Mechanisms For p38 MAP Kinase DependentT Gene Induction

p38 MAP 激酶依赖性 T 基因诱导机制

• 批准号: 6799254
• 负责人: Betty Sue Pace
• 金额: $ 34.61万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6799254
• 关键词: amidohydrolases; butyrates; chemical structure function; chromatin; clinical research; disease /disorder model; enzyme induction /repression; enzyme inhibitors; free radical oxygen; gamma globulin; gel mobility shift assay; gene induction /repression; genetic regulation; genetic regulatory element; genetically modified animals; hemoglobin F; human tissue; hydroxamate; immunoprecipitation; laboratory mouse; mitogen activated protein kinase; nucleic acid structure; sickle cell anemia; sodium; thalassemia; transcription factor

DESCRIPTION (provided by applicant): This application is aimed at identifying inducible transcription factors that activate fetal hemoglobin (HbF) expression, for the development of novel therapies for Sickle cell disease and Cooley's anemia. The rationale for this strategy is based on clinical, genetic and biochemical evidence illustrating the beneficial effect of HbF in both genetic disorders. Histone deacetylase inhibitors (HDAIs) are validated HbF inducers but available analogues are not widely used due to anti-proliferative effects and poor bioavailability. HDAIs alter DNA-protein interactions in the fetal globin gene promoter, which indicates initial activation of hitherto unknown transactivator molecules, which in turn activate fetal globin. Based on this deduction, we initiated studies with two HDAIs sodium butyrate and trichosatin A and found both to activate p38 mitogen activating protein kinase (p38 MAPK) and activating transcription factor 2 (ATF-2). We have confirmed this mechanism of g globin activation in experiments using a specific activator (anisomycin) and inhibitor (SB203580) of p38 MAPK. From these findings we have developed a hypothesis to be tested in four major specific aims, 1) Determine the mechanism for p38 MAPK activation by histone deacetylase inhibitors. We will establish whether HDAI activation of p38 MAPK is direct or through reactive oxygen species in the mitochondria and test novel HDAIs currently in Phase I clinical trials for g globin inducibility. 2) Define the cis-regulatory element(s) required for p38 MAPK dependent g gene induction. These studies will include functional analysis of the g promoter using reporter and expression vectors. 3) Identify transcription factor(s) required for p38 MAPK dependent g gene activation. Cognate binding transcription factors for the elements identified in Aim 2 will be characterized by gel shift analysis and immunoprecipitation studies. Experiments in this Aim will include validation that the identified transactivation or co-activator proteins activate endogenous g globin. 4) Determine the ability of novel HDAIs to induce g gene activity in vivo in transgenic lines. Novel HDAIs found to be effective in Aim 1 will be tested in sickle transgenic mice. Successful completion of the experiments described in the Specific Aims will provide the basis for developing effective drug or gene therapy protocols for Sickle cell disease and Cooley's anemia.

描述（由申请人提供）： 该应用程序旨在确定可诱导的转录因子 激活胎儿血红蛋白（HBF）表达，以发展新颖 镰状细胞疾病和库利贫血的疗法。理由 策略是基于临床，遗传和生化证据来说明的 HBF在两个遗传疾病中的有益作用。组蛋白脱乙酰基酶 抑制剂（HDAI）是验证的HBF诱导剂，但可用的类似物不是 由于抗增殖作用和生物利用度差而广泛使用。 Hdais 改变胎儿球蛋白基因启动子中DNA蛋白相互作用 表明迄今未知的反式激活分子的初始激活， 这反过来激活胎儿球蛋白。基于这种扣除，我们发起了 对两个HDAIS丁酸钠和trichosatin A进行的研究，并发现这两者都 激活p38有丝分裂激活蛋白激酶（p38 MAPK）并激活 转录因子2（ATF-2）。我们已经确认了G Globin的这种机制 使用特定激活剂（砷霉素）和 P38 MAPK的抑制剂（SB203580）。从这些发现，我们开发了 要在四个主要特定目标中进行检验的假设，1）确定 组蛋白脱乙酰基酶抑制剂p38 MAPK激活的机制。我们将 确定p38 MAPK的HDAI激活是直接的还是通过反应性的 线粒体中的氧气和测试新的HDAI当前处于I期 G球蛋白诱导性的临床试验。 2）定义顺式调节 p38 MAPK依赖性G基因诱导所需的元素。这些研究 将使用记者对G启动子进行功能分析，并 表达向量。 3）确定p38 MAPK所需的转录因子 依赖G基因激活。 同源结合转录因子 AIM 2中确定的要素将以凝胶转移分析和 免疫沉淀研究。此目标的实验将包括验证 确定的反式激活或共激活蛋白激活 内源G球蛋白。 4）确定新型HDAI诱导G基因的能力 转基因线中的体内活性。 新颖的Hdais发现有效 AIM 1将在镰状转基因小鼠中进行测试。成功完成 特定目的中描述的实验将为 开发有效的药物或基因治疗方案 和库利的贫血。