MAP Kinase Regualtion of Cardiac Nuclear Receptors

描述（申请人提供）：心脏肥大和心力衰竭是全球发病和死亡的主要原因。这些综合征是由多种血液动力学和体液病理生理刺激引起的，包括高血压、缺血和心肌梗死。在细胞水平上，蛋白激酶信号传导将细胞外信号级联为心肌细胞中的特征性分子和结构变化，其通常包括通过线粒体脂肪酸氧化（FAO）失调而改变正常能量产生。心脏中FAO酶基因的主要转录调节因子，过氧化物酶体增殖物激活受体a（PPARa），已被证明在心肌肥大期间被细胞外信号调节激酶（ERK）依赖性过程失活，导致FAO酶表达降低和脂肪酸氧化能力丧失。相反，p38丝裂原活化蛋白激酶（MAPK）活化通过直接磷酸化事件增加PPARa转录活性并增强心肌细胞中的FAO。该提议旨在了解MAPK途径差异调节PPARa活性以响应心脏中细胞应激的机制。i）利用EMSA、配体结合测定和蛋白质-蛋白质相互作用测定鉴定PPARa上的特异性p38 MAPK磷酸化位点; ii）通过体内磷酸化研究、EMSA、配体结合测定、蛋白质-蛋白质相互作用测定、肌细胞中的免疫共沉淀测定来确定ERK MAPK使PPARa失活的机制，iii）定义体外和体内PPARa靶基因表达的途径特异性调节，并阐明非选择性MAPK活化对PPARa活性的影响; iv）通过酵母双杂交和蛋白质组学方法鉴定新的心脏表达的PPARa相互作用蛋白。MAPK家族成员对PPARa基因调控通路的调节提供了理解多个信号通路整合在单个转录因子上的基本机制的可能性。我们推测，PPARa信号的调节是细胞应激反应的一个组成部分，并对细胞能量代谢的下游调节产生影响。

DESCRIPTION (provided by applicant): Cardiac hypertrophy and heart failure are major causes of morbidity and mortality worldwide. These syndromes result from a variety of hemodynamic and humoral pathophysiologic stimuli, including hypertension, ischemia, and myocardial infarction. At the cellular level, protein kinase signaling cascades transduce extracellular signals into characteristic molecular and structural changes in the cardiac myocyte, which frequently includes alterations in normal energy production via dysregulation of mitochondrial fatty acid ¿-oxidation (FAO). The principal transcriptional regulator of FAO enzyme genes in heart, the peroxisome proliferator-activated receptor a (PPARa), has been shown to be deactivated by an extracellular signal-regulated kinase (ERK)-dependent process during cardiac hypertrophy, contributing to decreased FAO enzyme expression and loss of fatty acid oxidative capacity. Conversely, p38 mitogen activated protein kinase (MAPK) activation increases PPARa transcriptional activity via a direct phosphorylation event and enhances FAO in cardiac myocytes. This proposal is designed to understand the mechanisms whereby MAPK pathways differentially regulate the activity of PPARa in response to cellular stressors in the heart. This will be accomplished by i) identification of specific p38 MAPK phosphorylation sites on PPARa utilizing EMSA, ligand-binding assays, and protein-protein interaction assays; ii) determination of the mechanism by which ERK MAPK deactivates PPARa via in vivo phosphorylation studies, EMSA, ligand-binding assays, protein-protein interaction assays, co-immunoprecipitation assays in myocytes, and cotransfection assays with candidate repressors of PPARa activity; iii) definition of pathway specific regulation of PPARa target gene expression in vitro and in vivo and elucidation of the consequences of non-selective MAPK activation on PPARa activity; iv) identification of novel cardiac-expressed PPARa-interacting proteins via yeast two-hybrid and proteomics approaches. Modulation of the PPARa gene regulatory pathway by MAPK family members offers the potential to understand fundamental mechanisms whereby multiple signaling pathways integrate on a single transcription factor. We speculate that regulation of PPARa signaling is an integral component of the cellular stress response with implications for downstream modulation of cellular energy metabolism.