Endosteal Bone Volume Regulation and Osteoporosis

描述(由申请人提供)：我们在这项资助(AR31062-25A2)中的长期目标是确定有助于调节骨内膜骨体积的控制分子及其信号通路，因为与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导致了一种新的分子，claudin-18(cldn-18)，作为调节骨内膜骨体积的重要参与者。CldN-18是跨膜蛋白大家族中的一员，已被确定为紧密连接链的重要组成部分。尽管最近有研究表明，包括CldN-18在内的几种Cldns在骨细胞中都有表达，但对Cldns在骨中的作用尚不清楚。在初步研究中，我们发现，cldn-18功能受损的小鼠表现出严重的骨小梁体积缺陷(&gt；50%)，这不是由体重或骨大小的变化引起的。相反，cldn-18基因敲除小鼠的这种骨小梁体积的减少是由骨吸收增加引起的，骨吸收增加反映为破骨细胞(OC)数量的增加，而不是由成骨细胞数量的相应增加或骨形成变化来补偿。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。根据我们的初步数据和已知的Cldns在其他组织中的作用，我们在本研究中提出了以下假设：1)CldN-18作用于OC谱系的承诺细胞，抑制分化。2)CLDN-18对RANKL信号转导的影响部分是通过CLDN-18与含有PDZ结构域的蛋白Occluden-2(ZO-2)相互作用来调节NFATc1对RANKL靶基因的调控。3)PDZ结构域介导的CLDN-18/ZO-2相互作用的调节部分是通过RANKL诱导的CLDN-18磷酸化的改变来实现的。为了验证假设1，我们将从cldn-18基因敲除小鼠和野生型小鼠中分离OC前体，并评估RANKL对OCS增殖、活性和凋亡的影响。我们将在CLDN-18基因敲除小鼠的OC前体中过表达CLDN-18，以确定过表达是否损害OC的发育。我们将检测标记基因和转录因子在卵巢癌不同发育阶段的表达水平，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OCS作用的特异性，我们将确定Cldn-18是否影响祖细胞向成熟粒细胞和巨噬细胞的分化。为了检验假设2，我们将测试CLDN-18与OCS中含有ZO-2的PDZ结构域之间的相互作用，这是我们的初步数据所建议的。为了确定ZO-2在OC功能调节中的作用，我们将评估ZO-2的过度表达或阻断对RANKL诱导的OC分化的影响。由于已知ZO-2被移位到细胞核中调节转录，我们还将确定Cldn-18是否调节ZO-2的核运输，以调节NFATc1介导的对OC分化至关重要的基因的转录调控。为了验证假设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的细胞定位和CLDN-18在OC分化中的生物学效应中的作用。我们成功地建立了RANKL与CLDN-18/ZO-2信号通路之间的相互作用，为RANKL调控OC分化提供了新的机制，并为了解其他CLDN家族成员在骨和其他组织中的作用提供了线索。 公共卫生相关性：制定诊断和治疗骨质疏松症的策略，这是一种主要的公共健康威胁，需要彻底了解骨吸收过程中涉及的分子途径和基因，因为骨吸收增加是导致骨质疏松症发病的主要因素。成功完成体内动物研究和体外机制研究的转基因方法，新的紧密连接蛋白，claudin-18，应导致阐明该基因的作用，调节骨吸收，从而骨内膜骨体积，根据我们的初步数据。由于claudin-18序列在小鼠和人类之间是保守的，未来对claudin-18在人类中的作用的确认最终将有助于更好地理解为什么一些人有高骨转换率，以及纠正这些患者过度骨吸收的治疗选择。

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.