PARP-1 is a Negative Modulator of the Heart Shock Response

PARP-1 是心脏休克反应的负调节剂

• 批准号: 7020897
• 负责人: RAJESH K. ANEJA
• 金额: $ 12.29万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-03 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020897
• 关键词: DNA; cell line; cytogenetics; enzyme activity; fibroblasts; gene induction /repression; genetically modified animals; heat shock proteins; immunoprecipitation; laboratory mouse; mutant; pathologic process; pentosyltransferase; protein binding; septicemia; small interfering RNA; transcription factor; transfection

DESCRIPTION (provided by applicant): I am a junior faculty member in the Department of Critical Care Medicine at the University of Pittsburgh. I am trained in Pediatric Critical Care Medicine and I recently completed a T-32 training grant at Cincinnati Children's Hospital. My short-term goal is to acquire additional training in molecular biology and learn additional techniques relevant to my long term goal: conduct translational research related to the biology of sepsis to improve outcomes for critically ill children. With the guidance of my mentor Dr. Mitchell P. Fink, (Chairman of my department at University of Pittsburgh) and key co-mentors, I have developed a program of formal coursework and tutorials that will form the educational and training core necessary to achieve my goals. My research project "Poly (ADP-ribose) polymerase (PARP) -1 is a negative modulator of the heat shock response" is designed to both provide vital new information and serve as an educational opportunity that compliments the other components of my career development plan. PARP-1 is a nuclear enzyme that upon activation mediates progression of septic shock. Genetic deletion or pharmacological inhibition of PARP is protective in animal models of sepsis. Another major cytoprotective stress response that is upregulated in critical care illness is the heat shock response (HSR). My preliminary data suggests that PARP-1 inhibition or genetic absence of PARP-1 is associated with upregulation of the HSR. DNA binding by the main transcription factor responsible for the HSR, heat shock factor (HSF)-1 in wild type (wt) cells is increased when heat-shocked cells are pretreated with a PARP-1 inhibitor (DIQ). Heat shocked PARP-/- cells demonstrate abundant HSP-70 expression and HSP-70 promoter activity, hallmarks of the HSR. However, DNA binding of HSF-1 in heat-shocked PARP-1-/- cells is not detectable by electrophoretic mobility shift assay (EMSA). Some of the experiments outlined in the present proposal should shed some light on this paradox (i.e. increased transcription of a HSF-1-dependent gene but absent HSF-1 DNA binding in heat shocked PARP-1-/- cells). Thus, in Aim 1,1 will confirm the data obtained in PARP-/- cells by studying wt cells transfected with small interfering RNA (siRNA) directed against PARP-1. Aim 2 examines the effect of PARP-1 inhibition on HSF-1-mediated transactivation. Aim 3 seeks to study these phenomena in an in vivo setting, specifically examining the effects of heat shock in wt and PARP-/- animals.

描述（由申请人提供）：我是匹兹堡大学重症监护医学系的一名初级教员。我接受过儿科重症监护医学培训，最近在辛辛那提儿童医院完成了T-32培训补助金。我的短期目标是获得分子生物学方面的额外培训，并学习与我的长期目标相关的其他技术：进行与脓毒症生物学相关的转化研究，以改善危重患儿的预后。在我的导师Mitchell P. Fink博士（我在匹兹堡大学的系主任）和主要合作导师的指导下，我开发了一个正式的课程和教程计划，这将形成实现我的目标所需的教育和培训核心。我的研究项目“聚（ADP-核糖）聚合酶（PARP）-1是热休克反应的负调节剂”旨在提供重要的新信息，并作为一个教育机会，补充我的职业发展计划的其他组成部分。PARP-1是一种核酶，激活后介导败血性休克的进展。PARP的遗传缺失或药理学抑制在脓毒症动物模型中具有保护作用。另一个在重症监护疾病中上调的主要细胞保护性应激反应是热休克反应（HSR）。我的初步数据表明，PARP-1抑制或PARP-1的遗传缺失与HSR的上调有关。当用PARP-1抑制剂（DIQ）预处理热休克细胞时，野生型（wt）细胞中负责HSR的主要转录因子热休克因子（HSF）-1的DNA结合增加。热休克的PARP-/-细胞表现出丰富的HSP-70表达和HSP-70启动子活性，这是HSR的标志。然而，热休克PARP-1-/-细胞中HSF-1的DNA结合不能通过电泳迁移率变动分析（EMSA）检测到。本提案中概述的一些实验应该对这种悖论有所启发（即，在热休克的PARP-1-/-细胞中，HSF-1依赖性基因的转录增加，但HSF-1 DNA结合缺失）。因此，在目的1，1中，将通过研究用针对PARP-1的小干扰RNA（siRNA）转染的wt细胞来证实在PARP-/-细胞中获得的数据。目的2检测PARP-1抑制对HSF-1介导的反式激活的影响。目的3旨在研究体内环境中的这些现象，特别是检查热休克对WT和PARP-/-动物的影响。