Retinoid Mediated Protection Against Reactive Oxygen Species Induced Cytotoxicity

项目摘要 我们已经证明，维甲酸信号在11-脱氧-16，16-二甲基前列腺素E_2(DDM-PGE_2)介导的过程中参与 对活性氧(ROS)诱导的坏死/肿胀细胞死亡的细胞保护。蛋白质组学分析 研究表明，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DDM-PGE2介导的细胞保护，更重要的是， 单剂量全反式维甲酸(1 mg/kg，6h预处理)对小鼠肾缺血再灌注损伤的保护作用 受伤。此外，在这个治疗剂量下，ATRA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。 修改后的应用旨在确定ATRA提供的分子机制 体外细胞保护，以及这种细胞保护机制(S)在体内的简要介绍。我们的 中心假说是ATRA诱导的细胞保护是由类似于缺血的机制介导的 预适应。在具体目标1中，我们建议确定ATRA在IN中提供细胞保护的能力 体外(人肾上皮HK-2细胞)缺氧/复氧损伤模型的建立 全反式维甲酸在体内缺血再灌流模型中的细胞保护作用我们亦会确定是否会 保护作用至少部分是通过上调抗氧化酶来实现的。生物学的 维甲酸的作用通常是通过与其同源核受体，即维甲酸的相互作用来实现的。 酸性受体(RAR)和维甲酸X受体(RXR)。RAR和/或RXR参与ATRA的程度- 在体外和体内模型中，介导的细胞保护尚不清楚，特异性靶点2将确定。 ATRA介导的细胞保护是否需要与RAR和/或RXR相互作用。具体目标1和2 因此被设计用来确定维甲酸信号的招募作为一种潜在的治疗干预 ROS在疾病病理中起重要作用的情况，如涉及缺血的情况 再灌注损伤(特异靶1)，并初步表征该作用的药理学基础(特异靶1 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，每个子目标都集中在已在 初步研究认为在细胞保护反应中起着重要作用。具体地说，这些调解人是 Nrf2、GRP78和p38MAPK，其中每一个也是缺血预适应的关键介质。具体目标3 因此将决定(I)ATRA介导的抗氧化剂应激反应的诱导是否依赖 (Ii)全反式维甲酸(ATRA)介导的细胞保护需要内质网(GRP78)介导的应激的招募 反应途径；(Iii)ATRA对Nrf2的募集依赖于p38 MAPK-GRP78相互作用；以及(Iv) 体外模型中确定的细胞保护机制(S)在体内模型中通过测试概括 全反式维甲酸对NRF2-/-小鼠的肾保护作用。当前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，这可以 随后提供了对化学治疗期间临床干预有效的新治疗策略的见解 诱导组织损伤或缺氧/缺血-再灌注损伤。

Project Summary 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.