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.

抽象的 许多分解代谢疾病状态，包括败血症，严重损伤和癌症，以肌肉为特征 浪费，主要反映出肌原纤维（收缩）蛋白的崩溃增加。肌原纤维蛋白水解 肌肉浪费在很大程度上受泛素 - 蛋白酶体途径调节，尤其是表达 泛素连接酶Atrogin-1和Murf1的活性。因为肌肉浪费的特征是 泛素 - 蛋白酶体和其他蛋白水解途径中多个基因的上调，很可能很可能 转录因子和核辅助因子（例如组蛋白乙酰转移酶）的表达和活性 P300参与分解代谢条件下肌肉质量的损失。当前项目将测试 假设败血症期间的肌肉浪费至少部分受转录因子C/EBP的调节。 p300依赖性的乙酰化和转录因子的MAPK依赖性磷酸化是 涉及C/EBP浪费的肌肉浪费。实验是在盲肠中败血的大鼠中进行的 连接和穿刺（CLP）和假手术对照大鼠。因为糖皮质激素很重要 败血症引起的肌肉浪费的介质，右美索松治疗的培养的肌管被用作体外 肌肉浪费的模型，可以进行详细的机械研究。具体而言，以下假设是 测试：1）至少部分受C/EBP的调节，败血症和糖皮质激素诱导的肌肉浪费至少部分受到调节； 2） 大鼠败血症会导致糖皮质激素介导的p300/组蛋白乙酰转移酶（HAT）和 组蛋白脱乙酰基酶（HDAC）3和6表达和活性的下调； 3）败血症 大鼠和地塞米松治疗培养的肌肉细胞会导致p300依赖性乙酰化c/eBP。 4）大鼠败血症和地塞米松治疗培养的肌肉细胞会导致有丝分裂原激活的蛋白 激酶（MAPK）依赖性磷酸化的磷酸化。 p300，HDAC3和 6，总计，乙酰化和磷酸化的C/EBP取决于实时PPCR，Western blotting和Co- 免疫沉淀。 p300和c/eBp¿在败血症和地塞米松诱导的肌肉蛋白水解中的作用， 通过转染大鼠伸肌长肌或 具有p300或c/ebp¿siRNA构建体的培养肌肉细胞。 c/ebp¿乙酰化和 地塞米松诱导的蛋白质降解和Atrogin-1和Murf1的表达中的磷酸化IS 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测试 特异性乙酰化或磷酸化位点的突变（分别为Lys39和Thr188）。提议 实验很重要，因为它们将增加对肌肉分子调节的理解 浪费。改善了对肌肉浪费的机制的洞察力，将有助于开发更好的治疗方法 这种令人衰弱的状况。