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

简介（由申请人提供）：我是匹兹堡大学重症医学系的一名初级教员。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。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额外技术：进行脓毒症生物学相关的转化研究，以改善危重儿童的预后。在我的导师Mitchell P. Fink博士（我在匹兹堡大学的系主任）和主要的共同导师的指导下，我制定了一个正式的课程和教程计划，这些课程和教程将构成实现我的目标所必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因子”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组成部分。PARP-1是一种核酶，激活后介导感染性休克的进展。基因缺失或药物抑制PARP对脓毒症动物模型具有保护作用。在重症监护疾病中上调的另一个主要的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）细胞中负责热休克的主要转录因子热休克因子(HSF)-1的DNA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，这是高热休克的标志。然而，在热休克的PARP-1-/-细胞中，HSF-1的DNA结合无法通过电泳迁移转移试验（EMSA）检测到。本提案中概述的一些实验应该能够揭示这一悖论（即，在热休克PARP-1-/-细胞中，HSF-1依赖性基因的转录增加，但缺乏HSF-1 DNA结合）。因此，在Aim 1中，我将通过研究转染了针对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检验PARP-1抑制对hsf -1介导的转激活的影响。目的3试图在体内环境中研究这些现象，特别是研究热休克对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.