Deptor Knockdown Enhances mTOR Activity and Protein Synthesis in Myocytes and Ameliorates Disuse Muscle Atrophy
Deptor is an mTOR binding protein that affects cell metabolism. We hypothesized that knockdown (KD) of Deptor in C2C12 myocytes will increase protein synthesis via stimulating mTOR-S6K1 signaling. Deptor KD was achieved using lentiviral particles containing short hairpin (sh)RNA targeting the mouse Deptor mRNA sequence, and control cells were transfected with a scrambled control shRNA. KD reduced Deptor mRNA and protein content by 90%, which increased phosphorylation of mTOR kinase substrates, 4E-BP1 and S6K1, and concomitantly increased protein synthesis. Deptor KD myoblasts were both larger in diameter and exhibited an increased mean cell volume. Deptor KD increased the percentage of cells in the S phase, coincident with an increased phosphorylation (S807/S811) of retinoblastoma protein (pRb) that is critical for the G1 to S phase transition. Deptor KD did not appear to alter basal apoptosis or autophagy, as evidenced by the lack of change for cleaved caspase-3 and light chain (LC)3B, respectively. Deptor KD increased proliferation rate and enhanced myotube formation. Finally, in vivo Deptor KD (∼50% reduction) by electroporation into gastrocnemius of C57/BL6 mice did not alter weight or protein synthesis in control muscle. However, Deptor KD prevented atrophy produced by 3 d of hindlimb immobilization, at least in part by increasing protein synthesis. Thus, our data support the hypothesis that Deptor is an important regulator of protein metabolism in myocytes and demonstrate that decreasing Deptor expression in vivo is sufficient to ameliorate muscle atrophy.
We thank Margaret Shumate and Robert Frost for discussions and critical readings of the manuscript. We thank Danuta Huber, Anne Pruznak and Maithili Navaratnarajah for technical support. We also thank David Stanford of the Penn State Flow Cytometry Core Facility for help with cell cycle analysis imaging. This work was supported in part by grants from the National Institutes of Health (GM38032 and AA11290) (to CH Lang) and Pennsylvania Department of Health using Tobacco Settlement Funds (to AA Kazi). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.
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