Molecular Mechanisms Underlying Tak1 Function in Osteoclasts

描述(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、破骨细胞的严格调控。因此，了解破骨细胞形成的机制对于解决骨丢失的病理机制至关重要。破骨细胞的分化受RANK配体的调控，RANK配体激活多个信号转导通路，包括MAP激酶和NF-κB通路。近端激活需要将TRAF6和包括转化生长因子活化蛋白-1(TAK1)在内的其他关键蛋白重新招募到受体的等级。TRAF6、Tak1和其他信号伙伴经历了广泛的翻译后修改，旨在稳定RAN信号，并使正确的下游信号能够精确调节和执行，主要是NF-κB激活。精确调节核因子-κB的活性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会导致有害的炎症性骨溶解。事实上，我们最近发现，IKK2的结构性激活足以在体外诱导RANKL非依赖性破骨细胞生成。更令人信服的是，我们报告说，敲入具有结构性活性的IKK2会导致小鼠严重的骨丢失。鉴于IKK2的磷酸化和激活是由TAK1控制的，TAK1是一种MAP激酶，与RANK-TRAF6复合体的多泛素化和稳定性以及下游信号密切相关，我们决定研究其在破骨细胞形成中的分子作用。因此，我们产生了含有髓系特异性TAK1缺失的小鼠。这些小鼠表现出骨化病的所有特征，主要是破骨细胞生成缺陷。从机制上讲，我们观察到Tak1缺失的前体细胞不能产生破骨细胞。更重要的是，我们发现关键的破骨细胞生成蛋白包括TRAF6、NEMO和Notch-NICD的表达减少。这种现象与NUMBL的积累有关，NUMBL是一种先前描述的神经元蛋白。与这些观察一致，我们建立了外源表达NUMBL诱导TRAF6，NEMO，NOTCH1-NICD的降解，并抑制体外破骨细胞的形成。在野生型细胞中，使用显性负PTB-磷酸酪氨酸结合的NUMBL和NUMBL的shRNAs敲除抑制NUMBL可促进TRAF6和NEMO的表达，但不抑制破骨细胞的形成。此外，用NUMBL的显性负PTB抑制NUMBL和外源表达NOTCH1-NICD可以恢复TAK1缺失细胞的破骨细胞生成。基于这些观察结果，我们推测“NUMBL是破骨细胞生成的抑制因子，其表达受TAK1的调控。TAK1的缺失导致NUMBL蛋白的积聚，从而导致TRAF6、NEMO和NICD蛋白的降解，从而阻止破骨细胞的生成。”为了验证这一假说，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缺失调控和阻碍TRAF6、NEMO和NICD表达的机制。3)检测基因消融NUMBL对TAK1基因缺失小鼠的骨化表型的影响。

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-κB pathways. Proximal activation entails recruitment of TRAF6 and other key proteins including TGF-ß-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-κB activation. Precise regulation of NF-κB 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.