Regulator of Calcineurin (RCAN1)-a Novel Regulator of Osteoclastogenesis

描述(申请人提供)：破骨细胞(OCs)是生理性骨吸收所必需的，来源于造血祖细胞对M-CSF和RANKL的反应。体外实验检测了暴露于RANKL和M-CSF的OC前体的基因表达谱，结果表明NFATc1是OC形成和功能的关键调节因子。这些实验是用培养在组织培养塑料上的细胞进行的，而OCs在体内是在骨基质的背景下发展起来的。我们比较了骨上培养的OCs和塑料上同一细胞获得的OCs的基因表达谱，并鉴定了在骨上生成的OCs中强烈且唯一诱导的多个基因。为了确定介导OC终末分化和激活的分子机制和信号通路，我们重点研究了钙调神经磷酸酶1(RCAN1)的调节基因，它是骨调节OC基因的典型成员，也是rcan家族中特征最好和表达最高的成员。RCAN1控制钙调神经磷酸酶的活性，进而调节NFATc1的激活，NFATc1在多个OC相关基因的转录调控中发挥关键作用。我们对RCAN1基因调节区的分析表明，几种潜在的转录和转录后调节机制可能介导骨特异性RCAN1的表达。缺乏RCAN1的小鼠表现出骨表型，特征是小梁骨增多，OC介导的吸收减少。此外，RCAN1还促进了多个OC基因的转录，表明RCAN1在调控OC分化和激活的遗传程序和信号通路中发挥着重要作用。基于这些数据，我们假设：RCAN1的表达和活性受到OCS与骨的相互作用的上调，并且RCAN1通过调节钙调神经磷酸酶-NFATc1途径在调节OC的形成和激活中发挥核心作用。为了追求我们的新发现，我们提出了以下具体目标：目的1确定在RANKL诱导的骨表面OC形成过程中RCAN1被激活的信号通路。目的2定义RCAN1在OC生物学中发挥作用的各个结构域。我们还将在骨质疏松的小鼠模型中测试RCAN1缺失将使动物对与雌激素缺乏相关的骨丢失产生抵抗力的假设。目的3将剖析RANKL和骨基质调节RCAN1表达的转录和转录后机制。在骨骼疾病患者中，仍然需要更有效的治疗方法来减少骨丢失，防止潜在的衰弱并发症，但仍未得到满足。利用在骨上分化的OCs进行的实验将为开发更有效和特异的治疗方法提供新的方向和机会，以防止骨质疏松症中OC介导的骨丢失和相关形式的病理性骨丢失。

DESCRIPTION (provided by applicant): Osteoclasts (OCs), which are essential for physiologic bone resorption, are derived from hematopoietic precursors in response to M-CSF and RANKL. In vitro experiments examining gene expression profiles of OC precursors exposed to RANKL and M-CSF led to identification of NFATc1 as a key regulator of OC formation and function. These experiments were performed with cells cultured on tissue culture plastic, whereas OCs develop in vivo in the context of a bone substrate. We compared the gene expression profiles of OCs cultured on bone with those obtained from the same cells on plastic and have identified multiple genes that are strongly and uniquely induced in OCs generated on bone. To identify the molecular mechanisms and signal pathways that mediate terminal OC differentiation and activation, we focused on regulator of calcineurin 1 (RCAN1), which is a prototypical member of the bone-regulated OC genes and is the best characterized and most highly expressed member of the RCAN family. RCAN1 controls the activity of calcineurin, which in turn regulates the activation of NFATc1, which plays a pivotal role in the transcriptional regulation of multiple OC-associated genes. Our analysis of the regulatory regions of the RCAN1 gene indicates several potential transcriptional and posttranscriptional regulatory mechanisms that may mediate bone-specific RCAN1 expression. Mice lacking RCAN1 exhibit a bone phenotype, characterized by increased trabecular bone with reduced OC-mediated resorption. Moreover, RCAN1 enhances transcription of multiple OC genes, indicating that RCAN1 plays an important role in controlling the genetic program and signal pathways of OC differentiation and activation. Based on these data we hypothesize that: RCAN1 expression and activity are up-regulated by interaction of OCs with bone and that RCAN1 plays a central role in regulating OC formation and activation by modulating the calcineurin-NFATc1 pathway. To pursue our novel findings, we propose the following specific aims: Aim 1 Identify the signaling pathways by which RCAN1 is activated during RANKL-induced OC formation on bone surfaces. Aim 2 Define the individual domains of RCAN1 that contribute to its role in OC biology. We will also test the hypothesis that the absence of RCAN1 will render animals resistant to the bone loss associated with estrogen deficiency in a mouse model of osteoporosis. Aim 3 will dissect the transcriptional and post-transcriptional mechanisms by which RANKL and bone substrate regulate expression of RCAN1. There remains an unmet need for more effective therapies to reduce bone loss and prevent potentially debilitating complications in patients with skeletal disorders. The proposed experiments utilizing OCs differentiated on bone will provide new directions and opportunities for developing more effective and specific therapies to prevent OC-mediated bone loss in osteoporosis and related forms of pathologic bone loss.