C/EBP, atrogin-1, and muscle wasting

C/EBP、atrogin-1 和肌肉萎缩

基本信息

批准号：
6769694
负责人：
PER-OLOF J HASSELGREN
金额：
$ 37.4万
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/6769694
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）：脓毒症和其他分解代谢疾病的特点是肌肉萎缩，主要反映了肌原纤维蛋白降解的增加。分解代谢条件下肌肉萎缩对发病率和死亡率的影响尚不完全清楚。糖皮质激素与促炎细胞因子一起是肌肉蛋白水解的主要介质。肌肉萎缩至少部分是由泛素蛋白酶体依赖性蛋白水解引起的。泛素-蛋白酶体途径的几个组成部分（包括泛素连接酶）的基因表达在分解代谢肌肉中增加，但对肌肉萎缩中基因激活的机制知之甚少。特别是，转录因子 C/EBP 和核辅因子 p300/CBP 在泛素-蛋白酶体途径中基因调节中的作用尚未明确。具体目的是检验以下假设：1）肌肉蛋白水解作用增加导致败血症期间的死亡率； 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 激活和 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 基因激活级联至少部分地导致肌肉萎缩。将通过创建在骨骼肌中选择性过度表达 11lbeta-HSDt 或 11beta-HSD2 的转基因小鼠来测试肌肉蛋白水解增加对脓毒症死亡率的作用，从而分别创造高和低肌肉皮质酮水平的条件。在其他实验中，用地塞米松和/或糖皮质激素受体拮抗剂RU38486治疗大鼠，然后测定C/EBP、p300/CBP和atrogin-1的表达和活性。在其他实验中，用地塞米松和促炎细胞因子处理培养的 L6 肌管。 C/EBP beta 和 delta 的 DNA 结合活性通过 EMSA 和 supershift 分析来确定。 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 测定。免疫共沉淀用于检查 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 激活和蛋白质降解中的作用通过用 C/EBPbeta 或 delta 和 p300/CBP 或反义寡核苷酸的表达质粒转染肌细胞来确定。该项目很重要，因为它将测试肌肉消耗对脓毒症死亡率的影响，并将转录因子和核辅助因子的激活与泛素蛋白酶体途径和肌肉蛋白水解中基因的激活联系起来。