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Transcriptional Regulation of Skeletal Muscle Atrophy‐induced Gene Expression by Muscle‐Specific RING Finger 1 (MuRF1)
Author(s) -
Kakareka Karina,
Waddell David
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1009.12
Subject(s) - reporter gene , myogenin , transfection , gene expression , mutant , microbiology and biotechnology , chemistry , muscle atrophy , myod , ectopic expression , regulation of gene expression , gene , biology , skeletal muscle , biochemistry , anatomy
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 neurogenic 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 C 2 C 12 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 (MRF), 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. To further characterize the role of the MuRF1 gene product in repression of MuRF1 expression, a MuRF1 RING domain mutant was created. The RING mutant construct was then co‐transfected along with MRF expression plasmids and the MuRF1 or MAFbx reporter gene into C 2 C 12 cells and reporter gene activity was assessed. The MuRF1 RING mutant failed to reverse MRF activation of the reporter gene, suggesting that ubiquitin ligase activity is required for MuRF1 transcriptional regulatory effects. To further investigate the potential regulation of MRFs by MuRF1, Western blot analysis was performed in order to determine the impact on MRF protein levels in C 2 C 12 cells in response to overexpression of MuRF1. Cells with ectopic expression of MuRF1 showed reduced levels of myogenin protein. In addition, cells overexpressing MuRF1 were treated with MG132, a general proteasome inhibitor, followed by Western blot analysis. The results of the Western blot revealed that cells treated with MG132 alone had increased myogenin protein levels, while cells ectopically expressing MuRF1 still exhibited reduced myogenin levels. MuRF1 repression of myogenin expression, with or without 26S proteasome inhibition, suggests that MuRF1 may be acting by a previously uncharacterized mechanism to regulate MRF expression, providing additional support that MuRF1 may act as a transcriptional regulator of atrophy‐induced gene expression. These data offer exciting evidence of a potential new function for MuRF1 as a transcriptional modulator of atrophy‐mediated changes in gene expression. Support or Funding Information University of North Florida Transformational Learning Opportunity Grant