ATF4 and Osteoclastogenesis

ATF4 和破骨细胞生成

• 批准号: 8452448
• 负责人: Guozhi Xiao
• 金额: $ 21.1万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8452448
• 关键词: ATF4; Osteoclastogenesis

DESCRIPTION (provided by applicant): Osteoclasts (OCLs) are the only bone-resorbing cells that are essential for normal skeletal development and bone remodeling throughout life. Abnormal OCL number and/or activity result in a number of bone diseases such as osteoporosis, Paget's disease of bone, metastatic osteolytic lesions, and rheumatoid arthritis. However, the molecular mechanisms underlying osteoclastogenesis are not well understood. Our preliminary studies demonstrate that activating transcription factor 4 (ATF4) is a key factor that regulates osteoclastogenesis. Our data indicate that ATF4 mediates M-CSF-induced expression of RANK, a critical molecular event required for early differentiation of OCLs. Furthermore, ATF4 directs RANKL-dependent gene expression of NFATc1, a master regulator of OCL differentiation. In this study, we hypothesize that ATF4 plays a critical role in regulating osteoclastogenesis by two distinct mechanisms: 1) ATF4 modulates M-CSF induction of RANK expression via PI3K/AKT-dependent phosphorylation and protein stabilization and/or activation; 2) ATF4 mediates RANKL induction of NFATc1 gene by binding to the NFATc1 gene P1 promoter via interactions with other key factors. To address our hypothesis, we will pursue the following specific aims: Aim 1 will confirm that ATF4 is phosphorylated and up-regulated by M-CSF signaling via PI3K/AKT in OCL precursors. We will identify the AKT and M-CSF responsive phosphorylation site(s) and assess their functional significance in regulating ATF4 protein stability and activity in support of osteoclastogenesis. Aim 2 will determine whether ATF4 mediates RANKL induction of the NFATc1 gene via cooperative interaction with other key factors on the NFATc1 gene P1 promoter. We will also identify ATF4 heterodimerization partners and assess the functional significance of interactions between ATF4 and partners in RANKL-induced NFATc1 expression. Aim 3 will determine whether OCL-specific transgenic expression of NFATc1 can rescue the defect in OCL differentiation in Atf4-/- mice. We will use transgenic mice in which the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter drives the expression of a constitutively active form of NFATc1 (NFATc1-CA) selectively in OCLs in Atf4-/- mice. Biochemical and histomorphometric parameters for OCL differentiation and bone resorption will be determined in the following mice groups: i) wt, ii) NFATc1- CA-tg, iii) Atf4-/-, and iv) Atf4-/-; NFATc1-CA-tg. Successful completion of these proposed aims will 1) significantly advance understanding of the molecular mechanisms underlying OCL differentiation, and 2) provide a molecular basis for development of new and more specific antiresorptive agents for treating patients with devastating osteoporosis, hypercalcemia of malignancy, and Paget's disease of bone. PUBLIC HEALTH RELEVANCE: Skeletal integrity requires a delicate balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCLs). Abnormally increased OCL number and/or activity result in a number of bone diseases such as osteoporosis, osteolytic lesions induced by many metastatic cancers, Paget's disease of bone, and rheumatoid arthritis. Conversely, reduced OCL number and/or activity causes osteopetrosis, a disorder characterized by significantly increased skeletal mass. Defining the molecular mechanisms underlying osteoclastogenesis is essential to advance understanding of the molecular basis for the pathogenesis of OCL-based or involved bone diseases and improve the prevention and treatment of these diseases. We demonstrate that ATF4 is a key transcription factor for osteoclastogenesis and present data revealing its importance in the regulation of both early and late OCL differentiation. ATF4 plays an intrinsic role in OCL precursors that is indispensable for RANKL-induced OCL differentiation. The important role of ATF4 in osteoclastogenesis is underscored by its requirements for M-CSF-induced RANK gene expression, a key molecular event for early OCL differentiation, as well as for RANKL-induced NFATc1 gene expression, required for OCL differentiation. This proposal intends to elucidate the mechanisms whereby M-CSF activates/upregulates ATF4 via the PI3K/AKT pathway, to examine how ATF4 mediates RANKL induction of NFATc1 via activation of the NFATc1 gene P1 promoter, and to assess the importance of ATF4 in OCL differentiation in vivo using a specific NFATc1-CA transgenic mouse model. The information obtained from these studies will significantly enhance our understanding of the molecular mechanism involved in normal osteoclastogenesis and bone resorption during skeletal development and throughout life. Furthermore, bisphosphonates (zolendronic acid and pamidronate), the most widely prescribed antiresorptive agents that are intravenously administrated to reduce bone pain, hypercalcemia and skeletal complications in patients with multiple myeloma, breast, prostate, lung and other cancers and Paget's disease of bone, have a severe side effect called bisphosphonate- associated osteonecrosis of the jaw via undefined mechanism(s). Successful completion of this study will provide a molecular basis for the development of new and more specific antiresorptive agents for treating these devastating diseases.

描述(申请人提供)：破骨细胞(OCL)是唯一的骨吸收细胞，对于正常的骨骼发育和一生中的骨重建是必不可少的。OCL数量和/或活性的异常会导致许多骨骼疾病，如骨质疏松症、骨派杰氏病、转移性溶骨性病变和类风湿性关节炎。然而，破骨细胞发生的分子机制还不是很清楚。我们的初步研究表明，激活转录因子4(ATF4)是调节破骨细胞发生的关键因子。我们的数据表明，ATF4介导了M-CSF诱导的RANK的表达，RANK是OCL早期分化所需的关键分子事件。此外，ATF4还指导OCL分化的主要调节因子NFATc1依赖于RANKL的基因表达。在本研究中，我们假设ATF4通过两种不同的机制在调节破骨细胞生成中起关键作用：1)ATF4通过依赖PI3K/AKT的磷酸化和蛋白稳定和/或激活来调节M-CSF对RANK表达的诱导；2)ATF4通过与其他关键因子相互作用与NFATc1基因P1启动子结合而介导RANKL对NFATc1基因的诱导。为了解决我们的假设，我们将追求以下具体目标：目标1将证实在OCL前体中，ATF4被磷酸化，并通过PI3K/AKT的M-CSF信号上调。我们将确定AKT和M-CSF应答的磷酸化位点(S)，并评估它们在调节ATF4蛋白稳定性和活性以支持破骨细胞形成中的功能意义。目的2确定ATF4是否通过与NFATc1基因P1启动子上的其他关键因子协同作用来介导RANKL对NFATc1基因的诱导。我们还将确定ATF4异源二聚伙伴，并评估ATF4与伙伴之间的相互作用在RANKL诱导的NFATc1表达中的功能意义。目的3确定OCL特异性转基因表达NFATc1能否修复ATF4-/-小鼠OCL分化缺陷。我们将使用转基因小鼠，在转基因小鼠中，小鼠耐酒石酸酸性磷酸酶(TRAP)基因启动子驱动组成活性形式的NFATc1(NFATc1-CA)选择性地在ATF4-/-小鼠的OCL中表达。OCL分化和骨吸收的生化和组织形态计量学参数将在以下小鼠中确定：i)wt，ii)NFATc1-CA-TG，iii)ATF4-/-，以及iv)ATF4-/-；NFATc1-CA-TG。这些拟议目标的成功完成将1)显著提高对OCL分化的分子机制的了解，2)为开发新的、更特异的抗吸收药物提供分子基础，用于治疗毁灭性的骨质疏松症、恶性肿瘤的高钙血症和Paget病患者。 公共卫生相关性：骨骼完整性需要在成骨细胞和骨吸收破骨细胞(OCL)之间保持微妙的平衡。OCL数量和/或活性的异常增加会导致许多骨骼疾病，如骨质疏松症、许多转移性癌症引起的溶骨性病变、骨派杰氏病和类风湿性关节炎。相反，OCL数量和/或活性的减少会导致骨化症，这是一种以骨骼质量显著增加为特征的疾病。明确破骨细胞形成的分子机制对于加深对OCL基础或累及的骨病发病机制的认识和改善这些疾病的预防和治疗是至关重要的。我们证明ATF4是破骨细胞形成的关键转录因子，目前的数据揭示了它在调节OCL早期和晚期分化中的重要性。ATF4在RANKL诱导OCL分化所必需的OCL前体细胞中发挥着内在的作用。ATF4对M-CSF诱导的RANK基因表达(OCL早期分化的关键分子事件)和RANKL诱导的NFATc1基因表达(OCL分化所必需的)的要求，突显了ATF4在破骨细胞形成中的重要作用。本研究旨在阐明M-CSF通过PI3K/AKT途径激活/上调ATF4的机制，研究ATF4如何通过激活NFATc1基因P1启动子来介导RANKL对NFATc1的诱导，并利用特定的NFATc1-CA转基因小鼠模型评价ATF4在体内OCL分化中的重要性。从这些研究中获得的信息将极大地增强我们对正常破骨细胞发生和骨吸收的分子机制的理解。此外，双膦酸类(唑伦磷酸和帕米磷酸钠)是最广泛使用的抗吸收药物，可通过静脉给药来缓解多发性骨髓瘤、乳腺癌、前列腺癌、肺癌和其他癌症以及Paget病患者的骨痛、高钙血症和骨骼并发症，但其机制尚不清楚，可引起严重的副作用，称为双磷酸盐相关性颌骨坏死(S)。这项研究的成功完成将为开发新的、更特异的抗吸收药物治疗这些破坏性疾病提供分子基础。