C/EBP, atrogin-1, and muscle wasting

描述(由申请人提供)： 脓毒症和其他分解代谢状态的特点是肌肉萎缩，主要反映肌原纤维蛋白降解增加。在分解代谢条件下，肌肉萎缩对发病率和死亡率的作用尚不完全清楚。糖皮质激素和促炎细胞因子是肌肉蛋白分解的主要介质。肌肉萎缩至少在一定程度上是由泛素-蛋白酶体依赖的蛋白分解引起的。在分解代谢的肌肉中，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增加，但在肌肉萎缩中导致基因激活的机制尚不清楚。特别是，转录因子C/EBP和核辅助因子p300/CBP在泛素-蛋白酶体途径基因调控中的作用尚未确定。其具体目的是验证以下假设：1)肌肉蛋白分解增加导致脓毒症死亡；2)糖皮质激素和细胞因子上调骨骼肌C/EBP的表达和活性；3)糖皮质激素和细胞因子上调骨骼肌p300/CBP的表达和活性；4)糖皮质激素和细胞因子依赖的C/EBP激活和C/EBP-p300/CBP相互作用激活泛素连接酶阿托品-1；5)糖皮质激素和细胞因子诱导的C/EBP-阿托品-1基因激活级联反应至少部分与肌肉萎缩有关。肌肉蛋白分解增加对脓毒症死亡率的作用将通过在骨骼肌中选择性地过度表达111β-HSDt或11β-HSD2的转基因小鼠来测试，从而分别创造肌肉皮质酮水平高和低的条件。在其他实验中，用地塞米松和/或糖皮质激素受体拮抗剂RU38486处理大鼠，然后测定C/EBP、p300/CBP和阿托金-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处理。用EMSA和超位移分析测定了C/EBPβ和Delta的DNA结合活性。Western blotting和Real-time PCR分别检测C/EBPβ和Delta、p300/CBP和阿托金-1的蛋白和基因表达。免疫共沉淀法检测p300/CBP和C/EBP转录因子之间的蛋白质相互作用。C/EBP和p300/CBP在糖皮质激素和细胞因子诱导的阿托金-1激活和蛋白质降解中的作用是通过将C/EBPbeta或Delta和p300/CBP或反义寡核苷酸的表达载体导入心肌细胞来确定的。该项目之所以重要，是因为它将测试肌肉萎缩在脓毒症死亡率中的作用，并将转录因子和核辅助因子的激活与泛素-蛋白酶体途径中的基因激活和肌肉蛋白分解联系起来。

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.