Advertisement

Molecular Medicine

, Volume 17, Issue 9–10, pp 925–936 | Cite as

Deptor Knockdown Enhances mTOR Activity and Protein Synthesis in Myocytes and Ameliorates Disuse Muscle Atrophy

  • Abid A. Kazi
  • Ly Hong-Brown
  • Susan M. Lang
  • Charles H. Lang
Research Article

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 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.

Notes

Acknowledgments

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.

Supplementary material

10020_2011_1709925_MOESM1_ESM.pdf (554 kb)
Deptor Knockdown Enhances mTOR Activity and Protein Synthesis in Myocytes and Ameliorates Disuse Muscle Atrophy

References

  1. 1.
    Lin TA, et al. (1994) PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science. 266:653–6.CrossRefPubMedGoogle Scholar
  2. 2.
    Smith MR, et al. (1991) Modulation of the mitogenic activity of eukaryotic translation initiation factor-4E by protein kinase C. New Biol. 3:601–7.PubMedGoogle Scholar
  3. 3.
    Kimball SR. (2006) Interaction between the AMP-activated protein kinase and mTOR signaling pathways. Med. Sci. Sports Exerc. 38:1958–64.CrossRefPubMedGoogle Scholar
  4. 4.
    Kazi AA, Pruznak AM, Frost RA, Lang CH. (2011) Sepsis-induced alterations in protein-protein interactions within Mtor complex 1 and the modulating effect of leucine on muscle protein synthesis. Shock. 35:117–25.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kazi AA, Lang CH. (2010) PRAS40 regulates protein synthesis and cell cycle in C2C12 myoblasts. Mol. Med. 16:359–71.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hong-Brown LQ, et al. (2010) Alcohol and PRAS40 knockdown decrease mTOR activity and protein synthesis via AMPK signaling and changes in mTORC1 interaction. J. Cell. Biochem. 109:1172–84.PubMedPubMedCentralGoogle Scholar
  7. 7.
    Pruznak AM, Kazi AA, Frost RA, Vary TC, Lang CH. (2008) Activation of AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside prevents leucinestimulated protein synthesis in rat skeletal muscle. J. Nutr. 138:1887–94.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Anthony JC, et al. (2002) Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine. Am. J. Physiol. Endocrinol. Metab. 282:E1092–101.CrossRefPubMedGoogle Scholar
  9. 9.
    Anthony JC, et al. (2000) Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J. Nutr. 130:2413–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Pham PT, et al. (2000) Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes. J. Cell. Biochem. 79:427–41.CrossRefPubMedGoogle Scholar
  11. 11.
    Kim DH, et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 110:163–75.CrossRefGoogle Scholar
  12. 12.
    Kim DH, et al. (2003) GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell. 11:895–904.CrossRefPubMedGoogle Scholar
  13. 13.
    Hay N, Sonenberg N. (2004) Upstream and downstream of mTOR. Genes. Dev. 18:1926–45.CrossRefPubMedGoogle Scholar
  14. 14.
    Kim DH, Sabatini DM. (2004) Raptor and mTOR: subunits of a nutrient-sensitive complex. Curr. Top. Microbiol. Immunol. 279:259–70.PubMedGoogle Scholar
  15. 15.
    Peterson TR, et al. (2009) DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell. 137:873–86.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zoncu R, Efeyan A, Sabatini DM. (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell. Biol. 12:21–35.CrossRefPubMedGoogle Scholar
  17. 17.
    Liu M, et al. (2010) Resveratrol inhibits mTOR signaling by promoting the interaction between mTOR and DEPTOR. J. Biol. Chem. 285:36387–94.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Frost RA, Lang CH, Gelato MC. (1997) Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis. Endocrinology. 138:4153–9.CrossRefPubMedGoogle Scholar
  19. 19.
    Williamson DL, Bolster DR, Kimball SR, Jefferson LS. (2006) Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR. Am. J. Physiol. Endocrinol. Metab. 291:E80–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Sancak Y, et al. (2007) PRAS40 is an insulinregulated inhibitor of the mTORC1 protein kinase. Mol. Cell. 25:903–15.CrossRefPubMedGoogle Scholar
  21. 21.
    Das A, Desai D, Pittman B, Amin S, El-Bayoumy K. (2003) Comparison of the chemopreventive efficacies of 1,4-phenylenebis(methylene)selenocyanate and selenium-enriched yeast on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced lung tumorigenesis in A/J mouse. Nutr. Cancer. 46:179–85.CrossRefPubMedGoogle Scholar
  22. 22.
    Le Doux JM. (2008) Gene Therapy Protocols. Humana Press, Totowa, N.J.Google Scholar
  23. 23.
    Corovic S, et al. (2010) The influence of skeletal muscle anisotropy on electroporation: in vivo study and numerical modeling. Med. Biol. Eng. Comput. 48:637–48.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Gissel H. (2010) Effects of varying pulse parameters on ion homeostasis, cellular integrity, and force following electroporation of rat muscle in vivo. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298: R918–29.CrossRefPubMedGoogle Scholar
  25. 25.
    Tuckow AP, Vary TC, Kimball SR, Jefferson LS. (2010) Ectopic expression of eIF2Bepsilon in rat skeletal muscle rescues the sepsis-induced reduction in guanine nucleotide exchange activity and protein synthesis. Am. J. Physiol. Endocrinol. Metab. 299:E241–8.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Krawiec BJ, Frost RA, Vary TC, Jefferson LS, Lang CH. (2005) Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis. Am. J. Physiol. Endocrinol. Metab. 289:E969–80.CrossRefPubMedGoogle Scholar
  27. 27.
    Tucker KR, Seider MJ, Booth FW. (1981) Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization. J. Appl. Physiol. 51:73–7.CrossRefPubMedGoogle Scholar
  28. 28.
    Vary TC, Lang CH. (2008) Assessing effects of alcohol consumption on protein synthesis in striated muscles. Methods Mol. Biol. 447:343–55.CrossRefPubMedGoogle Scholar
  29. 29.
    Lang CH, Lynch CJ, Vary TC. (2010) BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299:R935–44.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Lang CH, Frost RA, Jefferson LS, Kimball SR, Vary TC. (2000) Endotoxin-induced decrease in muscle protein synthesis is associated with changes in eIF2B, eIF4E, and IGF-I. Am. J. Physiol. Endocrinol. Metab. 278:E1133–43.CrossRefPubMedGoogle Scholar
  31. 31.
    Vary TC, Kimball SR. (1992) Sepsis-induced changes in protein synthesis: differential effects on fast- and slow-twitch muscles. Am. J. Physiol. 262:C1513–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Garlick PJ, McNurlan MA, Essen P, Wernerman J. (1994) Measurement of tissue protein synthesis rates in vivo: a critical analysis of contrasting methods. Am. J. Physiol. 266:E287–97.PubMedGoogle Scholar
  33. 33.
    Srinivas V, Bohensky J, Shapiro IM. (2009) Autophagy: a new phase in the maturation of growth plate chondrocytes is regulated by HIF, mTOR and AMP kinase. Cells Tissues Organs. 189:88–92.CrossRefPubMedGoogle Scholar
  34. 34.
    Zeng M, Zhou JN. (2008) Roles of autophagy and mTOR signaling in neuronal differentiation of mouse neuroblastoma cells. Cell Signal. 20:659–65.CrossRefPubMedGoogle Scholar
  35. 35.
    Boyd KD, et al. (2010) High expression levels of the mammalian target of rapamycin inhibitor DEPTOR are predictive of response to thalidomide in myeloma. Leuk. Lymphoma. 51:2126–9.CrossRefPubMedGoogle Scholar
  36. 36.
    Thoreen CC, et al. (2009) An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J. Biol. Chem. 284:8023–32.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Schmelzle T, Hall MN. (2000) TOR, a central controller of cell growth. Cell. 103:253–62.CrossRefPubMedGoogle Scholar
  38. 38.
    Thomas G, Hall MN. (1997) TOR signalling and control of cell growth. Curr. Opin. Cell. Biol. 9:782–7.CrossRefPubMedGoogle Scholar
  39. 39.
    Fingar DC, et al. (2004) mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol. Cell. Biol. 24:200–16.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Fingar DC, Salama S, Tsou C, Harlow E, Blenis J. (2002) Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes. Dev. 16:1472–87.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    de la Rubia J, Such E. (2010) DEPTOR expression and response to thalidomide: toward a new therapeutic target in multiple myeloma? Leuk. Lymphoma. 51:1960–1.CrossRefPubMedGoogle Scholar
  42. 42.
    Proud CG. (2009) Dynamic balancing: DEPTOR tips the scales. J. Mol. Cell. Biol. 1:61–3.CrossRefPubMedGoogle Scholar
  43. 43.
    Hentges KE, et al. (2001) FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse. Proc. Natl. Acad. Sci. U. S. A. 98:13796–801.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Peponi E, et al. (2006) Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma. Am. J. Pathol. 169:2171–80.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Rosner M, Fuchs C, Siegel N, Valli A, Hengstschlager M. (2009) Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle. Hum. Mol. Genet. 18:3298–310.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Wang X, Proud CG. (2009) Nutrient control of TORC1, a cell-cycle regulator. Trends Cell Biol. 19:260–7.CrossRefPubMedGoogle Scholar
  47. 47.
    Sehgal SN. (1998) Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin. Biochem. 31:335–40.CrossRefPubMedGoogle Scholar
  48. 48.
    Crespo JL, Hall MN. (2002) Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 66:579–91.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Asnaghi L, Bruno P, Priulla M, Nicolin A. (2004) mTOR: a protein kinase switching between life and death. Pharmacol. Res. 50:545–9.CrossRefPubMedGoogle Scholar
  50. 50.
    Oldham S, Hafen E. (2003) Insulin/IGF and target of rapamycin signaling: a TOR de force in growth control. Trends Cell Biol. 13:79–85.CrossRefPubMedGoogle Scholar
  51. 51.
    Jacinto E, Hall MN. (2003) Tor signalling in bugs, brain and brawn. Nat. Rev. Mol. Cell. Biol. 4:117–26.CrossRefPubMedGoogle Scholar
  52. 52.
    White RJ. (1997) Regulation of RNA polymerases I and III by the retinoblastoma protein: a mechanism for growth control? Trends Biochem. Sci. 22:77–80.CrossRefPubMedGoogle Scholar
  53. 53.
    Hashemolhosseini S, et al. (1998) Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol. Chem. 273:14424–9.CrossRefPubMedGoogle Scholar
  54. 54.
    Luo Y, et al. (1996) Rapamycin resistance tied to defective regulation of p27Kip1. Mol. Cell. Biol. 16:6744–51.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Ren S, Rollins BJ. (2004) Cyclin C/cdk3 promotes Rb-dependent G0 exit. Cell. 117:239–51.CrossRefPubMedGoogle Scholar
  56. 56.
    Munger K, Howley PM. (2002) Human papillomavirus immortalization and transformation functions. Virus Res. 89:213–28.CrossRefPubMedGoogle Scholar
  57. 57.
    Muise-Helmericks RC, et al. (1998) Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 273:29864–72.CrossRefPubMedGoogle Scholar
  58. 58.
    Ong CS, Zhou J, Ong CN, Shen HM. (2010) Luteolin induces G1 arrest in human nasopharyngeal carcinoma cells via the Akt-GSK-3beta-Cyclin D1 pathway. Cancer Lett. 298:167–75.CrossRefPubMedGoogle Scholar
  59. 59.
    Yeste-Velasco M, et al. (2007) Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells. Neuropharmacology. 53:295–307.CrossRefPubMedGoogle Scholar
  60. 60.
    Diehl JA, Cheng M, Roussel MF, Sherr CJ. (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 12:3499–511.CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Faber AC, Chiles TC. (2007) Inhibition of cyclindependent kinase-2 induces apoptosis in human diffuse large B-cell lymphomas. Cell Cycle. 6:2982–9.CrossRefPubMedGoogle Scholar
  62. 62.
    Delston RB, Matatall KA, Sun Y, Onken MD, Harbour JW. (2010) p38 phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers Rb-Hdm2 interaction and apoptosis. Oncogene. 30:588–99.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Mammucari C, Schiaffino S, Sandri M. (2008) Downstream of Akt: FoxO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy. 4:524–6.CrossRefPubMedGoogle Scholar
  64. 64.
    Annovazzi L, et al. (2009) mTOR, S6 and AKT expression in relation to proliferation and apoptosis/autophagy in glioma. Anticancer Res. 29:3087–94.PubMedGoogle Scholar
  65. 65.
    Jung CH, Ro SH, Cao J, Otto NM, Kim DH. (2010) mTOR regulation of autophagy. FEBS Lett. 584:1287–95.CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Ravikumar B, et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36:585–95.CrossRefPubMedGoogle Scholar

Copyright information

© The Feinstein Institute for Medical Research 2011
www.feinsteininstitute.org

Authors and Affiliations

  • Abid A. Kazi
    • 1
  • Ly Hong-Brown
    • 1
  • Susan M. Lang
    • 1
  • Charles H. Lang
    • 1
  1. 1.Department of Cellular and Molecular Physiology (H166)Pennsylvania State University College of MedicineHersheyUSA

Personalised recommendations