Endosteal Bone Volume Regulation and Osteoporosis

骨内骨量调节和骨质疏松症

• 批准号: 8500197
• 负责人: SUBBURAMAN MOHAN
• 金额: $ 31.15万
• 依托单位: LOMA LINDA VETERANS ASSN RESEARCH & EDUC
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500197
• 关键词: Affect; Age; Aging; Animals; Apoptosis; Binding; Biological; Biological Assay; Biology; Body Weight; Bone Resorption; C-terminal; CSF3 gene; Cell Count; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cell Separation; Cell physiology; Cells; Chromatin; Co-Immunoprecipitations; Collaborations; Commit; Complex; Cyclin D1; Cytoplasm; Cytoplasmic Tail; DNA; Data; Deterioration; Development; Diagnosis; Drug or chemical Tissue Distribution; Elements; Epithelium; Evaluation; Exhibits; FOS gene; Factor-42; Family; Family member; Fracture; Future; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Grant; Growth Factor; Health; Herpes zoster disease; Histology; Hormones; Human; ITGAM gene; Immunofluorescence Immunologic; In Vitro; Individual; Integral Membrane Protein; Knock-out; Knockout Mice; Label; Lead; Length; Lentivirus Vector; Ligands; Macrophage Colony-Stimulating Factor; Measurement; Measures; Mediating; Membrane; Modeling; Molecular; Mus; Mutate; NF-kappa B; Nuclear; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; PXXP Motif; Pathogenesis; Pathway interactions; Patients; Pharmacologic Substance; Phenotype; Phosphorylation; Phosphorylation Site; Plasma; Play; Process; Proline; Proteins; Public Health; Publishing; Regulation; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; SH3 Domains; Scientist; Senile Osteoporosis; Serum; Signal Pathway; Signal Transduction; Site; Specificity; Staging; Stem cells; Stomach; Surface; TNFSF11 gene; TNFSF5 gene; Tail; Tertiary Protein Structure; Testing; Text; Thick; Tight Junctions; Time; Tissues; Transcription factor genes; Transcriptional Regulation; Transgenic Organisms; Western Blotting; Wild Type Mouse; Work; age related; base; bone; bone cell; bone loss; bone mass; bone turnover; cellular targeting; claudin-1 protein; granulocyte; human SAFB protein; in vivo; long bone; macrophage; member; migration; novel; nucleocytoplasmic transport; osteoporosis with pathological fracture; overexpression; peripheral blood; progenitor; promoter; protein complex; protein protein interaction; research study; skeletal; small hairpin RNA; substantia spongiosa; transcription factor

DESCRIPTION (provided by applicant): Our long term goal in this grant (AR31062-25A2) is to identify the control molecules and their signaling pathways that contribute to the regulation of endosteal bone volume since age-related loss of endosteal bone is a major cause of senile osteoporosis. To this end, our studies during the past grant period have led to the identification of a novel molecule, claudin-18 (Cldn-18), as an important player in the regulation of endosteal bone volume. Cldn-18 is a member of a large family of transmembrane proteins that have been identified as important components of tight junction strands. Although several Cldns, including Cldn-18, have recently been shown to be expressed in bone cells, nothing is known on the role of any Cldns in bone. In preliminary studies, we have found that mice with disruption of Cldn-18 function exhibit a severe deficit in trabecular bone volume (>50%) that is not caused by changes in body weight or bone size. Instead, this reduction in trabecular bone volume in Cldn-18 knockout mice is caused by increased bone resorption as reflected by increased osteoclast (OC) number that is not compensated by a corresponding increase in osteoblast number or bone formation changes. Our studies on the mechanism by which Cldn-18 regulates OC functions have revealed exciting preliminary data to suggest that Cldn-18 effects on RANKL signaling are mediated by a novel mechanism that is independent of its known tight junction function. Based on our preliminary data and what is known on the actions of Cldns in other tissues, we propose the following hypotheses in this study: 1) Cldn-18 acts on committed cells of OC lineage to inhibit differentiation. 2) Cldn-18 effects on RANKL signaling are mediated in part via Cldn-18 interaction with a PDZ domain containing protein, Zona Occluden-2 (ZO-2), to modulate NFATc1 regulation of RANKL target genes. 3) Regulation of PDZ domain-mediated Cldn-18/ZO-2 interaction is mediated in part by RANKL-induced changes in Cldn-18 phosphorylation. To test hypothesis 1, we will isolate OC precursors from Cldn-18 knockout and wild type mice and evaluate the effects of RANKL on proliferation, activity and apoptosis of OCs. We will overexpress Cldn-18 in OC precursors from Cldn-18 knockout mice to determine if Cldn-18 overexpression impairs OC development. We will measure expression levels of marker genes and transcription factors at different stages of OC development to determine the stage of the cell and target genes influenced by Cldn-18. To evaluate the specificity of Cldn-18 action on OCs, we will determine if Cldn-18 influences the differentiation of progenitors into mature granulocytes and macrophages. To test hypothesis 2, we will test for interaction between Cldn-18 and PDZ domain containing ZO-2 in OCs as suggested by our preliminary data. To determine the role for ZO-2 in regulating OC functions, we will evaluate the consequence of overexpression or of blockade of ZO-2 on RANKL-induced OC differentiation. Because ZO-2 is known to be translocated into the nucleus to regulate transcription, we will also determine if Cldn-18 regulates nuclear transport of ZO-2 to modulate NFATc1-mediated transcriptional regulation of genes that are critical for OC differentiation. To test hypothesis 3, we will determine the role of PDZ binding motif in Cldn-18 in binding to ZO-2 and evaluate the role of RANKL-induced phosphorylation changes in Cldn-18 in regulating cellular localization of ZO-2 and Cldn-18 biological effects on OC differentiation. Our successful establishment of interaction between RANKL and Cldn-18/ZO-2 signaling pathways will provide a novel mechanism for RANKL regulation of OC differentiation and provide clues to understanding the actions of other Cldn family members in bone as well as in other tissues. PUBLIC HEALTH RELEVANCE: Developing strategies to diagnose and treat osteoporosis, a major public health threat, would require a thorough understanding of the molecular pathways and the genes involved in the bone resorption process since increased bone resorption is a major contributor to the pathogenesis of osteoporosis. Successful completion of the proposed in vivo animal studies and in vitro mechanistic studies using transgenic approaches on a novel tight junction protein, claudin-18, should lead to elucidation of the role for this gene in regulating bone resorption and thereby endosteal bone volume as suggested by our preliminary data. Because claudin-18 sequence is conserved between mice and humans, future confirmation of a role for claudin-18 in humans will eventually lead to a better understanding of why some people have high bone turnover and treatment options to correct excess bone resorption in those patients.

描述（由申请人提供）：本基金（AR 31062 - 25 A2）的长期目标是鉴定有助于调节骨内膜骨体积的控制分子及其信号通路，因为年龄相关的骨内膜骨丢失是老年性骨质疏松症的主要原因。为此，我们在过去的资助期间的研究已经鉴定出一种新的分子，claudin-18（Cldn-18），作为骨内膜骨体积调节的重要参与者。Cldn-18是一个跨膜蛋白大家族的成员，已被确定为紧密连接链的重要组成部分。尽管最近已经显示包括Cldn-18在内的几种Cldns在骨细胞中表达，但对任何Cldns在骨中的作用一无所知。在初步研究中，我们发现具有Cldn-18功能破坏的小鼠表现出小梁骨体积的严重缺陷（>50%），这不是由体重或骨大小的变化引起的。相反，Cldn-18敲除小鼠中的这种骨小梁体积的减少是由骨吸收增加引起的，如破骨细胞（OC）数量增加所反映的，其没有被成骨细胞数量或骨形成变化的相应增加所补偿。我们对Cldn-18调节OC功能的机制的研究揭示了令人兴奋的初步数据，表明Cldn-18对RANKL信号传导的影响是由一种新的机制介导的，该机制独立于其已知的紧密连接功能。基于我们的初步数据和已知的Cldn在其他组织中的作用，我们在本研究中提出以下假设：1）Cldn-18作用于OC谱系的定向细胞以抑制分化。2)Cldn-18对RANKL信号传导的影响部分通过Cldn-18与含PDZ结构域的蛋白质Zona Occluden-2（ZO-2）相互作用介导，以调节RANKL靶基因的NFATc 1调节。3)PDZ结构域介导的Cldn-18/ZO-2相互作用的调节部分由RANKL诱导的Cldn-18磷酸化变化介导。为了检验假设1，我们将从Cldn-18敲除和野生型小鼠中分离OC前体，并评估RANKL对OC增殖、活性和凋亡的影响。我们将在来自Cldn-18敲除小鼠的OC前体中过表达Cldn-18以确定Cldn-18过表达是否损害OC发育。我们将在OC发育的不同阶段测量标记基因和转录因子的表达水平，以确定受Cldn-18影响的细胞和靶基因的阶段。为了评估Cldn-18对OC作用的特异性，我们将确定Cldn-18是否影响祖细胞分化为成熟粒细胞和巨噬细胞。为了检验假设2，我们将检验如我们的初步数据所建议的在OC中Cldn-18和含有ZO-2的PDZ结构域之间的相互作用。为了确定ZO-2在调节OC功能中的作用，我们将评估ZO-2过表达或阻断对RANKL诱导的OC分化的影响。由于ZO-2被转移到细胞核中调节转录，我们还将确定Cldn-18是否调节ZO-2的核转运，以调节NFATc 1介导的对OC分化至关重要的基因的转录调节。为了检验假设3，我们将确定Cldn-18中PDZ结合基序在与ZO-2结合中的作用，并评估RANKL诱导的Cldn-18磷酸化变化在调节ZO-2细胞定位中的作用，以及Cldn-18对OC分化的生物学效应。我们成功建立了RANKL和Cldn-18/ZO-2信号通路之间的相互作用，这将为RANKL调节OC分化提供一种新的机制，并为理解其他Cldn家族成员在骨和其他组织中的作用提供线索。 公共卫生相关性：骨质疏松症是一个主要的公共卫生威胁，发展战略来诊断和治疗骨质疏松症，将需要一个彻底的了解的分子途径和基因参与骨吸收过程，因为增加骨吸收是骨质疏松症的发病机制的主要贡献者。成功完成拟议的体内动物研究和体外机制的研究，使用转基因方法的一种新的紧密连接蛋白，claudin-18，应导致阐明该基因在调节骨吸收的作用，从而骨内膜骨体积所建议的我们的初步数据。由于claudin-18序列在小鼠和人类之间是保守的，未来对claudin-18在人类中作用的确认将最终导致更好地理解为什么有些人具有高骨转换和治疗选择，以纠正这些患者的过度骨吸收。