Role and Mechanism of Claudin-11 Action and Signaling in Bone

Claudin-11 作用和信号传导在骨中的作用和机制

基本信息

批准号：
10678629
负责人：
SUBBURAMAN MOHAN
金额：
$ 32.23万
依托单位：
LOMA LINDA VETERANS ASSN RESEARCH & EDUC
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10678629
关键词：
Address Affect Anabolic Agents Applications Grants Ascorbic Acid Binding Bone Resorption Bone callus Bone remodeling Breeding Calvaria Cell Differentiation process Cell Line Cells Conserved Sequence Data Defect Development Dominant-Negative Mutation Evaluation Exhibits Family Family member Femoral Fractures Financial Hardship Fracture Future Gender Gene Expression Genes Grant Histology Human Immunohistochemistry Impairment In Vitro Integral Membrane Protein Knock-out Knockout Mice Male Sterility Male Sterilizations Mechanics Mediating Membrane Modeling Molecular Mus Myelin Natural regeneration Oligodendroglia Osteoblasts Osteoclasts Osteogenesis Osteoporosis Pathogenesis Pathway interactions Persons Pharmaceutical Preparations Phenotype Prevention Process Protein Family Proteins Public Health Regulation Reporter Resolution Role Signal Pathway Signal Transduction Signaling Molecule Site TNFSF11 gene Testing Testis Tight Junctions Time Tissues Transgenic Mice Transgenic Organisms Yeasts age related bisphosphonate bone bone cell bone fracture repair bone loss bone mass healing in vivo inhibitor knock-down member microCT new therapeutic target notch protein novel novel strategies novel therapeutics osteoblast differentiation overexpression parathyroid hormone (1-34)recruit sertoli cell skeletal small molecule substantia spongiosa transgene expression yeast two hybrid system

项目摘要

Project Summary/Abstract Osteoporosis is a significant public health problem in the U.S. and poses a substantial financial burden. The pathogenesis of osteoporosis is known to involve increased destruction of bone, not compensated by parallel increases in the synthesis of new tissue. The only approved anabolic drug for osteoporosis, PTH, also increases bone resorption, thereby limiting its long term use. Thus, there is an urgent need for development of novel anabolic therapies for the treatment of osteoporosis. In our effort to identify control molecules and their signaling pathways that contribute to the regulation of osteogenesis, we have discovered a novel role for the Claudin (Cldn) family of tight junction proteins. In this RO1 application, our focus is on elucidating the role and mechanism of action of Cldn11 in regulating functions of osteoblasts (OBs) based on our exciting new data that mice with targeted disruption of the Cldn11 gene exhibit a severe deficit in trabecular bone volume (40%). Additionally, Cldn11 expression is increased several-fold during fracture healing. While the traditional role of Cldns is to regulate paracellular transport of small molecules, we have new exciting preliminary data that suggests that Cldn11 acts on OBs non-canonically via interacting with a transmembrane protein, Tetraspanin3 (Tspan3), to regulate ADAM10-mediated Notch signaling. We will test this model of Cldn11 action as follows: 1) To test the hypothesis that the Cldn11 expressed in OBs regulates trabecular bone formation, we will characterize the skeletal phenotype of OB-specific Cldn11 transgenic (Tg) mice by micro-CT, histology, mechanical testing and gene expression and determine if the reduced bone formation in Cldn11 KO mice can be rescued by transgenic expression of Cldn11 in OBs. 2) To test the hypothesis that Cldn11 effects on OBs are mediated via its interaction with Tspan3, we will determine the functional consequence of Cldn11/Tspan3 interactions by evaluating if knockdown of Tspan3 abolishes Cldn11-mediated differentiation in OBs, in vitro and in vivo. 3) To test the hypothesis that Cldn11/Tspan3 effects on OBs are mediated via ADAM10-mediated regulation of Notch signaling, we will evaluate the consequence of disruption of the Cldn11/Tspan3 interaction on ADAM10 maturation and activity via overexpression of a dominant negative mutant Cldn11 encoding the Tspan3 binding domain and determine if the Cldn11/Tspan3 interaction regulates Notch signaling and OB differentiation via modulation of ADAM10 activity. 4) To test the hypothesis that Cldn11 promotes fracture healing via regulating Notch signaling, we will determine the fracture phenotype in Cldn11 Tg and/or Tspan3 KO and control mice using a stabilized closed femoral fracture model. We will use notch signaling reporter mice to evaluate if Notch signaling is activated at the fracture site in the Cldn11 Tg mice and determine if treatment with an ADAM10 inhibitor blocks activated Notch signaling. Successful completion of our proposed studies will provide important information on the pathway by which Cldn11 regulates OB functions and could provide novel drug targets to promote anabolic activities of OBs for treatment of osteoporosis.

项目概要/摘要骨质疏松症是美国的一个重大公共卫生问题，并造成沉重的经济负担。这众所周知，骨质疏松症的发病机制涉及骨骼破坏增加，而不是通过平行的补偿来补偿。新组织的合成增加。唯一获批治疗骨质疏松症的合成代谢药物 PTH，也是增加骨吸收，从而限制其长期使用。因此，迫切需要开发用于治疗骨质疏松症的新型合成代谢疗法。在我们努力识别控制分子及其有助于调节成骨的信号通路，我们发现了一种新的作用紧密连接蛋白 Claudin (Cldn) 家族。在此 RO1 应用程序中，我们的重点是阐明角色和基于我们令人兴奋的新数据，Cldn11 调节成骨细胞 (OB) 功能的作用机制靶向破坏 Cldn11 基因的小鼠表现出骨小梁体积严重不足 (40%)。此外，Cldn11 表达在骨折愈合过程中增加数倍。虽然传统角色 Cldns 是调节小分子的旁细胞转运，我们有新的令人兴奋的初步数据表明 Cldn11 通过与跨膜蛋白 Tetraspanin3 相互作用以非规范方式作用于 OB (Tspan3)，调节 ADAM10 介导的 Notch 信号传导。我们将测试这个 Cldn11 动作模型如下： 1) 为了检验 OB 中表达的 Cldn11 调节骨小梁形成的假设，我们将通过显微 CT、组织学、机械测试和基因表达，并确定 Cldn11 KO 小鼠骨形成的减少是否可以通过在 OB 中转基因表达 Cldn11 可以挽救这种情况。 2) 检验Cldn11对OB影响的假设通过与 Tspan3 的相互作用介导，我们将确定 Cldn11/Tspan3 的功能结果通过评估 Tspan3 的敲低是否会消除 Cldn11 介导的 OB 分化，体外进行相互作用和体内。 3) 检验 Cldn11/Tspan3 对 OB 的影响是通过 ADAM10 介导的假设 Notch 信号传导的调节，我们将评估 Cldn11/Tspan3 相互作用中断的后果通过编码 ADAM10 的显性失活突变体 Cldn11 的过表达来影响 ADAM10 的成熟和活性 Tspan3 结合域并确定 Cldn11/Tspan3 相互作用是否调节 Notch 信号传导和 OB 通过调节 ADAM10 活性进行分化。 4) 检验Cldn11促进骨折的假设通过调节 Notch 信号传导来愈合，我们将确定 Cldn11 Tg 和/或 Tspan3 中的骨折表型 KO 小鼠和对照小鼠使用稳定的闭合股骨骨折模型。我们将使用Notch信号报告器评估 Cldn11 Tg 小鼠骨折部位的 Notch 信号是否被激活，并确定是否 ADAM10 抑制剂治疗可阻断激活的 Notch 信号传导。成功完成我们提出的研究将提供有关 Cldn11 调节 OB 功能途径的重要信息，并可能提供新的药物靶点来促进 OB 的合成代谢活性，从而治疗骨质疏松症。