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Response of Skeletal Muscle to Steroids: A Systems Biology Approach Open Access

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Cortisol (adrenal glucocorticoid) and testosterone (gonadal sex hormone) both have significant, yet opposite, effects on skeletal muscle. Cortisol, and the highly prescribed synthetic derivatives (prednisone, dexamethasone) are considered catabolic (muscle wasting), yet have an enigmatic muscle anabolic effect in some neuromuscular disorders. Testosterone is anabolic to muscle, and is commonly prescribe specifically for this beneficial effect. The best studied molecular pharmacology of both prednisone and testosterone involve their `hormonal' transcriptional effects, mediated by binding to soluble receptors (glucocorticoid receptor and androgen receptor, respectively). There is a less well-characterized non-transcriptional response involving extensive protein signaling within minutes of steroid exposure. The goal of this dissertation was to develop methods that were able to survey the signaling downstream of drug exposure, developing time series data on the `minutes' time scale, and building integrative models for the effects of steroids on muscle (systems biology). We developed a novel application for the proteomic quantitative technique stable isotope labeling of amino acids in cell culture (SILAC) to study the acute temporal subcellular protein translocation in response to prednisone and testosterone in myogenic cells (C2C12 myotubes). For prednisone, significant increases in ribosomal proteins in the cytosol and translocation changes in additional proteins involved in translational machinery suggested acute changes to protein translation; possibly involving quiescent pools of mRNA. This hypothesis was confirmed by identification of altered de novo protein translation rates in 5 proteins all containing 5'TOP motifs characteristic of inactive mRNAs. In addition to alteration of translational machinery, proteomic profiling following prednisone exposure showed perturbations to the glycolytic pathway occurring within minutes. The acute effects on glycolysis were validated by a reduction of intracellular ATP and decreased phosphorylation of the energy sensor heat shock protein 90. The integration of the data with temporal gene expression studies and literature allowed us to generate a multi-scale model describing the effect of glucocorticoids on muscle metabolism and its role in muscle atrophy. We used a similar approach to investigate the anabolic response of testosterone on muscle. There was a significant movement of actin and actin binding proteins into the soluble fractions of the myotubes in response to testosterone, suggestive of actin cytoskeleton remodeling. The effect on actin cytoskeleton was validated by changes in de novo protein translation of actin binding proteins MSN and CAPG. Taken together, the studies described here provide novel insight into the affects of steroids on skeletal muscle and allows us to model the mechanisms facilitating muscle atrophy or hypertrophy.

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