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病症的特征在于肌肉萎缩，主要反映肌原纤维蛋白的降解增加。肌肉萎缩在分解代谢条件下的发病率和死亡率中的作用尚未完全了解。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-蛋白酶体途径的几种组分的基因表达，包括泛素连接酶，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-蛋白酶体途径中调控基因的作用尚未确定。本研究旨在验证以下假设：1）脓毒症时肌肉蛋白水解增加导致死亡; 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）糖皮质激素依赖的C/EBP激活和C/EBP-p300/CBP相互作用激活泛素连接酶atrogin-1; 5）糖皮质激素和精氨酸诱导的C/EBP-atrogin-1基因激活级联反应至少部分导致肌肉萎缩。将通过建立在骨骼肌中选择性过表达11 β-HSDt或11 β-HSD2的转基因小鼠，从而分别产生高和低肌肉皮质酮水平的条件，来测试增加的肌肉蛋白水解对脓毒症死亡率的作用。在其它实验中，用地塞米松和/或糖皮质激素受体拮抗剂RU 38486处理大鼠，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。通过EMSA和超位移分析测定C/EBP β和δ的DNA结合活性。C/EBP β和δ、p300/CBP和atrogin-1的蛋白质和基因表达分别通过Western印迹和实时PCR测定。免疫共沉淀用于检测p300/CBP和C/EBP转录因子之间的蛋白质-蛋白质相互作用。通过用C/EBP β或δ和p300/CBP或反义寡核苷酸的表达质粒转染肌细胞，确定C/EBP和p300/CBP在糖皮质激素和阿托品诱导的atrogin-1活化和蛋白降解中的作用。该项目很重要，因为它将测试肌肉萎缩对脓毒症死亡率的作用，并将转录因子和核辅因子的激活与泛素-蛋白酶体途径和肌肉蛋白水解中基因的激活联系起来。

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.