Molecular Mechanisms Underlying Tak1 Function in Osteoclasts

破骨细胞中 Tak1 功能的分子机制

基本信息

批准号：
8501884
负责人：
YOUSEF ABU-AMER
金额：
$ 32.3万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-07 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8501884
关键词：
Ablation Adaptor Signaling Protein Address Albers-Schonberg disease Area Binding Biology Bone Development Bone remodeling Cells Complex Coupled Development Dominant-Negative Mutation Genes Genetic Growth Homeostasis In Vitro Inflammatory Knock-in Mouse Knockout Mice Link Lysine MAPK14 gene MAPK8 gene Macrophage Colony-Stimulating Factor Maintenance Marrow Mediating Mitogen-Activated Protein Kinases Molecular Mus Myelogenous NOTCH1 gene Neurons Null Lymphocytes Osteoblasts Osteoclasts Osteolysis PTB Domain Pathology Pathway interactions Phenotype Phosphorylation Phosphotransferases Phosphotyrosine Post-Translational Protein Processing Proteins Regulation Reporting Role Signal Pathway Signal Transduction Signal Transduction Pathway Skeletal Development Stream TNFSF11 gene TRAF6 gene TRANCE protein Testing Thick Tooth eruption Transcriptional Regulation Ubiquitination base bone bone loss cell type notch protein novel osteoclastogenesis protein degradation public health relevance receptor response restoration substantia spongiosa transcription factor ubiquitin-protein ligase upstream kinase

项目摘要

DESCRIPTION (provided by applicant): Bone development is tightly regulated by bone forming cells, osteoblasts, and bone resorbing cells, osteoclasts. Therefore, understanding the mechanisms governing osteoclastogenesis is crucial for addressing bone loss pathologies. Differentiation of osteoclasts is governed by RANK ligand which activates several signal transduction pathways, including MAP kinases and NF-?B pathways. Proximal activation entails recruitment of TRAF6 and other key proteins including TGF-¿-activated kinase-1 (TAK1) to the receptor RANK. TRAF6, TAK1 and other signaling partners undergo extensive post-translational modifications aimed at stabilizing RANK signaling and enabling precise regulation and execution of proper down stream signals, primarily NF-?B activation. Precise regulation of NF-?B activity is crucial to maintain normal osteoclast activity and bone homeostasis. Conversely, abnormal activity of this transcription factor causes deleterious inflammatory osteolysis. In fact, we discovered recently that constitutive activation of IKK2 is sufficient to induce RANKL-independent osteoclastogenesis in vitro. More convincingly, we reported that knock-in of constitutively active IKK2 causes severe bone loss in mice. Given that IKK2 phosphorylation and activation is governed by TAK1, a MAP kinase heavily implicated in poly-ubiquitination and stabilization of RANK-TRAF6 complexes and down-stream signaling, we decided to investigate its molecular role in osteoclastogenesis. Thus, we generated mice harboring myeloid-specific deletion of TAK1. These mice displayed all hallmarks of osteopetrosis primarily defective osteoclastogenesis. Mechanistically, we observed that Tak1-null precursors fail to generate osteoclasts. More importantly, we discovered diminished expression of key osteoclastogenic proteins including TRAF6, NEMO and Notch-NICD. This phenomenon was associated with accumulation of NUMBL, a previously described neuron protein. Consistent with these observations, we established that exogenous expression of NUMBL induces degradation of TRAF6, NEMO, NOTCH1-NICD, and inhibits osteoclastogenesis in vitro. Inhibition of NUMBL using a dominant negative PTB-phosphotyrosine-binding of NUMBL and shRNAs knockdown of NUMBL enhanced expression of TRAF6 and NEMO and did not inhibit osteoclastogenesis in wild-type cells. In addition, inhibition of NUMBL using a dominant negative PTB of NUMBL and exogenous expression of NOTCH1-NICD restored osteoclastogenesis in TAK1-null cells. Based on these observations we hypothesize that "NUMBL is a repressor of osteoclastogenesis and its expression is regulated by TAK1. Deletion of TAK1 leads to accumulation of NUMBL protein which induces degradation of TRAF6, NEMO and NICD proteins, and subsequently blocks osteoclastogenesis." To test this hypothesis, we propose to investigate the following specific aims: 1) Determine the mechanism by which TAK1 regulates NUMBL expression. 2) Determine the mechanism by which TAK1 deletion regulates and impedes expression of TRAF6, NEMO and NICD. 3) Determine the effect of genetic ablation of NUMBL on the osteopetrotic phenotype of TAK1-null mice.

描述（由申请人提供）：骨发育受到骨形成细胞、成骨细胞和骨吸收细胞、破骨细胞的严格调节，因此，了解控制破骨细胞生成的机制对于解决骨丢失病理学至关重要。破骨细胞的分化受RANK配体控制。激活多种信号转导途径，包括 MAP 激酶和 NF-κB 途径。近端激活需要招募 TRAF6 和其他关键蛋白，包括 TGF-¿ -激活激酶-1 (TAK1) 与受体 TRAF6、TAK1 和其他信号传导伴侣进行广泛的翻译后修饰，旨在稳定 RANK 信号传导并实现正确下游信号（主要是 NF-κB 激活）的精确调节和执行。 NF-κB 活性的精确调节对于维持正常的破骨细胞活性和骨稳态至关重要，该转录因子的异常活性会导致有害的炎症性骨溶解。我们最近发现，IKK2 的组成型激活足以在体外诱导不依赖 RANKL 的破骨细胞生成，更令人信服的是，我们报道，鉴于 IKK2 磷酸化和激活受 TAK1 控制，敲入组成型活性 IKK2 会导致小鼠严重骨质流失。 MAP 激酶与 RANK-TRAF6 复合物的多泛素化和稳定以及下游信号传导密切相关，我们决定研究其分子因此，我们培育了具有 TAK1 骨髓特异性缺失的小鼠。从机制上讲，我们观察到 TAK1 缺失的前体细胞无法产生破骨细胞。关键的破骨细胞蛋白，包括 TRAF6、NEMO 和 Notch-NICD，这种现象与 NUMBL（一种先前描述的神经元蛋白）的积累有关。与这些观察结果一致，我们确定 NUMBL 的外源表达会诱导 TRAF6、NEMO、NOTCH1-NICD 降解，并在体外通过 NUMBL 的显性失活 PTB-磷酸酪氨酸结合和 shRNA 敲低 NUMBL 增强表达来抑制 NUMBL。 TRAF6 和 NEMO 的作用，并且不抑制野生型细胞中的破骨细胞生成。此外，使用显性失活 PTB 抑制 NUMBL。 NUMBL 和 NOTCH1-NICD 的外源表达恢复了 TAK1 缺失细胞中的破骨细胞生成，基于这些观察，我们发现“NUMBL 是破骨细胞生成的抑制因子，其表达受 TAK1 调节。TAK1 的缺失会导致 NUMBL 蛋白的积累，从而诱导降解。 TRAF6、NEMO 和 NICD 蛋白，并随后阻断破骨细胞生成。”为了检验这一假设，我们建议研究以下具体目标：1)确定 TAK1 调节 NUMBL 表达的机制 2) 确定 TAK1 缺失调节和阻碍 TRAF6、NEMO 和 NICD 表达的机制 3) 确定 NUMBL 基因消除对 TAK1 缺失小鼠骨硬化表型的影响。