Skeletal muscle wasting is a consequence of numerous physiological conditions, including denervation, corticosteroid treatment, immobilization, and aging. The E3 ubiquitin ligases, MuRF1 and MAFbx, are induced under nearly all atrophy conditions and are believed to play a key role in protein degradation in atrophying muscle. However, the preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of atrophy-induced gene activity, including the regulation of MAFbx and MuRF1 expression. To characterize the transcriptional regulation of MuRF1 and MAFbx, reporter gene constructs containing fragments of the proximal promoter regions of these genes were developed, transfected into C2C12 cells with or without a MuRF1 expression plasmid and monitored for differences in reporter gene activity. The MuRF1 and MAFbx reporters each showed repressed activity in cells ectopically expressing MuRF1 compared to cells that did not overexpress MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRFs), MyoD1 and myogenin, caused significant activation of the MuRF1 and MAFbx reporter constructs. However, co-overexpression of MuRF1 with MyoD1 or myogenin resulted in reversal of MRF induction of reporter gene activity, and synergistic repression of a constructed E-box reporter system. To further characterize the role of the MuRF1 gene product in repression of MuRF1 expression, a MuRF1 RING domain mutant and a MuRF1 c-terminal mutant were created. The mutant constructs were then co-transfected along with MRF expression plasmids and the MuRF1 reporter construct into C2C12 cells and reporter gene activity was assessed. The MuRF1 RING mutant failed to reverse MRF activation of the reporter gene, while the c-terminal mutant successfully reversed activation of the reporter gene. These findings suggest that ubiquitin ligase activity is required for MuRF1 transcriptional regulatory effects. These data offer exciting evidence of a potential new function for MuRF1 as a transcriptional modulator of atrophy-induced changes in gene expression.
| Identifer | oai:union.ndltd.org:unf.edu/oai:digitalcommons.unf.edu:etd-1505 |
| Date | 01 January 2014 |
| Creators | Olson, Theodore |
| Publisher | UNF Digital Commons |
| Source Sets | University of North Florida |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | UNF Theses and Dissertations |
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