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

描述(由申请人提供)：我是匹兹堡大学重症监护医学系的初级教员。我接受过儿科重症监护医学的培训，最近在辛辛那提儿童医院完成了T-32培训助学金。我的短期目标是获得更多的分子生物学培训，并学习与我的长期目标相关的更多技术：进行与脓毒症生物学相关的翻译研究，以改善危重儿童的预后。在我的导师Mitchell P.Fink博士(匹兹堡大学我的系主任)和主要合作导师的指导下，我制定了一个正式课程和教程计划，这将成为实现我的目标所必需的教育和培训核心。我的研究项目“多聚(ADP-核糖)聚合酶(PARP)-1是热休克反应的负向调节器”，旨在提供重要的新信息，并作为一个教育机会，补充我职业发展计划的其他部分。PARP-1是一种核酶，激活后可介导感染性休克的进展。在脓毒症动物模型中，PARP的基因缺失或药物抑制具有保护作用。另一个在危重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的抑制或遗传缺失与HSR的上调有关。当热休克细胞用PARP-1抑制剂(DIQ)处理时，野生型(Wt)细胞中负责HSR的主要转录因子热休克因子(HSF)-1与DNA的结合增加。热休克PARP-/-细胞表现出丰富的HSP-70表达和HSP-70启动子活性，这是HSR的特征。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改变分析(EMSA)检测到。本提案中概述的一些实验应该能解释这一悖论(即在热休克的PARP-1-/-细胞中，依赖于HSF-1的基因转录增加，但没有HSF-1 DNA结合)。因此，在目标1中，1将通过研究导入针对PARP-1的小干扰RNA(SiRNA)的wt细胞来证实在PARP-/-细胞中获得的数据。目的2研究抑制PARP-1对HSF-1介导的反式激活的影响。目的3试图在活体环境中研究这些现象，特别是检测热休克对wt和PARP-/-动物的影响。

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.