, Volume 33, Issue 1–3, pp 63–70 | Cite as

Constant expression of mouse calpastatin isoforms during differentiation in myoblast cell line, C2C12

  • Kiyotaka Hitomi
  • Maki Murase
  • Toshimi Kawamura
  • Masatoshi Maki


C2C12 is a myoblast cell line which is used to studydifferentiation into multinucleated cells in vitro. Addition of calpain inhibitors, calpeptin orE-64d, to the culture medium prevented the myoblasticfusion of C2C12 cells. Immunoblot studies usingaffinity-purified antibody, revealed that the expressedlevels of mouse calpastatin remained unaltered duringC2C12 cell fusion. The detected calpastatin migratedas a protein of 130 kDa on SDS-polyacrylamide gelelectrophoresis. The estimated molecular mass wassomewhat greater than that in mouse liver anderythrocytes, and much greater than that reported inrat myoblasts. The 130 kDa isoform may contain anadditional N-terminal region designated XL domainfound in bovine calpastatin.

calpain calpastatin myoblast C2C12 


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  1. Adachi Y, Takahashi AI, Takahashi C, Takano E, Murachi T and Hatanaka M (1991) Phosphorylation and subcellular distribution ofcalpastatin. J Biol Chem 266: 3968–3972.Google Scholar
  2. Balcerzak D, Cottin P, Poussard S, Cucuron A, Brustis JJ and Ducastaing A (1998) Calpastatin-modulation of m-calpain activityis required for myoblast fusion. Eur J Cell Biol 75: 247–253.Google Scholar
  3. Balcerzak D, Poussard S, Brustis JJ, Elamrani N, Soriano M, Cottin P and Ducastaing A (1995) An antisense oligodeoxyribonucleotide tom-calpain mRNA inhibits myoblast fusion. J Cell Sci 108: 2077–2082.Google Scholar
  4. Barnoy S, Glaser T and Kosower NS (1997) Calpain and calpastatin in myoblast differentiation and fusion: effects of inhibitors. Biochim Biophys Acta 1358: 181–188.Google Scholar
  5. Barnoy S, Glaser T and Kosower NS (1998) The calpain-calpastatin system and protein degradation in fusing myoblasts. Biochim Biophys Acta 1402: 52–60.Google Scholar
  6. Barnoy S, Glaser T and Kosower NS (1996) The role of calpastatin (the specific calpain inhibitor) in myoblast differntiation andfusion. Biochem Biophys Res Commun 220: 933–938.Google Scholar
  7. Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, and Webster C (1985) Plasticity of the differentiated state. Science 230: 758–766.Google Scholar
  8. Blomgren K, Kawashima S, Saido TC, Karlsson JO, Elmered A and Hagbag H (1995) Fodrin degradation and subcellular distribution of calpains after neonatal. Brain Res 684: 143–149.Google Scholar
  9. Cong M, Thompson VF, Goll DE and Antin PB (1998) The bovine calpastatin gene promoter and a new N-terminal region of the protein are targets for cAMP-dependent protein kinase activity. J Biol Chem 273: 660–666.Google Scholar
  10. Cottin P, Brustis JJ, Poussard S, Elamrani N, Broncard S and Ducastaing BA (1994) Ca2+-dependent proteinases (calpains) and muscle cell differentiation. Biochim Biophys Acta 1223: 170–178.Google Scholar
  11. Dourdin N, Brustis JJ, Balcerzak D, Elamrani N, Poussard S, Cottin P and Ducastaing BA (1997) Myoblast fusion requires fibronectin degradation by exteriorized m-calpain. Exp Cell Res 235: 385–394.Google Scholar
  12. Emori Y, Kawasaki H, Imajoh S, Imahori K and Suzuki K (1987) Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites. Proc Natl Acad Sci USA 84: 3590–3594.Google Scholar
  13. Emori Y, Kawasaki H, Imajoh S, Minami Y and Suzuki K (1988) All four repeating domains of the endogenous inhibitor for calciumdependent protease independently retain inhibitory activity. J Biol Chem 263: 2364–2370.Google Scholar
  14. Hitomi K, Uchiyama Y, Ohkubo I, Kunimatsu M, Sasaki M and Maki M (1998a) Purification and characterization of the active-site-mutated recombinant human mu-calpain expressed in baculovirus-infected insect cells. Biochem Biophys Res Commun 246: 681–685.Google Scholar
  15. Hitomi K, Yokoyama A and Maki M (1998b) Expression of biologically active human calpastatin in baculovirus-infected insect cells andin Escherichia coli. Biosci Biotech Biochem 62: 136–141.Google Scholar
  16. Imajoh S, Kawasaki H, Emori Y and Suzuki K (1987) Calciumactivated neutral protease inhibitor from rabbit erythrocytes lacks N-terminal region of the liver inhibitor but retains three inhibitory units. Biochem Biophys Res Commun 146: 630–637.Google Scholar
  17. Inomata M, Nakamura M, Imajoh S and Kawashima S (1993) A variety of calpain/calpastatin systems in mammalian erythrocytes. Biochim Biophys Acta 1178: 207–214.Google Scholar
  18. Kwak KB, Chung SS, Kim OM, Kang MS, Ha DB and Chung CH (1993) Increase in the level of m-calpain correlates with the elevated cleavage offilamin during myogenic differentiation of embryonic muscle cells. Biochim Biophys Acta 1175: 243–249.Google Scholar
  19. Lee WJ, Hatanaka M and Maki M (1992) Multiple forms of rat calpastatin cDNA in the coding region of functionally unknown amino-terminal domain. Biochim Biophys Acta 1129: 251–253.Google Scholar
  20. Ma H, Yang QH, Takano E, Hatanaka M and Maki M (1994) Amino-terminal conserved region in proteinase inhibitor domain of calpastatin potentiates its calpain inhibitory activity by interaction with calmodulin-like domain of the protease. J Biol Chem 269: 24430–24436.Google Scholar
  21. Maki M, Takano E, Mori H, Kannagi R, Murachi T and Hatanaka M (1987a) Repetitive region of calpastatin is a functional unit of theproteinase inhibitor. Biochem Biophys Res Commun 143: 300–308.Google Scholar
  22. Maki M, Takano E, Mori H, Sato A, Murachi T and Hatanaka M (1987b) All four internally repetitive domains of pig calpastatin possess inhibitory activities against calpains I and II. FEBS Lett 223: 174–180.Google Scholar
  23. Niwa H, Yamamura K and Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193–200.Google Scholar
  24. Ostwald K, Hagberg H, Andine P and Karlsson JO (1993) Upregulation of calpain activity in neonatal rat brain after hypoxicischemia. Brain Res 630: 289–294.Google Scholar
  25. Pontremoli S, Viotti PL, Michetti M, Salamino F, Speratore B and B and Melloni E (1992) Modulation of inhibitory efficiency of ratskeletal muscle calpastatin by phosphorylation. Biochem BiophysRes Commun 187: 751–759.Google Scholar
  26. Saito K, Elce JS, Hamos JE and Nixon RA (1993) Widespread activation of calcium-activated neutral proteinase in the brain in Alzheimer disease:a potential molecular basis for neuronal degradation. Proc Natl Acad Sci USA 90: 2628–2632.Google Scholar
  27. Salamino F, Tullio RD, Michetti M, Mengotti P, Melloni E and Pontremoli S (1994) Modulation of calpastatin specificity in rat tissues by reversible phosphrylation and dephosphorylation. Biochem Biophys Res Commun 199: 1326–1332.Google Scholar
  28. Schollmeyer JE (1986) Role of Ca2+ and Ca2+-activated protease in myoblast fusion. Exp Cell Res 162: 411–422.Google Scholar
  29. Sorimachi H, Ishiura S and Suzuki K (1997) Structure and physiological function of calpains. Biochem J 328: 721–732.Google Scholar
  30. Studier FW, Rosenberg AH, Dunn JJ and Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 60–89.Google Scholar
  31. Suzuki K and Sorimachi H (1998) A novel aspect of calpain activation. FEBS Lett 433: 1–4.Google Scholar
  32. Takano E and Murachi T (1982) Purification and some properties of human erythrocyte calpastatin. J Biochem 92: 2021–2128.Google Scholar
  33. Takano E, Kitahara A, Sasaki T, Kannagi R and Murachi T (1986) Two different molecular species of pig calpastatin. Structural and functional relationship between 107 and 68 kDa molecules. Biochem J 235: 97–102.Google Scholar
  34. Takano E, Maki M, Mori H, Hatanaka M, Marti T, Titani K, Kannagi R, Ooi T and Murachi T (1988) Pig heart calpastatin: Identification of repetitive domain structure and anomalous behavior in polyacrylamide gel electrophoresis. Biochemistry 27, 1964–1972.Google Scholar
  35. Wakelam, MJ (1985) The fusion of myoblasts. Biochem J 228: 1–12.Google Scholar
  36. Yang HQ, Ma H, Takano E, Hatanaka M and Maki M (1994) Analysis of calcium-dependent interaction between aminoterminal conservedregion of calpastatin functional domain and calmodulin-like domain of μ-calpain large subunit. J Biol Chem 269: 18977–18984.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Kiyotaka Hitomi
    • 1
  • Maki Murase
    • 1
  • Toshimi Kawamura
    • 1
  • Masatoshi Maki
    • 2
  1. 1.Lab of Molecular and Cellular Regulation, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural SciencesNagoya UniversityChikusa, NagoyaJapan
  2. 2.Lab of Molecular and Cellular Regulation, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural SciencesNagoya UniversityChikusa, NagoyaJapan

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