Abstract
Studies on skeletal muscle cell specification and development have demonstrated in the past that calpains interact with various transcriptional factors in regulating the cellular function. It has therefore, been assumed that transcriptional factors like myogenin, MyoD, Myf5, and MRF4 that are active during the myogenic differentiation might be affected and degraded by calpains. Therefore, to examine the biochemical adaptations of myoblasts during myocyte formation and muscle development comprehensively, the current study was designed to identify the effect of calpeptin (calpain inhibitors) on protein expression during differentiation of C2C12 mouse myoblast. Cells were proliferated to near 80% confluence under Dulbecco's modified eagle medium and differentiated further in 2% HS with 50 μM calpeptin. Incubated cells were collected at 0, 12, and 72 h and later the cell proteins were focused onto pH 4–7 IEF strip, followed by 12.5% SDS-PAGE. Obtained spots on the gels were compared and matched using commercial 2-DE analysis software and matched spots were identified by MALDI-ToF and/or Q-Tof systems. Conclusively, cell differentiation was observed to be active from 12 to 72 h however, calpeptin affected the differentiation process and cut down the rate of fusion by approximately 50%. Out of 41 proteins identified, 12 proteins were found to be upregulated where as 29 proteins were downregulated.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adinolfi S.; Iannuzzi C.; Prischi F.; Pastore C.; Iametti S.; Martin S. R.; Bonomi F.; Pastore A. Bacterial frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS. Nat. Struct. Mol. Biol. 16(4): 390–396; 2009.
Arcuri C.; Giambanco I.; Bianchi R.; Donato R. Annexin V, annexin VI, S100A1 and S100B in developing and adult avian skeletal muscles. Neuroscience 109: 371–388; 2002.
Blackwood R. A.; Ernst J. D. Characterization of Ca2 (+)-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins. Biochem. J. 266: 195–200; 1999.
Blomgren K.; Zhu C.; Wang X.; Karlsson J. O.; Leverin A. L.; Bahr B. A.; Mallard C.; Hagberg H. Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of “pathological apoptosis”? J. Biol. Chem. 276: 10191–10198; 2001.
Borges J. C.; Ramos C. H. Protein folding assisted by chaperones. Protein Pept. Lett. 12(3): 257–261; 2005.
Carvajal J. J.; Pook M. A.; Santos M.; Doudney K.; Hillermann R.; Minogue S.; Williamson R.; Hsuan J. J.; Chamberlain S. The Friedreich’s ataxia gene encodes a novel phosphatidylinositol-4- phosphate 5-kinase. Nat. Genet. 14(2): 157–162; 1996.
Daly K. A.; Lefévre C.; Nicholas K.; Deane E.; Williamson P. Characterization and expression of Peroxiredoxin 1 in the neonatal tammarwallaby (Macropus eugenii). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 49: 108–119; 2008.
De Maio A. Heat shock proteins: facts, thoughts, and dreams. Shock (Augusta, Ga.) 11(1): 1–12; 1999.
Debiasi R. L.; Pike B.; Squier M. K. Reovirus-induced apoptosis is preceded by increased cellular calpain activity and is blocked by calpain inhibitors. J. Virol. 73: 695–701; 1999.
Dedieu S.; Mazeres G.; Cottin P.; Brustis J. J. Involvement of myogenic regulator factors during fusion in the cell line C2C12. Int. J. Dev. Biol. 46: 235–241; 2002.
Dedieu S.; Poussard S.; Mazeres G.; Grise F.; Dargelos E.; Cottin P.; Brustis J. J. Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization. Exp. Cell Res. 292: 187–200; 2004.
Delgado I.; Huang X.; Zhang L.; Hatcher R.; Gao B.; Zhang P. Dynamic gene expression during the onset of myoblast differentiation in vitro. Genomics 82(2): 109–121; 2003.
Dodson M. V.; Mathison B. A. Comparison of ovine and rat muscle-derived satellite cells: response to insulin. Tis. Cell 20: 909–918; 1988.
Fernando P.; Kelly J. F.; Balazsi K.; Slack R. S.; Megeney L. A. Caspase 3 activity is required for skeletal muscle differentiation. Proc Natl Acad Sci USA 99: 11025–11030; 2002.
Franco A. A.; Odom R. S.; Rando T. A. Regulation of antioxidant enzyme gene expression in response to oxidative stress and during differentiation of mouse skeletal muscle. Free Radic. Biol. Med. 27: 1122–1132; 1999.
Fusaro G.; Dasgupta P.; Rastogi S.; Joshi B.; Chellappan S. Prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling. J. Biol. Chem. 278(48): 47853–47861; 2003.
Goll G. E.; Thompson V. F.; Li H.; Wei W.; Cong J. The calpain system. Physiol. Rev. 83: 731–801; 2003.
Hartman D. J.; Hoogenraad N. J.; Condron R.; Hoj P. B. Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric ornithine transcarbamoylase in vitro. Proc Natl Acad Sci USA 89: 3394–3398; 1992.
Kim J.; Jung Y. K. Calpeptin suppresses tumor necrosis factor-α-induced death and accumulation of p53 in L929 mouse sarcoma cells. Apoptosis 7: 115–121; 2002.
Knepper-Nicolai B.; Savill J.; Brown S. B. Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases. J. Biol. Chem. 273: 30530–30536; 1998.
Kulkarni S.; Saido T. C.; Suzuki K.; Fox J. E. Calpain mediates integrin-induced signaling at a point upstream of Rho family members. J. Biol. Chem. 274: 21265–21275; 1999.
Lebart M. C.; Benyamin Y. Calpain involvement in the remodeling of cytoskeletal anchorage complexes. FEBS J. 273: 3415–3426; 2006.
Lee Y. M.; Park S. H.; Shin D. I.; Hwang J. Y.; Park B.; Park Y. J.; Lee T. H.; Chae H. Z.; Jin B. K.; Oh T. H.; Oh Y. J. Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease. J. Biol. Chem. 283: 9986–9998; 2008.
Lodish H.; Berk A.; Matsudaira P.; Kaiser C. A.; Krieger M.; Scott M. P.; Zipursky S. L.; Darnell J. “3”. Molecular cell biology. 5th ed. W.H. Freeman and CO, New York, pp 66–72; 2004.
Mazeres G.; Leloup L.; Daury L.; Cottin P.; Brustis J. J. Myoblast attachment and spreading are regulated by different patterns of ubiquitous calpains. Cell Motil. Cytoskeleton 63: 193–207; 2006.
Molinari M.; Carafoli E. Calpains: a cytosolic proteinase active at the membranes. J. membr. Biology 156: 1–8; 1997.
Moyen C.; Goudenege S.; Poussard S.; Ssassi A. H.; Brustis J. J.; Cottin P. Involvement of microcalpain (CAPN1) in muscle cell differentiation. The international. J. Biochem. Cell Biol. 36(4): 728–743; 2004.
Pariat M.; Salvat C.; Bebien M.; Brockly F.; Altieri E.; Carillo S.; Jariel-Encontre I.; Piechaczyk M. The sensitivity of c-Jun and c-Fosproteins to calpains depends on conformational determinants of the mono-mers and not on formation of dimers. Biochem. J. 345: 129–138; 2000.
Parmar H.; Melov S.; Samper E.; Ljung B. M.; Cunha G. R.; Benz C. C. Hyperplasia, reduced E-cadherin expression, and developmental arrest in mammary glands oxidatively stressed by loss of mitochondrial superoxide dismutase. Breast 14: 256–263; 2005.
Perrin B. J.; Huttenlocher A. Calpain. Int. J. Biochem. Cell Biol. 34: 722–725; 2002.
Petit V.; Thiery J. P. Focal adhesions: structure and dynamics. Biol. Cell 92: 477–494; 2000.
Runembert I.; Queffeulou G.; Federici P.; Vrtovsnik F.; Colucci-Guyon E.; Babinet C. Vimentin affects localization and activity of sodium- glucose cotransporter SGLT1 in membrane rafts. J. Cell Sci. 115: 713–724; 2002.
Salinthone S.; Tyagi M.; Gerthoffer W. T. Small heat shock proteins in smooth muscle. Pharmacol. Ther. 119(1): 44–54; 2008.
Schoenwaelder S. M.; Burridge K. Evidence for a calpeptin protein-tyrosine phosphatase upstream of the small GTPase rho. J. Biol. Chem. 274: 14359–14367; 2000.
Shimokawa T.; Kato M.; Ezaki O.; Hashimoto S. Transcritional regulation of muscle specific genes during myoblast differentiation. Biochem. Biophys. Res. Communic 246: 287–292; 1998.
Sorimachi H.; Suzuki K. The structure of calpain. J. Biochem. 129: 653–664; 2001.
Suzuki K.; Sorimachi H. A novel aspect of calpain activation. FEBS Lett. 433: 1–4; 1998.
Tannu N. S.; Rao V. K.; Chaudhary R. M.; Giorgianni F.; Saeed A. E.; Gao Y.; Raghow R. Comparative proteomes of the proliferating C2C12 myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program. Mol. Cell. Proteomics 3: 1065–1082; 2004.
Tarricone E.; Ghirardello A.; Zampieri S.; Elisa R. M.; Doria A.; Gorza L. Cell stress response in skeletal muscle myofibers. Ann. N. Y. Acad. Sci. 1069: 472–476; 2006.
Wu C. Heat shock transcription factors: structure and regulation. Annu. Rev. Cell Dev. Biol. 11: 441–469; 1995.
Yajima Y.; Kawashima S. Calpain function in the differentiation of mesenchymal stem cells. Biol. Chem. 383: 757–764; 2002.
Zhao S. H.; Nettleton D.; Liu W.; Fitzsimmons C.; Ernst C. W.; Raney N. E. Complementary DNA macroarray analyses of differential gene expression in porcine fetal and postnatal muscle. J. Anim. Sci. 81: 2179–2188; 2003.
Acknowledgment
This work has been conducted and partly supported by a grant from Next-Generation BioGreen 21 Program (no. PJ008191), Rural Development Administration, Republic of Korea.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: T. Okamoto
Rights and permissions
About this article
Cite this article
Singh, N.K., Shiwani, S. & Hwang, I.H. Proteomic study of calpeptin-induced differentiation on calpain-interacting proteins of C2C12 myoblast. In Vitro Cell.Dev.Biol.-Animal 48, 175–185 (2012). https://doi.org/10.1007/s11626-012-9484-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-012-9484-1