Cells Chemicals Chimera organism Coupled Cytoplasmic Tail Data Disease Dyes Exhibits Extracellular Domain Fibronectins Genes Hematopoietic Homeostasis Immune response In Vitro Inflammation Inflammatory Inflammatory Response Integrins Label Ligature Measures Mediating Mediator of activation protein Membrane Mesenchymal Modeling Molecular Mononuclear Mus Oncoproteins Osteoblasts Osteoclasts Osteogenesis Pathologic Pathology Pathway interactions Patients Periodontitis Physiological Physiological Processes Protein Family Protein Isoforms Protein phosphatase Proteins Publishing Regulation Role Screening Result Signal Pathway Signal Transduction Slice Small Interfering RNA Stains Stimulus TNFSF11 gene Testing Tracer Vesicle Work bisphosphonate bone bone loss bone strength cell motility improved in vivo inflammatory bone loss inhibitor/antagonist interest knock-down lipophilicity member microCT molecular marker osteoclastogenesis receptor rho GTP-Binding Proteins screening side effect therapeutic target trafficking

项目摘要

Inflammation is known to cause bone destruction by excessive osteoclast (OC) activity in patients with inflammatory diseases, such as periodontitis. To address the underlying causes of such inflammation-related bone loss, it is important to understand how the cellular and molecular mechanisms of bone homeostasis maintained by bone-forming osteoblasts (OBs) and bone-resorbing OCs are perturbed by inflammatory stimuli. Targeting OC maturation rather than differentiation is of particular interest and provides an added benefit of avoiding unintentionally inhibiting new bone formation. However, identifying promising therapeutic targets of OC maturation will require greater understanding of its mechanisms of regulation. Cell adhesion is a physiologic process critical to both OC maturation and its hallmark feature, multinucleation. In the course of screening potential genes that regulate OC maturation in vitro, we identified a cell adhesion-related gene, Pcdh7, a protocadherin member of the cadherin superfamily. We have now generated Pcdh7-/- mice for the purpose of further studying Pcdh7 in OC maturation and inflammatory responses, and therefore propose the following specific aims: 1. Investigate the role of Pcdh7 in OC differentiation, function, and inflammatory bone loss. We will employ Pcdh7-/- bone marrow (BM) cells to examine expression of known biological markers and cell biological functions, including adhesion, motility, actin ring formation, ruffled border formation, and vesicle trafficking. Pcdh7floxed mice and BM chimeras will be generated for the purpose of more precisely interrogating OC- versus OB-specific (or other) Pcdh7 functions in the context of bone homeostasis. These mice will also be employed to confirm the importance of OC-expressed Pcdh7 in the context of inflammatory bone loss and immune responses that occur after LPS treatment or ligature-induced periodontitis. Together, these studies should elucidate the cell-specific roles of Pcdh7 in OC maturation and pathologic bone loss. 2. Investigate mechanisms of Pcdh7 molecular function within OC biology. To investigate how OC-expressed Pcdh7 protein regulates cell adhesion and/or signal transduction, we will test a four-step model. For each step, we will test OC maturation, cell adhesion, and activation of signaling pathways, and will employ both physiologically- activated and hCD3-inducible retroviral (RV) Pcdh7 constructs. First, we will test whether Pcdh7 mediates cell- cell interactions that activate Pcdh7 intracellular signaling by separately track WT and Pcdh7-/- OCs in mixed heterotypic OC cultures. Second, we will test the effects of cytoplasmic domain truncation isoforms of Pcdh7 by assaying physiologic expression in OCs and then by RV-expressing isoforms in OCs. Third, we will test whether and, if so, how Pcdh7 mediates intracellular signaling via the oncoprotein SET. Fourth, we will employ siRNA and chemical inhibitors to test the relative contributions of Pcdh7-dependent activation of various signaling pathways to Pcdh7-mediated OC adhesion and maturation. Together, these studies will improve our understanding of the function of Pcdh7 protein generally, and more specifically, how it controls OC maturation.

已知炎症会因破骨细胞 (OC) 活性过高而导致骨质破坏炎症性疾病，例如牙周炎，解决此类炎症相关的根本原因。骨质流失，了解骨稳态的细胞和分子机制非常重要由骨形成成骨细胞 (OB) 维持，而骨吸收 OC 则受到炎症刺激的干扰。以 OC 成熟而非分化为目标特别令人感兴趣，并且提供了额外的好处避免无意中抑制新骨形成然而，确定有希望的治疗靶点。 OC 的成熟需要对其细胞粘附的调节机制有更多的了解。 OC 成熟及其标志性特征（多核化）至关重要的生理过程。体外筛选调节OC成熟的潜在基因，我们鉴定了一个细胞粘附相关基因， Pcdh7，钙粘蛋白超家族的原钙粘蛋白成员我们现在已经生成了 Pcdh7-/- 小鼠。进一步研究 Pcdh7 在 OC 成熟和炎症反应中的目的，因此提出具体目标如下： 1. 研究 Pcdh7 在 OC 分化、功能和炎症骨中的作用我们将使用 Pcdh7-/- 骨髓 (BM) 细胞来检查已知生物标志物的表达和细胞生物学功能，包括粘附、运动、肌动蛋白环形成、褶皱边界形成和囊泡将生成 Pcdh7floxed 小鼠和 BM 嵌合体，以便进行更精确的审讯。 OC 与 OB 特异性（或其他）Pcdh7 在骨稳态方面的功能。这些小鼠也将用于确认 OC 表达的 Pcdh7 在炎性骨质流失背景下的重要性 LPS 治疗或结扎引起的牙周炎后发生的免疫反应。应阐明 Pcdh7 在 OC 成熟和病理性骨丢失中的细胞特异性作用 2. 研究。 OC 生物学中 Pcdh7 分子功能的机制。研究 OC 如何表达 Pcdh7 蛋白调节细胞粘附和/或信号转导，我们将测试一个四步模型，对于每个步骤，我们将进行测试。 OC 成熟、细胞粘附和信号通路激活，并将采用生理学- 首先，我们将测试 Pcdh7 是否介导细胞-激活和 hCD3 诱导的逆转录病毒 (RV) Pcdh7 构建体。通过单独跟踪混合中的 WT 和 Pcdh7-/- OC 来激活 Pcdh7 细胞内信号传导的细胞相互作用其次，我们将测试 Pcdh7 胞质结构域截短亚型的影响。通过分析 OC 中的生理表达，然后通过 OC 中的 RV 表达亚型进行第三步，我们将进行测试。是否以及如果是的话，Pcdh7 如何通过癌蛋白 SET 介导细胞内信号传导。 siRNA 和化学抑制剂测试 Pcdh7 依赖性激活的相对贡献 Pcdh7 介导的 OC 粘附和成熟的信号通路一起，这些研究将改善我们的研究。了解 Pcdh7 蛋白的总体功能，更具体地说，了解它如何控制 OC 成熟。