Repression of ARE-Mediated Gene Expression

描述（由申请人提供）：本申请的总体目标是确定负责响应TGF-β信号传导的活性氧（ROS）水平升高的分子途径，以帮助开发治疗这一重大临床问题所需的治疗策略。ROS促进接受放射治疗的胸部恶性肿瘤患者退行性肺损伤的进展。II期解毒蛋白负责清除ROS。转录因子Nrf 2是II期基因表达的主要调节因子。Nrf 2信号传导的缺失消除II期基因表达，升高ROS，并促进肺纤维化的进展。我们发现TGF-β信号抑制Nrf 2调节的II期基因表达并升高ROS。我们假设Nrf 2调节的转录抑制是ATF 3介导的，是TGF-β通过SmadS信号传导的结果。目的1将确定ATF 3抑制Nrf 2调节的基因表达的机制。该方法将确定所需的结构域ATF 3 Nrf 2在体外和体内的相互作用，并确定这种相互作用是否改变Nrf 2的功能，无论是Nrf 2 DNA结合活性或转录激活。将使用缺失构建体与GST融合分子的结合、免疫共沉淀、双杂交系统、DNA沉淀和ChIP测定。目的2将确定ATF 3的缺失是否消除TGF-β介导的Nrf 2调节的II期基因表达的抑制并降低ROS水平。将使用两种方法来下调ATF 3：显性负性ATF 3分子和针对ATF 3的siRNA。目的3将检验TGF-β介导的信号转导抑制体内Nrf 2调节的基因表达以及Nrf 2影响辐射诱导的肺纤维化的假设。放射诱导的纤维化的良好表征的模型将用于确定Nrf 2信号传导的损失是否增强肺损伤。将使用Nrf 2野生型和无效动物。

DESCRIPTION (provided by applicant): The overall goal of the application is to identify the molecular pathways responsible for elevated reactive oxygen specie (ROS) levels that follow in response to TGF-beta signaling in order to aid the development of therapeutic strategies needed to treat this significant clinical problem. ROS contribute to the progression of degenerative pulmonary injury in patients with thoracic malignancies who undergo radiation therapy. Phase II detoxification proteins are responsible for removal of ROS. The transcription factor Nrf2 is a master regulator of Phase II gene expression. Loss of Nrf2 signaling ablates Phase II gene expression, elevates ROS, and enhances progression of pulmonary fibrosis. We show that TGF-beta signaling suppresses Nrf2- regulated Phase II gene expression and elevates ROS. We hypothesize that suppression of Nrf2-regulated transcription is ATF3 mediated, a consequence of TGF-beta signaling through SmadS. Aim 1 will identify the mechanism by which ATF3 suppresses Nrf2-regulated gene expression. The approach will identify the domains required for ATF3 Nrf2 interaction in vitro and in vivo and determine if such an interaction alters Nrf2 function in terms of either Nrf2 DNA binding activity or transcriptional activation. Binding of deletion constructs to GST fusion molecules, co-immunoprecipitation, two hybrid systems, DNA precipitation and ChIP assays will be used. Aim 2 will determine if loss of ATF3 abrogates TGF-beta-mediated suppression of Nrf2-regulated Phase II gene expression and lowers ROS levels. Two approaches will be used to down regulate ATF3: Dominant negative ATF3 molecules and siRNA directed against ATF3. Aim 3 will test the hypothesis that TGF-beta-mediated signaling suppresses Nrf2-regulated gene expression in vivo and that Nrf2 impacts radiation-induced pulmonary fibrosis. The well characterized model of radiation-induced fibrosis will be used to determine if loss of Nrf2 signaling enhances pulmonary injury. Nrf2 wild type and null animals will be used.