Retinoid Mediated Protection Against Reactive Oxygen Species Induced Cytotoxicity

描述(由申请人提供)：我们已经证明，在11-脱氧-16，16-二甲基前列腺素E_2(DDM-PGE_2)介导的细胞保护中，维甲酸信号参与了对活性氧(ROS)诱导的坏死/肿胀细胞死亡的保护作用。蛋白质组学分析表明，细胞保护与特定数量的蛋白质合成增加有关，包括视黄醇结合蛋白(RBP)、肌动蛋白和葡萄糖调节蛋白78(GRP78)。我们随后证实，全反式维甲酸(ATRA)在体外复制了DDM-PGE2介导的细胞保护作用，更重要的是，单次给药(1 mg/kg，6h预处理)完全保护小鼠肾脏缺血/再灌注(I/R)损伤。此外，在此治疗剂量下，ATRA可诱导Nrf2反应的抗氧化剂HO-1和NQO1基因，以及核维甲酸受体RAR？、RAR？2、RAR？2和维甲酸X受体RXR？在肾脏里。修订后的应用旨在确定全反式维甲酸在体外提供细胞保护的分子机制，以及这种细胞保护机制(S)在体内的概括程度。我们的中心假设是，ATRA诱导的细胞保护是由类似于缺血预适应的机制介导的。在特定目的1中，我们建议在体外缺氧/复氧损伤模型(人肾上皮HK-2细胞)中确定ATRA提供细胞保护的能力，并在体内缺血/再灌流模型(IR)中优化ATRA介导的细胞保护方案。我们还将确定这种保护作用是否至少部分是通过抗氧化酶的上调来介导的。维甲酸的生物学效应通常是通过与其同源核受体，即维甲酸受体(RAR)和维甲酸X受体(RXR)的相互作用来实现的。RAR和/或RXR在多大程度上参与了ATRA介导的细胞保护尚不清楚，在体外和体内I/R模型中，特异性靶点2将决定ATRA介导的细胞保护是否需要与RAR和/或RXR相互作用。因此，特定的目标1和2旨在确定在ROS在疾病的病理中发挥重要作用的情况下，如涉及缺血再灌注损伤的情况下，维甲酸信号的招募作为一种潜在的治疗干预措施(特定目标1)，并初步表征这种作用的药理学基础(特定目标2)。第三个也是最后一个特定目标是确定ATRA完成细胞保护的分子机制，每个子目标都集中在一个在初步研究中已经确定的在细胞保护反应中发挥重要作用的靶点。具体地说，这些介质是Nrf2、GRP78和p38MAPK，它们中的每一个也是缺血预适应的关键介质。因此，特定目的3将决定是否(I)全反式维甲酸介导的诱导抗氧化应激反应依赖于Nrf2；(Ii)全反式维甲酸介导的细胞保护需要ER(GRP78)介导的应激反应通路的募集；(Iii)全反式维甲酸对Nrf2的募集依赖于p38MAPK-GRP78的相互作用；以及(Iv)通过检测全反式维甲酸诱导的NRF2-/-小鼠的肾保护，体外模型中确定的细胞保护机制(S)被概述。目前这些研究的意义在于它们有可能在分子和细胞水平上加强我们对维甲酸介导的细胞保护的理解，从而为临床治疗化学诱导的组织损伤或缺氧/缺血再灌注损伤提供有效的新的治疗策略。

DESCRIPTION (provided by applicant): We have shown that retinoid signaling is engaged during 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) mediated cytoprotection against reactive oxygen species (ROS) induced necrotic/oncotic cell death. Proteomics analyses revealed that cytoprotection is associated with the increased synthesis of a select number of proteins, including retinol binding protein (RBP), actin, and glucose-regulated protein 78 (Grp78). We subsequently confirmed that all-trans-retinoic acid (aTRA) replicates DDM-PGE2-mediated cytoprotection in vitro, and more importantly, a single dose of aTRA (1 mg/kg, 6h pretreatment) completely protects mice from renal ischemia/reperfusion (I/R) injury. Furthermore, at this therapeutic dose, aTRA induces Nrf2-responsive antioxidant HO-1 and NQO1 genes, as well as nuclear retinoic acid receptors RAR?, RAR?2, RAR?2, and retinoid X receptors RXR? in the kidney. The revised application is designed to determine the molecular mechanisms by which aTRA affords cytoprotection in vitro, and the extent to which this mechanism(s) of cytoprotection is recapitulated in vivo. Our central hypothesis is that aTRA-induced cytoprotection is mediated by mechanisms similar to ischemic preconditioning. In Specific Aim 1 we propose to determine the ability of aTRA to offer cytoprotection in an in vitro model (human renal epithelial HK-2 cells) of hypoxia/reoxygenation injury, and to optimize protocols for aTRA-mediated cytoprotection in an in vivo ischemia/reperfuson model (IR). We will also ascertain whether the protective effects are mediated, at least in part, via the upregulation of anti-oxidant enzymes. The biological effects of retinoids are typically mediated via interaction with their cognate nuclear receptors, namely, retinoic acid receptors (RAR) and retinoid X receptors (RXR). The extent to which RAR and/or RXR participate in aTRA-mediated cytoprotection is not known, and Specific Aim 2 will determine, in both the in vitro and in vivo models of I/R, whether aTRA-mediated cytoprotection requires interaction with RAR and/or RXR. Specific Aims 1 and 2 are therefore designed to establish the recruitment of retinoid signaling as a potential therapeutic intervention in conditions where ROS play an important role in the pathology of the disease, such as those involving ischemia reperfusion injury (Specific Aim 1), and to initially characterize the pharmacological basis of this effect (Specific Aim 2). The third and final Specific Aim is designed to identify the molecular mechanisms by which aTRA accomplishes cytoprotection, with each sub- aim focusing on a target that has already been identified in preliminary studies as playing an important role in the cytoprotective response. Specifically, those mediators are Nrf2, Grp78, and p38 MAPK, each of which is also a key mediator of ischemia preconditioning. Specific Aim 3 will therefore determine whether (i) aTRA- mediated induction of the anti-oxidant stress response is dependent upon Nrf2; (ii) aTRA-mediated cytoprotection requires the recruitment of the ER (Grp78) mediated stress response pathway; (iii) the recruitment of Nrf2 by aTRA is dependent on p38 MAPK-Grp78 interactions; and (iv) the mechanism(s) of cytoprotection identified in the in vitro model are recapitulated in the in vivo model by testing aTRA induced renoprotection in Nrf2-/- mice. The significance of the current studies resides in their potential to enhance our understanding of retinoid mediated cytoprotection at the molecular and cellular level, which can subsequently provide insights into novel therapeutic strategies effective for clinical interventions during chemical induced tissue injury or hypoxia/ischemia-reperfusion injury.