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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：
9764134
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/9764134
关键词：
Address Affect Anabolic Agents Applications Grants Ascorbic Acid Binding Bone Resorption Bone callus Bone remodeling Calvaria Cell Differentiation process Cells Conserved Sequence Data Defect Development Dominant-Negative Mutation Evaluation Exhibits Family Family member Femoral Fractures Financial Hardship Fracture Fracture Healing Future Gender Gene Expression Genes Grant Histology Human Immunohistochemistry Impairment In Vitro Integral Membrane Protein Knock-out Knockout Mice Lead Male Sterility Mechanics Mediating Membrane Modeling Molecular Mus Myelin Natural regeneration Oligodendroglia Osteoblasts Osteoclasts Osteogenesis Osteoporosis Pathogenesis Pathway interactions 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 base bisphosphonate bone bone cell bone loss bone mass healing in vivo inhibitor/antagonist 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 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.

项目总结/摘要骨质疏松症在美国是一个严重的公共卫生问题，并造成了巨大的经济负担。的已知骨质疏松症的发病机制涉及骨破坏的增加，而不是由平行的补偿。增加新组织的合成。唯一批准的骨质疏松症合成代谢药物，PTH，也增加骨吸收，从而限制其长期使用。因此，迫切需要发展用于治疗骨质疏松症的新的合成代谢疗法。在我们努力识别控制分子及其信号通路，有助于调节成骨，我们已经发现了一个新的作用，紧密连接蛋白（Cldn）家族。在这个RO 1应用程序中，我们的重点是阐明角色，基于我们令人兴奋的新数据，Cldn 11在调节成骨细胞（OBs）功能中的作用机制有针对性地破坏Cldn 11基因的小鼠表现出严重的骨小梁体积缺陷（40%）。此外，Cldn 11表达在骨折愈合期间增加数倍。虽然传统的角色 Cldns是调节小分子的细胞旁转运，我们有新的令人兴奋的初步数据，表明Cldn 11通过与跨膜蛋白Tetraspanin 3相互作用而非规范地作用于OB （Tspan 3），以调节ADAM 10介导的Notch信号传导。我们将测试这个Cldn 11动作模型如下： 1)为了检验OBs中表达的Cldn 11调节松质骨形成的假设，我们将通过显微CT，组织学，机械测试和基因表达，并确定在Cldn 11 KO小鼠中减少的骨形成是否可以 2）验证Cldn 11基因对OBs的作用通过与Tspan 3的相互作用介导，我们将确定Cldn 11/Tspan 3的功能后果通过评估Tspan 3的敲低是否在体外消除了OB中Cldn 11介导的分化，和体内。3)为了验证Cldn 11/Tspan 3对OB的作用是通过ADAM 10介导的 Notch信号的调节，我们将评估Cldn 11/Tspan 3相互作用的破坏的后果通过过表达显性负性突变体Cldn 11对ADAM 10成熟和活性的影响， Tspan 3结合结构域，并确定Cldn 11/Tspan 3相互作用是否调节Notch信号传导和OB 通过调节ADAM 10活性来分化。4)为了检验Cldn 11促进骨折的假设，通过调节Notch信号传导愈合，我们将确定Cldn 11 Tg和/或Tspan 3中的骨折表型 KO和对照小鼠使用稳定的闭合性股骨骨折模型。我们将使用notch信号报告器以评估Notch信号传导是否在Cldn 11 Tg小鼠的骨折部位被激活，并确定是否用ADAM 10抑制剂治疗阻断活化的Notch信号传导。成功完成我们的建议研究将提供Cldn 11调节OB功能的途径的重要信息，提供新的药物靶点以促进OB的合成代谢活性用于治疗骨质疏松症。