Molecular Mechanisms Underlying Tak1 Function in Osteoclasts

破骨细胞中 Tak1 功能的分子机制

• 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
• 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

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（由适用提供）：骨发育受到骨形成细胞，成骨细胞和骨分辨细胞，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质损失病理至关重要。破骨细胞的分化受秩配体的控制，该秩配体激活了几种信号转导途径，包括MAP激酶和NF-？B途径。近端激活需要募集TRAF6和其他关键蛋白，包括TGF-activated激活的激酶-1（TAK1）到接收器等级。 TRAF6，TAK1和其他信号合作伙伴进行了广泛的翻译后修改，旨在稳定秩信号传导并实现精确调节和执行适当的下流信号，主要NF-？b激活。 NF-？B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。相反，这种转录因子的异常活性实际上是 我们最近发现，IKK2的组成型激活足以在体外诱导RANKL非依赖性的骨质碎裂发生。更令人信服的是，我们报道说，组成型活性IKK2的敲击会导致小鼠严重的骨质流失。鉴于IKK2磷酸化和激活受TAK1的控制，TAK1是一种在多泛素化和稳定级别TRAF6复合物和下游信号传导方面强烈实现的MAP激酶，我们决定研究其在骨质现代发生的分子作用。那，我们产生了带有髓样特定于TAK1的小鼠。这些小鼠表现出骨外骨的全部标志，主要有缺陷的破骨细胞生成。从机械上讲，我们观察到tak1-null前体无法产生破骨细胞生成。更重要的是，我们发现关键破骨细胞生成的表达减少，包括TRAF6，Nemo和Notch-Nicd。该现象与先前描述的神经元蛋白Numbl的积累有关。与这些观察结果一致，我们确定NUMBL的表达表达会诱导Traf6，Nemo，Notch1-Nicd的降解，并在体外抑制破骨细胞生成。使用NUMBL和NUMBL增强TRAF6和NEMO表达的NUMBL和SHRNAS敲低的主要负PTB-磷酸酪氨酸结合对NUMBL的抑制，并且不抑制野生型细胞中的骨质核层生成。此外，使用Numbl的主要负PTB抑制NumBL，Notch1-NICD的表达性表达恢复了Tak1-Null细胞中的骨质质发生。基于这些观察结果，我们假设“ Numbl是破骨构成的复制品，其表达受到TAK1的调节。TAK1的缺失导致Numbl蛋白的积累，诱导Traf6，Nemo和Nicd蛋白的降解，以及随后的阻塞骨骼生成。”为了检验该假设，我们建议研究以下特定目的：1）确定TAK1调节NUMBL表达的机制。 2）确定TAK1删除调节和阻碍Traf6，Nemo和Nicd的表达的机制。 3）确定Numbl遗传消融对Tak1-Null小鼠骨质原理表型的影响。