ATF4 and Osteoclastogenesis

描述（由申请人提供）：破骨细胞（ocl）是唯一的骨吸收细胞，在整个生命过程中对正常骨骼发育和骨重塑至关重要。异常的OCL数量和/或活动导致许多骨疾病，如骨质疏松症、骨佩吉特病、转移性溶骨病变和类风湿性关节炎。然而，破骨细胞发生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）是调节破骨细胞发生的关键因子。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还指导OCL分化的主要调控因子NFATc1的rankl依赖性基因表达。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发生中起关键作用：1)ATF4通过PI3K/ akt依赖性磷酸化和蛋白稳定和/或激活调节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。为了验证我们的假设，我们将追求以下具体目标：Aim 1将证实ATF4在OCL前体中通过PI3K/AKT被M-CSF信号磷酸化和上调。我们将确定AKT和M-CSF响应磷酸化位点，并评估其在调节ATF4蛋白稳定性和活性以支持破骨细胞发生中的功能意义。Aim 2将确定ATF4是否通过与NFATc1基因P1启动子上的其他关键因子的协同相互作用介导NFATc1基因的RANKL诱导。我们还将鉴定ATF4异二聚化伙伴，并评估ATF4和伙伴之间相互作用在rankl诱导的NFATc1表达中的功能意义。Aim 3将确定OCL特异性转基因表达NFATc1是否可以挽救Atf4-/-小鼠OCL分化缺陷。我们将使用转基因小鼠，其中小鼠抗酒石酸酸性磷酸酶（TRAP）基因启动子在Atf4-/-小鼠的ocl中选择性地驱动组成型活性形式的NFATc1 （NFATc1- ca）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。成功完成这些目标将1)显著推进对OCL分化的分子机制的理解，2)为开发新的和更特异性的抗吸收药物提供分子基础，用于治疗破坏性骨质疏松症、恶性肿瘤高钙血症和骨Paget病。

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.