C/EBP, atrogin-1, and muscle wasting

C/EBP、atrogin-1 和肌肉萎缩

基本信息

批准号：
7112427
负责人：
PER-OLOF J HASSELGREN
金额：
$ 36.52万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7112427
关键词：
cytokine enhancer binding protein gel mobility shift assay genetic regulation glucocorticoids laboratory mouse ligase muscle disorders muscle necrosis northern blottings protein structure function proteolysis septicemia transcription factor western blottings

项目摘要

DESCRIPTION (provided by applicant): Sepsis and other catabolic conditions are characterized by muscle wasting, mainly reflecting increased degradation of myofibrillar proteins. The role of muscle wasting for morbidity and mortality in catabolic conditions is not fully understood. Glucocorticoids, together with proinflammatory cytokines, are major mediators of muscle proteolysis. Muscle wasting is at least in part caused by ubiquitin-proteasome-dependent proteolysis. The gene expression of several components of the ubiquitin-proteasome pathway, including ubiquitin ligases, is increased in catabolic muscle but mechanisms responsible for gene activation in muscle wasting is poorly understood. In particular, the role of the transcription factor C/EBP and nuclear co-factors p300/CBP for regulation of genes in the ubiquitin-proteasome pathway has not been defined. The specific aims are to test the hypotheses that 1) increased muscle proteolysis contributes to mortality during sepsis; 2) glucocorticoids and cytokines upregulate the expression and activity of C/EBP in skeletal muscle; 3) glucocorticoids and cytokines upregulate the expression and activity of p300/CBP in skeletal muscle; 4) glucocorticoid- and cytokine-dependent C/EBP activation and C/EBP-p300/CBP interaction activate the ubiquitin ligase atrogin-1; 5) the glucocorticoid- and cytokine-induced C/EBP-atrogin-1 gene activation cascade is at least in part responsible for muscle wasting. The role of increased muscle proteolysis for mortality in sepsis will be tested by creating transgenic mice overexpressing 11lbeta-HSDt or 11beta-HSD2 selectively in skeletal muscle, thereby creating conditions with high and low muscle corticosterone levels, respectively. In other experiments, rats are treated with dexamethasone and/or the glucocorticoid receptor antagonist RU38486 followed by determination of expression and activity of C/EBP, p300/CBP and atrogin-1. In other experiments, cultured L6 myotubes are treated with dexamethasone and proinflammatory cytokines. DNA binding activity of C/EBP beta and delta is determined by EMSA and supershift analysis. Protein and gene expression of C/EBP beta and delta, p300/CBP, and atrogin-1, is determined by Western blotting and real-time PCR, respectively. Coimmunoprecipitation is used to examine protein-protein interaction between p300/CBP and C/EBP transcription factors. The role of C/EBP and p300/CBP in glucocorticoid- and cytokine-induced activation of atrogin-1 and protein degradation is determined by transfecting myocytes with expression plasmids for C/EBPbeta or delta and p300/CBP or antisense oligonucleotides. The project is important because it will test the role of muscle wasting for mortality in sepsis and will link the activation of a transcription factor and nuclear co-factors to the activation of a gene in the ubiquitin-proteasome pathway and muscle proteolysis.

描述（由申请人提供）：败血症和其他分解代谢条件的特征是肌肉浪费，主要反映了肌原纤维蛋白降解的增加。肌肉浪费在发病率和死亡率中在分解代谢条件下的作用尚不完全了解。糖皮质激素以及促炎细胞因子是肌肉蛋白水解的主要介质。肌肉浪费至少部分是由泛素 - 蛋白酶体依赖性蛋白水解引起的。在分解代谢肌肉中增加了泛素 - 蛋白酶体途径的几个成分的基因表达，包括泛素连接酶，但对肌肉浪费的基因激活的机制却鲜为人知。特别是，尚未定义转录因子C/EBP和核副因素P300/CBP在泛素 - 蛋白酶体途径中调节基因的作用。具体目的是检验假设1）增加肌肉蛋白水解会导致败血症死亡率； 2）糖皮质激素和细胞因子上调骨骼肌中C/EBP的表达和活性； 3）糖皮质激素和细胞因子上调骨骼肌中P300/CBP的表达和活性； 4）糖皮质激素和细胞因子依赖性C/EBP激活以及C/EBP-P300/CBP相互作用激活泛素连接酶Atrogin-1； 5）糖皮质激素和细胞因子诱导的C/EBP-ATROGIN-1基因激活级联反应至少部分负责肌肉浪费。肌肉蛋白水解在败血症中增加死亡率的作用将通过创建过表达11LBETA-HSDT或11BETA-HSD2的转基因小鼠在骨骼肌中有选择性，从而分别创造出具有高和低肌肉皮质酮水平的疾病。在其他实验中，大鼠用地塞米松和/或糖皮质激素受体拮抗剂RU38486处理，然后确定C/EBP，p300/cbp和Atrogin-1的表达和活性。在其他实验中，培养的L6肌管用地塞米松和促炎细胞因子进行治疗。 C/EBP Beta和Delta的DNA结合活性由EMSA和SuperShift分析确定。 C/EBP Beta和Delta，P300/CBP和Atrogin-1的蛋白质和基因表达分别通过蛋白质印迹和实时PCR确定。共免疫沉淀用于检查p300/cbp和C/EBP转录因子之间的蛋白质 - 蛋白质相互作用。 C/EBP和p300/CBP在糖皮质激素和细胞因子诱导的Atrogin-1和蛋白质降解的激活中的作用是通过用C/EBPBETA或DELTA和P300/CBP或Antensensensensensensensensensensemensens质粒转染的肌细胞来确定的。该项目很重要，因为它将测试肌肉浪费在败血症中死亡率的作用，并将转录因子和核辅助因子的激活与泛素蛋白 - 蛋白酶体途径和肌肉蛋白水解中基因的激活联系起来。