C/EBP, atrogin-1, and muscle wasting

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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.