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

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.

• Authors: Abid A Kazi, Ly Hong-Brown, Susan M Lang, Charles H Lang
• Journal: Molecular medicine (Cambridge, Mass.) (Mol Med) 2011 Sep-Oct Vol. 17 Issue 9-10 Pg. 925-36 ISSN: 1528-3658 [Electronic] England
• PMID: 21607293 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• Carrier Proteins
• Cell Cycle Proteins
• Eif4ebp1 protein, mouse
• Eukaryotic Initiation Factors
• Intracellular Signaling Peptides and Proteins
• Phosphoproteins
• Retinoblastoma Protein
• deptor protein, mouse
• mTOR protein, mouse
• Ribosomal Protein S6 Kinases, 90-kDa
• Rps6ka1 protein, mouse
• TOR Serine-Threonine Kinases

Topics

• Adaptor Proteins, Signal Transducing
• Animals
• Carrier Proteins (metabolism)
• Cell Cycle Proteins
• Cell Line
• Cell Proliferation
• Cell Size
• Eukaryotic Initiation Factors
• Female
• HEK293 Cells
• Hindlimb Suspension
• Humans
• Immunoblotting
• Intracellular Signaling Peptides and Proteins (genetics, metabolism)
• Male
• Mice
• Mice, Inbred C57BL
• Muscle Cells (cytology, metabolism)
• Muscle, Skeletal (growth & development, metabolism)
• Muscular Dystrophy, Animal (genetics, metabolism, pathology)
• Myoblasts (cytology, metabolism)
• Organ Size
• Phosphoproteins (metabolism)
• Phosphorylation
• Protein Biosynthesis
• RNA Interference
• Retinoblastoma Protein (metabolism)
• Ribosomal Protein S6 Kinases, 90-kDa (metabolism)
• S Phase
• TOR Serine-Threonine Kinases (genetics, metabolism)