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：
10430027
负责人：
YONGWON CHOI
金额：
$ 35.39万
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 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10430027
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 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.

已知炎症会导致破骨细胞（OC）活性过多的骨骼炎症性疾病，例如牙周炎。解决这种炎症有关的根本原因骨质流失，重要的是要了解骨体内稳态的细胞和分子机制如何炎症刺激通过骨形成的成骨细胞（obs）和骨呈现OC维持。针对OC成熟而不是差异化是特别感兴趣的，并为避免无意中抑制新的骨形成。但是，确定有希望的治疗靶标 OC成熟将需要对其调节机制有更多了解。细胞粘附是生理过程对OC成熟及其标志性功能至关重要。在筛选在体外调节OC成熟的潜在基因，我们确定了细胞粘合剂相关的基因，即 PCDH7，Cadherin超家族的协议杜德蛋白成员。我们现在已经生成了PCDH7 - / - 鼠标在OC成熟和炎症反应中进一步研究PCDH7的目的，因此提出以下特定目的：1。研究pCDH7在OC分化，功能和炎症骨中的作用损失。我们将员工PCDH7 - / - 骨髓（BM）细胞检查已知生物学标记和细胞生物学功能，包括粘附，运动，肌动蛋白环的形成，褶皱的边界形成和囊泡贩运。将生成PCDH7FLOX的小鼠和BM嵌合体，以进行更精确的询问在骨稳态的背景下，OC-os-ob特异性（或其他）PCDH7功能。这些老鼠也会是在炎症性骨质流失和 LPS治疗或结扎引起的牙周炎后发生的免疫反应。在一起，这些研究应该阐明PCDH7在OC成熟和病理骨质流失中的细胞特异性作用。 2。调查 OC生物学中PCDH7分子功能的机制。研究OC表达的PCDH7蛋白如何调节细胞粘附和/或信号转导，我们将测试一个四步模型。对于每个步骤，我们将测试 OC成熟，细胞粘附和信号通路的激活，并将在身体上雇用激活和HCD3诱导式逆转录病毒（RV）PCDH7构建体。首先，我们将测试PCDH7是否介导细胞 - 通过单独跟踪WT和PCDH7 - / - OC激活PCDH7细胞内信号传导的细胞相互作用异型OC文化。其次，我们将测试PCDH7的细胞质结构域截断同工型的影响通过在OC中分析生理表达，然后通过表达RV的同工型在OC中。第三，我们将测试是否并且如果是这样，PCDH7如何通过癌蛋白组介导细胞内信号传导。第四，我们将雇用 siRNA和化学抑制剂，以测试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。这些研究将共同改善我们的了解PCDH7蛋白的功能通常，更具体地说，它如何控制OC成熟。