ATF4 and Osteoclastogenesis

ATF4 和破骨细胞生成

• 批准号: 8314106
• 负责人: Guozhi Xiao
• 金额: $ 26.44万
• 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/8314106
• 关键词: Acids; Address; Adverse effects; Albers-Schonberg disease; Animals; Binding; Biochemical; Biological Assay; Bone Diseases; Bone Marrow; Bone Pain; Bone Resorption; Bone necrosis; Bone remodeling; Breast; Cells; Coculture Techniques; Data; Defect; Development; Disease; Disseminated Malignant Neoplasm; Equilibrium; Event; Gene Expression; Genes; Heterodimerization; Hypercalcemia; Hypercalcemia of Malignancy; In Vitro; Jaw; Life; Lung; Lytic Metastatic Lesion; Macrophage Colony-Stimulating Factor; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Multiple Myeloma; Mus; Osteitis Deformans; Osteoblasts; Osteoclasts; Osteoporosis; PI3K/AKT; Pathogenesis; Pathway interactions; Patients; Phosphorylation; Phosphorylation Site; Play; Prevention; Prostate; Proteins; Proto-Oncogene Proteins c-akt; Regulation; Rheumatoid Arthritis; Role; Signal Transduction; Skeletal Development; Skeletal bone; TNF Receptor-Associated Factors; TRANCE protein; Transgenic Mice; Transgenic Organisms; activating transcription factor 4; base; bisphosphonate; bone; design; improved; in vivo; macrophage; monocyte; mouse model; nuclear factors of activated T-cells; osteoclastogenesis; overexpression; pamidronate; precursor cell; promoter; public health relevance; receptor; research study; skeletal; tartrate-resistant acid phosphatase; transcription factor

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（由申请人提供）：破骨细胞（OCL）是唯一对整个生命过程中正常骨骼发育和骨重塑至关重要的骨吸收细胞。异常的OCL数量和/或活性导致许多骨疾病，例如骨质疏松症、佩吉特骨病、转移性溶骨性病变和类风湿性关节炎。然而，破骨细胞生成的分子机制尚不清楚。我们的初步研究表明，激活转录因子4（ATF4）是调节破骨细胞生成的关键因素。我们的数据表明，ATF4 介导 M-CSF 诱导的 RANK 表达，这是 OCL 早期分化所需的关键分子事件。此外，ATF4 指导 NFATc1 的 RANKL 依赖性基因表达，NFATc1 是 OCL 分化的主要调节因子。在这项研究中，我们假设 ATF4 通过两种不同的机制在调节破骨细胞生成中发挥关键作用：1）ATF4 通过 PI3K/AKT 依赖性磷酸化和蛋白质稳定和/或激活来调节 M-CSF 诱导 RANK 表达； 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，介导 NFATc1 基因的 RANKL 诱导。为了实现我们的假设，我们将追求以下具体目标：目标 1 将确认 OCL 前体中 ATF4 通过 PI3K/AKT 被 M-CSF 信号磷酸化并上调。我们将鉴定 AKT 和 M-CSF 反应性磷酸化位点，并评估它们在调节 ATF4 蛋白稳定性和活性以支持破骨细胞生成方面的功能意义。目标 2 将确定 ATF4 是否通过与 NFATc1 基因 P1 启动子上其他关键因子的协同相互作用来介导 NFATc1 基因的 RANKL 诱导。我们还将鉴定 ATF4 异二聚化伴侣，并评估 ATF4 和伴侣之间相互作用在 RANKL 诱导的 NFATc1 表达中的功能意义。目标 3 将确定 NFATc1 的 OCL 特异性转基因表达是否可以挽救 Atf4-/- 小鼠 OCL 分化的缺陷。我们将使用转基因小鼠，其中小鼠抗酒石酸酸性磷酸酶 (TRAP) 基因启动子选择性地驱动 Atf4-/- 小鼠 OCL 中 NFATc1 (NFATc1-CA) 的组成型活性形式的表达。将在以下小鼠组中测定 OCL 分化和骨吸收的生化和组织形态学参数：i) wt，ii) NFATc1-CA-tg，iii) Atf4-/-，和 iv) Atf4-/-； NFATc1-CA-tg。成功完成这些拟议目标将：1）显着促进对 OCL 分化分子机制的理解，2）为开发新的、更特异的抗骨吸收药物提供分子基础，用于治疗患有破坏性骨质疏松症、恶性肿瘤高钙血症和佩吉特骨病的患者。 公共健康相关性：骨骼完整性需要骨形成成骨细胞和骨吸收破骨细胞 (OCL) 之间的微妙平衡。 OCL数量和/或活性异常增加导致许多骨疾病，例如骨质疏松症、许多转移性癌症引起的溶骨性病变、佩吉特骨病和类风湿性关节炎。相反，OCL 数量和/或活性减少会导致骨硬化症，这是一种以骨骼质量显着增加为特征的疾病。明确破骨细胞生成的分子机制对于增进对基于 OCL 或相关骨疾病发病机制的分子基础的理解并改善这些疾病的预防和治疗至关重要。我们证明 ATF4 是破骨细胞生成的关键转录因子，并提供的数据揭示了其在调节早期和晚期 OCL 分化中的重要性。 ATF4 在 OCL 前体中发挥内在作用，这对于 RANKL 诱导的 OCL 分化是必不可少的。 ATF4 在破骨细胞生成中的重要作用通过其对 M-CSF 诱导的 RANK 基因表达（早期 OCL 分化的关键分子事件）以及 RANKL 诱导的 NFATc1 基因表达（OCL 分化所需）的要求得到强调。该提案旨在阐明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启动子介导RANKL诱导NFATc1，并使用特定的NFATc1-CA转基因小鼠模型评估ATF4在体内OCL分化中的重要性。从这些研究中获得的信息将显着增强我们对骨骼发育和整个生命过程中正常破骨细胞生成和骨吸收所涉及的分子机制的理解。此外，双膦酸盐（唑仑膦酸和帕米膦酸盐）是最广泛使用的抗骨吸收药物，通过静脉注射来减轻多发性骨髓瘤、乳腺癌、前列腺癌、肺癌和其他癌症以及佩吉特骨病患者的骨痛、高钙血症和骨骼并发症，但具有严重的副作用，称为双膦酸盐相关性骨坏死。 通过未定义的机制进行钳口。这项研究的成功完成将为开发新的、更特异的抗骨吸收剂来治疗这些破坏性疾病提供分子基础。