? 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. ? ?
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