Crosstalk Between Nephrogenesis and Ahr Signaling

肾发生和 Ahr 信号传导之间的串扰

• 批准号: 6889498
• 负责人: MOHAMMAD H FALAHATPISHEH
• 金额: $ 3.99万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-05-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6889498
• 关键词: apoptosis; aromatic hydrocarbon receptor; benzopyrenes; biological signal transduction; cell differentiation; cell growth regulation; dioxins; embryo /fetus cell /tissue; embryo /fetus toxicology; environmental toxicology; gene expression; genetically modified animals; immunoprecipitation; intermolecular interaction; laboratory mouse; microarray technology; nephrogenesis; oxidative stress; polymerase chain reaction; postdoctoral investigator; protein structure function; spliceosomes; transcription factor; tumor suppressor proteins; yeast two hybrid system

DESCRIPTION (provided by applicant): The Ramos laboratory has recently shown that activation of Ahr signaling by environmental hydrocarbons modulates wt1 splicing and disrupts nephrogenesis. The overall goal of the research proposed in this application is to evaluate molecular interactions between Ahr and wt1, and their role in hydrocarbon-induced deficits in nephrogenesis. Studies will be conducted to test the hypotheses that 1) activation of Ahr and redox signaling by benzo(a)pyrene (BaP), an Ahr activator, mediates alterations in spliceosome function that are coupled to deficits in nephroblast differentiation and nephrogenesis and, 2) that activation of Ahr signaling by BaP, modulates downstream effectors of WT1 during nephrogenesis and induces variations in global mRNA expression that cluster within mesenchymal phenotypes or "attractors". Metanephroi cultures will be established from 11.5 C57BL/6J embryos and challenged with vehicle (DMSO) or 3 muM BaP for 4 days. Cyp1a1 and 1b1 mRNA and protein levels will be examined by RT-PCR and Western analysis. To address the role of BaP-induced DNA adduction or mutation in modulation of wt1 splicing, mutational analysis and 32P post-labeling experiments will be conducted. The first approach to evaluate the ability of Ahr to interact directly with components of the splicing machinery will rely on yeast two-hybrid methodology. To determine if Ahr and U2AF65 form protein complexes, we will perform pull down assays and co-immunopercipitation experiments following two hybrid experiments. Transcriptional regulation of splicing factors following Ahr activation will be examined by real time or RT-PCR experiments. Microarray analysis will be performed using RNA isolated from both Ahr+/+ and Ahr-/- metahephroi in the presence and absence of Ahr ligands to identify critical targets of WT1 and functional networks of co-regulation. Gene expression profiles as outlined above will be evaluated using a multiple binary expression system. The rules of Boolean genetic networks will enable us to identify specific genes expressed in different stages of renal development as blastemal cells transition from mesenchymal to epithelial phenotypes, and the transcriptional contextual network in which these changes occur.

描述（由申请人提供）：Ramos 实验室最近表明，环境碳氢化合物激活 Ahr 信号传导可调节 wt1 剪接并破坏肾发生。本申请中提出的研究的总体目标是评估 Ahr 和 wt1 之间的分子相互作用，以及它们在碳氢化合物诱导的肾发生缺陷中的作用。将进行研究来测试以下假设：1) Ahr 激活剂苯并(a)芘 (BaP) 激活 Ahr 和氧化还原信号传导，介导剪接体功能的改变，而剪接体功能的改变与肾母细胞分化和肾发生的缺陷相关，2) BaP 激活 Ahr 信号传导，在肾发生过程中调节 WT1 的下游效应器，并诱导间充质细胞内聚集的全局 mRNA 表达的变化表型或“吸引子”。后肾培养物将从 11.5 个 C57BL/6J 胚胎中建立，并用载体 (DMSO) 或 3 muM BaP 攻击 4 天。 Cyp1a1 和 1b1 mRNA 和蛋白质水平将通过 RT-PCR 和 Western 分析进行检查。为了解决 BaP 诱导的 DNA 加合或突变在 wt1 剪接调节中的作用，将进行突变分析和 32P 后标记实验。评估 Ahr 与剪接机器组件直接相互作用的能力的第一种方法将依赖于酵母双杂交方法。为了确定 Ahr 和 U2AF65 是否形成蛋白质复合物，我们将在两次混合实验后进行 Pull Down 测定和免疫共沉淀实验。 Ahr 激活后剪接因子的转录调控将通过实时或 RT-PCR 实验进行检查。在存在和不存在 Ahr 配体的情况下，将使用从 Ahr+/+ 和 Ahr-/- 变肝中分离的 RNA 进行微阵列分析，以识别 WT1 的关键靶标和共同调节的功能网络。如上所述的基因表达谱将使用多个二元表达系统进行评估。布尔遗传网络的规则将使我们能够识别在胚细胞从间充质表型转变为上皮表型时在肾脏发育的不同阶段表达的特定基因，以及发生这些变化的转录背景网络。