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. 2011 Sep-Oct;17(9-10):925-36.
doi: 10.2119/molmed.2011.00070. Epub 2011 May 19.

Deptor knockdown enhances mTOR Activity and protein synthesis in myocytes and ameliorates disuse muscle atrophy

Abid A Kazi  1 , Ly Hong-Brown, Susan M Lang, Charles H Lang
Affiliations

Deptor knockdown enhances mTOR Activity and protein synthesis in myocytes and ameliorates disuse muscle atrophy

Abid A Kazi et al. Mol Med. 2011 Sep-Oct.

Abstract

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 G(1) 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.

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Figures

Figure 1
Figure 1
Effect of Deptor KD in C2C12 myoblasts. (A) Representative Western blot of Deptor protein in control (scramble) and Deptor KD myoblasts. (B) Quantification of Western blot data. Values are means ± SE; n = 12 per group. P < 0.05 compared with time-matched scramble control values. (C) Real-time PCR showing decreased Deptor mRNA in myoblasts transfected with lentiviruses targeting Deptor. rpL32 served as the internal reference control gene, and its expression did not differ between control and Deptor KD cell (Scramble control = 100 ± 5; Deptor KD = 107 ± 7; P = NS). Values are means ± SE; n = 12 per group. P < 0.05 compared with time-matched scramble control values. (D) Effect of Deptor KD on myocyte protein synthesis. Values are means ± SE for n = 12 for each condition. For bar graphs, *P < 0.05, compared with control values. (E) Effect of Deptor KD on mTORC1 and mTORC2 signaling in C2C12 myoblasts. Representative Western blots for protein substrates involved in the mTORC1 and mTORC2 complex mediated signal transduction. Where absent, standard error bars are too small to be visualized.
Figure 2
Figure 2
Effect of IGF-I and AICAR on scramble (control) and Deptor KD C2C12 myoblasts. Cells were transfected with scramble and Deptor KD–containing lentiviral particles and incubated with vehicle (control), IGF-I (100 ng/mL; 1 h) or AICAR (2 μmol/L; 8 h) and labeled with 35S-methionine. (A) Protein synthesis in myoblasts. Values are mean ± SE for n = 8–10 for each condition. Means not sharing the same superscript (a, b, c and d) are significantly different (P < 0.05). For quantification, data were normalized to scramble control values. (B) Representative Western blots for various total and phosphorylated proteins, where cells were treated as described above, except that the isotope was omitted. The blot is representative of at least three independent experiments with two to four replicates per experiment.
Figure 3
Figure 3
Effect of Deptor KD on cell size in C2C12 myoblasts. (A) Cell size, shown in parentheses, was measured using the Coulter Counter particle size analyzer; n = 8 for each condition. (B) Mean cell volume of myoblasts. Bar graph is mean ± SE; n = 7–9 for each condition, *P < 0.0001. Where absent, standard error bars are too small to be visualized.
Figure 4
Figure 4
Effect of Deptor KD on C2C12 myoblast proliferation. Proliferation rate was determined in stably transfected myoblasts with scramble and Deptor KD. Myoblasts were seeded at the same density and counted using the Coulter Counter, as described in Materials and Methods. Time intervals are indicated; n = 6 for each treatment time point; experiments were repeated at least three times. *P < 0.05, compared with the time-matched control value. (B) Proliferation rate was measured using an independent MTT assay. Cells were treated with MTT for 4 h, and formazan produced was measured colorimetrically. Bar graph is mean ± SE; n = 16–18 for each condition, *P < 0.0001; experiments were repeated at least three times.
Figure 5
Figure 5
Effect of Deptor KD on cell cycle in C2C12 myoblasts. Myoblasts were transfected with either control (scramble) shRNA or shRNA targeting Deptor. Myoblasts were grown in DMEM supplemented with 10% FBS for 18–24 h and stained with propidium iodide to assess cell cycle using FACS. Representative forward scatter histograms highlighting G1 and G2 phases of cell cycle for scramble control (A) and KD (B) are shown. The percentage of cells in each stage of the cell cycle for each treatment group is shown in the accompanying pie graphs. FL2-A, propidium iodide fluorescence. (C) Western blotting of samples (normalized to total protein content for loading) as described above. Blots were probed with total Deptor antibody to show the different groups (top band ) and with phospho-specific antibody to detect phosphorylation on S807/S811 of pRb protein (middle band ). The lower band shows β-tubulin to confirm equal loading. (D) Quantification of Western blot from C for phosphorylation on S807/S811 of the pRb protein.
Figure 6
Figure 6
Effect of Deptor KD on cell cycle regulation. Control and Deptor KD myoblasts were grown in DMEM supplemented with 10% FBS overnight to 50–60% confluency. Myoblasts were then serum starved for 18–24 h in serum-free DMEM to arrest them in the G1 to G0 phase. Serum-starved myocytes were then released from the cell cycle arrest by the addition of fresh media containing 10% FBS. At 16 h, these proliferating cells were fixed. (A) Bar graph shows the percent of cells in the S-phase of the cell cycle in serum-starved cells (0 h) and after addition of serum (16 h); mean ± SE; n = 5–6 for each condition. *P < 0.05, compared with time-matched control values. (B) Western blot of samples (normalized to total protein content for loading) as described above. Blots were probed with total Deptor antibody (top band) and with phospho-specific antibody to detect phosphorylation on S807/S811 of pRb protein (middle band). The lower band was probed with β-tubulin to confirm equal loading.
Figure 7
Figure 7
Effect of Deptor KD on C2C12 differentiation. (A) Myoblasts were transfected with either control (scramble) shRNA or shRNA targeting Deptor. Cells were plated at the same density and photographed daily (10× objective magnification) to visually record changes in cell proliferation (time to reach confluence) and formation of myotubes. On days 3–4, when the plates were confluent, the media were switched to 2% horse serum (DM = differentiation media) to induce myotube formation. Deptor KD in C2C12 myocytes enhances MHC protein expression. (B) Representative Western blots for Deptor protein, MHC and the muscle-specific transcription factor MyoD in samples treated as in A. β-Tubulin serves as a loading control.
Figure 8
Figure 8
Effect of in vivo Deptor KD on skeletal muscle weight and protein synthesis. Gastrocnemius of C57/BL6 mice were electroporated with plasmids containing either scramble (control) shRNA or shRNA targeting Deptor mRNA. (A) Three days after electroporation, the animals were anesthetized and the muscle was excised and homogenized, and mRNA in the homogenate was quantified using real-time PCR. The bar graph shows Deptor mRNA content normalized to rpL32, which serves as an endogenous control. Data are mean ± SE; n = 5–6 for each condition. *P < 0.05, compared with control values. (B) Animals were treated as described above, and then immediately after electroporation, one hindlimb was immobilized to induce disuse atrophy, as described in Materials and Methods. At day 3, muscles were excised. B shows muscle weight and C shows the in vivo–determined rate of protein synthesis. For (B) and (C), values are means ± SE; n = 9–10 for each condition. Means with different superscripts (a, b and c) are significantly different (P < 0.05). Immob, immobilization.
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