C/EBP, atrogin-1, and muscle wasting

C/EBP、atrogin-1 和肌肉萎缩

• 批准号: 7811815
• 负责人: PER-OLOF J HASSELGREN
• 金额: $ 42.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811815
• 关键词: Accounting; Acetylation; Animals; Atrophic; CCAAT-Enhancer-Binding Proteins; Co-Immunoprecipitations; Contractile Proteins; Dexamethasone; Disease; Down-Regulation; EP300 gene; Figs - dietary; Funding; Gene Proteins; Genes; Glucocorticoids; Grant; HDAC3 gene; Histone Deacetylase; Histones; Injury; Ligation; Malignant Neoplasms; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Molecular; Muscle; Muscle Cells; Muscle Fibers; Mutation; Nuclear; Pathway interactions; Phosphorylation; Phosphorylation Site; Plasmids; Proteolysis; Puncture procedure; Rattus; Regulation; Role; Sepsis; Skeletal Muscle; Small Interfering RNA; Testing; Transferase; Ubiquitin; Up-Regulation; Western Blotting; abstracting; base; cofactor; factor C; histone deacetylase 3; improved; in vitro Model; in vivo; insight; multicatalytic endopeptidase complex; muscle form; protein degradation; protein expression; research study; septic; transcription factor; ubiquitin ligase; wasting

ABSTRACT A number of catabolic disease states, including sepsis, severe injury, and cancer, are characterized by muscle wasting, mainly reflecting increased breakdown of myofibrillar (contractile) proteins. Myofibrillar proteolysis in muscle wasting is to a great extent regulated by the ubiquitin-proteasome pathway, in particular the expression and activity of the ubiquitin ligases atrogin-1 and MuRF1. Because muscle wasting is characterized by the upregulation of multiple genes in the ubiquitin-proteasome as well as other proteolytic pathways, it is likely that the expression and activity of transcription factors and nuclear cofactors, such as the histone acetyl transferase p300, are involved in the loss of muscle mass in catabolic conditions. The current project will test the hypothesis that muscle wasting during sepsis is at least in part regulated by the transcription factor C/EBP¿ and that p300-dependent acetylation and MAPK-dependent phosphorylation of the transcription factor are involved in C/EBP¿-regulated muscle wasting. Experiments are performed in rats made septic by cecal ligation and puncture (CLP) and in sham-operated control rats. Because glucocorticoids are important mediators of sepsis-induced muscle wasting, dexametasone-treated cultured myotubes are used as an in vitro model of muscle wasting, allowing for detailed mechanistic studies. Specifically, the following hypotheses are tested: 1) sepsis- and glucocorticoid-induced muscle wasting is, at least in part, regulated by C/EBP¿; 2) sepsis in rats results in glucocorticoid-mediated upregulation of p300/histone acetyl transferase (HAT) and downregulation of histone deacetylase (HDAC) 3 and 6 expression and activity in skeletal muscle; 3) sepsis in rats and dexamethasone treatment of cultured muscle cells result in p300-dependent acetylation of C/EBP¿; and 4) sepsis in rats and dexamethasone treatment of cultured muscle cells result in mitogen-activated protein kinase (MAPK)-dependent phosphorylation of C/EBP¿. The gene and protein expression of p300, HDAC3 and 6, total, acetylated, and phosphorylated C/EBP¿ is determined by real-timePCR, Western blotting, and co- immunoprecipitation. The role of p300 and C/EBP¿ in sepsis- and dexamethasone-induced muscle proteolysis, atrophy, and atrogin1 and MuRF1 expression is tested by transfecting rat extensor digitorum longus muscle or cultured muscle cells with p300 or C/EBP¿ siRNA constructs. The role of C/EBP¿ acetylation and phosphorylation in dexamethasone-induced protein degradation and expression of atrogin-1 and MuRF1 is tested by transfecting cultured muscle cells with plasmids expressing wild-type C/EBP¿ or C/EBP¿ with mutations of specific acetylation or phosphorylation sites (Lys39 and Thr188, respectively). The proposed experiments are important because they will increase the understanding of the molecular regulation of muscle wasting. Improved insight into mechanisms accounting for muscle wasting will help develop better treatments of this debilitating condition.

摘要 许多分解代谢疾病状态，包括败血症，严重损伤和癌症，其特征在于肌肉 消瘦，主要反映肌原纤维（收缩）蛋白的分解增加。肌原纤维蛋白水解 肌肉萎缩在很大程度上是由泛素-蛋白酶体途径调节的，特别是 以及泛素连接酶atrogin-1和MuRF 1的活性。因为肌肉萎缩的特点是 泛素-蛋白酶体以及其他蛋白水解途径中多个基因的上调，很可能 转录因子和核辅因子的表达和活性，如组蛋白乙酰转移酶 p300参与分解代谢条件下肌肉质量的损失。目前的项目将测试 脓毒症期间肌肉萎缩至少部分受转录因子C/EBP调节的假设 并且转录因子的p300依赖性乙酰化和MAPK依赖性磷酸化被 参与C/EBP调节的肌肉萎缩。实验在通过盲肠感染的大鼠中进行。 结扎和穿刺（CLP）和假手术对照大鼠。因为糖皮质激素很重要 介质脓毒症诱导的肌肉萎缩，地塞米松处理的培养肌管被用作体外 肌肉萎缩的模型，允许详细的机制研究。具体而言，以下假设是 测试：1）脓毒症和糖皮质激素诱导的肌肉萎缩至少部分受C/EBP调节; 2） 大鼠脓毒症导致糖皮质激素介导的p300/组蛋白乙酰转移酶（HAT）上调， 组蛋白去乙酰化酶（HDAC）3和6在骨骼肌中的表达和活性下调; 大鼠和地塞米松处理培养的肌细胞导致p300依赖的C/EBP乙酰化; 大鼠败血症和地塞米松处理培养的肌细胞导致丝裂原活化蛋白 C/EBP的MAPK依赖性磷酸化。p300、HDAC 3和p300蛋白表达 6，总的，乙酰化的，和磷酸化的C/EBP通过实时PCR，Western印迹和共沉淀法测定。 免疫沉淀。p300和C/EBP在脓毒症和地塞米松诱导的肌肉蛋白水解中的作用， 萎缩，通过分离大鼠趾长伸肌或 用p300或C/EBP？siRNA构建体培养肌细胞。C/EBP的乙酰化作用和 地塞米松诱导的蛋白质降解和atrogin-1和MuRF 1表达中的磷酸化是 通过用表达野生型C/EBP <$或C/EBP <$的质粒转染培养的肌肉细胞进行测试， 特定乙酰化或磷酸化位点（分别为Lys 39和Thr 188）的突变。拟议 实验是重要的，因为它们将增加对肌肉分子调节的理解。 浪费对肌肉萎缩机制的深入了解将有助于开发更好的治疗方法 这种衰弱的状况。