Redox-based Fas signaling in allergic airway disease

描述（由申请人提供）：死亡受体Fas在肺细胞上表达，它可以转导介导多种结果的信号。我们最近发现，氧化剂过氧化氢（H202）促进小鼠肺上皮细胞中Fas的聚集，并且H202利用Fas向c- jun - n末端激酶（JNK）发出信号，JNK是细胞感知和响应压力的关键信号模块。Fas信号转导的结果不是严格意义上的细胞凋亡，还包括细胞形状改变、炎症等。促生存因子NF-kB在变应性气道疾病小鼠的气道中被长期激活，这可以抵消Fas激活的促凋亡作用。在过敏性气道炎症期间，肺的氧化还原环境向促氧化状态改变，H202存在于过敏性气道疾病小鼠的肺中。因此，本提案需要验证的假设是，H202介导的JNK在变应性气道疾病小鼠肺上皮中的激活，通过Fas的氧化激活，导致气道高反应性（AHR）和重塑。在Specific Aim 1中，我们将通过过氧化氢酶过表达小鼠和使用降低H202的药物来确定H202是否促进过敏性气道疾病小鼠的JNK激活、AHR和重塑。我们还将直接给药H202，以确定H202是否足以激活JNK并引发AHR，以及这是否需要功能性Fas。在Specific Aim 2中，我们将确定过敏性肺部疾病小鼠中Fas是否被氧化和寡聚化，以及这种情况是否发生在气道上皮中，以及此处对H202的需求。在Specific Aim 3中，我们将通过使用全身缺乏Fas的LPR小鼠或通过创建表达CC 10-DN-Fas的小鼠来确定过敏性气道疾病小鼠气道中JNK激活、AHR和重塑对气道上皮Fas的需求。最后，在Specific Aim 4中，我们将通过在传导气道细胞中以四环素诱导的方式表达JNK1显性阴性版本的转基因小鼠的产生，确定气道上皮JNK激活在AHR中的贡献。这些方法对于确定对过敏性气道疾病的病理生理学至关重要的关键氧化靶点和上皮信号事件至关重要。

DESCRIPTION (provided by applicant): The death receptor Fas is expressed on lung cells where it can transduce signals that mediate diverse outcomes. We recently showed that the oxidant, hydrogen peroxide (H202) promotes clustering of Fas in mouse lung epithelial cells, and that H202 uses Fas to signal to c-Jun-N-terminal kinase, (JNK), a critical signaling module that allows cells to sense and respond to stress. The outcome of Fas signaling is not strictly apoptosis, but also including shape changes, inflammation etc. The pro-survival factor, NF-kB is activated in a prolonged fashion in airways of mice with allergic airway disease, which can counteract the pro-apoptotic effect of Fas activation. During allergic airways inflammation, the redox environment of the lung is changed towards a pro-oxidant state, and H202 is present in lungs of mice with allergic airways disease. Thus, the hypothesis to be tested in this proposal is that H202- mediated activation of JNK in lung epithelium of mice with allergic airways disease contributes airways hyper-responsiveness (AHR) and remodeling, through oxidative activation of Fas. In Specific Aim 1, we will determine whether H202 promotes JNK activation, AHR, and remodeling in mice with allergic airways disease via the use of catalase over-expressing mice, and the use of pharmacologic agents that lower H202. We will also directly administer a source of H202, to determine whether H202 is sufficient to activate JNK, and elicit AHR, and whether this requires functional Fas. In Specific Aim 2, we will determine whether Fas is oxidized and oligomerized in mice with allergic lung disease, and whether this occurs in airway epithelium, and the requirement for H202 herein. In Specific Aim 3, we will determine the requirement of airway epithelial Fas in JNK activation, AHR, and remodeling in the airways of mice with allergic airway disease by using LPR mice, which lack Fas systemically, or via the creation a CC 10-DN-Fas expressing mouse. Lastly, in Specific Aim 4 we will determine the contribution of airway epithelial JNK activation in AHR through the generation of a transgenic mouse expressing a dominant negative version of JNK1 in cells of the conducting airways, in a tetracycline inducible manner. These approaches are crucial in defining critical oxidant targets and epithelial signaling events crucial to the patho-physiology of allergic airways disease.