DOI QR코드

DOI QR Code

C2C12 근관세포에서 dexamethasone 및 hydrogen peroxide에 의한 근위축 유도

Induction of Muscle Atrophy by Dexamethasone and Hydrogen Peroxide in Differentiated C2C12 Myotubes

  • 박철 (동의대학교 자연과학대학 분자생물학과) ;
  • 정진우 (동의대학교 항노화연구소) ;
  • 최영현 (동의대학교 항노화연구소)
  • Park, Cheol (Department of Molecular Biology, College of Natural Sciences, Dongeui University) ;
  • Jeong, Jin-Woo (Anti-Aging Research Center and Blue Bio Industry RIC, Dongeui University) ;
  • Choi, Yung Hyun (Anti-Aging Research Center and Blue Bio Industry RIC, Dongeui University)
  • 투고 : 2017.08.22
  • 심사 : 2017.10.24
  • 발행 : 2017.12.30

초록

일반적으로 노화, 영양부족 및 다양한 만성질환에 의하여 유발되는 근위축은 근육 단백질 합성 억제 및 분해증가를 통하여 근섬유 및 근육의 밀도를 감소시키는 것으로 알려져 있다. 본 연구에서는 근위축과 관련된 in vitro 실험을 위한 C2C12 근아세포에서 근관세포로의 분화과정을 확립하고, 분화가 유발된 C2C12 근관세포를 대상으로 dexamethasone 및 hydrogen peroxide에 의한 근위축 유발 및 관련 단백질들의 발현 변화를 조사하였다. 먼저 C2C12 근아세포에 분화배지를 처리하였을 경우 근관세포로 분화가 유발되었으며, 분화와 관련된 단백질인 myogenin 및 myoD의 발현이 증가하는 것으로 나타났다. 분화가 유발된 C2C12 근관세포에 세포독성이 없는 조건의 dexamethasone 및 hydrogen peroxide를 처리하였을 경우 근관의 지름이 감소하였으며, 이러한 현상은 musclespecific ubiquitin ligases인 MAFbx/atrogin-1 및 MuRF1의 발현 증가와 함께 muscle-specific transcription factor인 myogenin 및 MyoD의 발현 감소와 관련이 있다는 것을 확인하였다. 본 연구 결과는 근위축과 관련된 in vitro 실험 모델의 구축을 위한 최적의 분화조건 확립과 함께 dexamethasone 및 hydrogen peroxide를 근위축 유도제로 사용할 수 있는 가능성 을 제시하는 것이다.

Muscle atrophy due to aging, starvation, and various chronic diseases leads to a decrease in muscle fiber area and density due to reduced muscle protein synthesis and increased protein breakdown. This study investigated the effect of dexamethasone and hydrogen peroxide on the induction of muscle atrophy and expression of atrophy-related genes in differentiated C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes in differentiation medium. During myoblast differentiation, muscle-specific transcription factors, such as myogenin, and MyoD expression increased. Differentiated C2C12 myotubes exposed to noncytotoxic levels of dexamethasone and hydrogen peroxide showed a decrease in myotube diameter, which was associated with up-regulation of muscle-specific ubiquitin ligases, such as muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and down-regulation of myogenin and MyoD. These results demonstrated that dexamethasone and hydrogen peroxide induced atrophy through regulation of muscle-specific ubiquitin ligases and muscle-specific transcription factors in C2C12 myotubes. In this study, we confirmed the process of differentiation of C2C12 myoblasts into myotubes in in vitro experiments in the presence of atrophy. This muscle atrophy model of C2C12 cells induced by dexamethasone or hydrogen peroxide seems suited to studies of the mechanism of muscle atrophy suppression and to exploit the experiment for excavating new muscle atrophy.

키워드

참고문헌

  1. Abe, S., Rhee, S., Iwanuma, O., Hiroki, E., Yanagisawa, N., Sakiyama, K. and Ide, Y. 2009. Effect of mechanical stretching on expressions of muscle specific transcription factors MyoD, Myf-5, myogenin and MRF4 in proliferated myoblasts. Anat. Histol. Embryol. 38, 305-310. https://doi.org/10.1111/j.1439-0264.2009.00945.x
  2. Attaix, D., Combaret, L., Bechet, D. and Taillandier, D. 2008. Role of the ubiquitin-proteasome pathway in muscle atrophy in cachexia. Curr. Opin. Support. Palliat. Care 2, 262-266. https://doi.org/10.1097/SPC.0b013e3283196ac2
  3. Bhatnagar, S., Mittal, A., Gupta, S. K. and Kumar, A. 2012. TWEAK causes myotube atrophy through coordinated activation of ubiquitin-proteasome system, autophagy, and caspases. J. Cell. Physiol. 227, 1042-1051. https://doi.org/10.1002/jcp.22821
  4. Bodine, S. C. and Baehr, L. M. 2014. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1. Am. J. Physiol. Endocrinol. Metab. 307, E469-484. https://doi.org/10.1152/ajpendo.00204.2014
  5. Chaudhary, P., Suryakumar, G., Prasad, R., Singh, S. N., Ali, S. and Ilavazhagan, G. 2012. Chronic hypobaric hypoxia mediated skeletal muscle atrophy: role of ubiquitin-proteasome pathway and calpains. Mol. Cell. Biochem. 364, 101-113. https://doi.org/10.1007/s11010-011-1210-x
  6. Du, J., Mitch, W. E., Wang, X. and Price, S. R. 2000. Glucocorticoids induce proteasome C3 subunit expression in L6 muscle cells by opposing the suppression of its transcription by NF-kappa B. J. Biol. Chem. 275, 19661-19666. https://doi.org/10.1074/jbc.M907258199
  7. Evenson, A. R., Fareed, M. U., Menconi, M. J., Mitchell, J. C. and Hasselgren, P. O. 2005. GSK-3beta inhibitors reduce protein degradation in muscles from septic rats and in dexamethasone-treated myotubes. Int. J. Biochem. Cell. Biol. 37, 2226-2238. https://doi.org/10.1016/j.biocel.2005.06.002
  8. Foletta, V. C., White, L. J., Larsen, A. E., Leger, B. and Russell, A. P. 2011. The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy. Pflugers Arch. 461, 325-335. https://doi.org/10.1007/s00424-010-0919-9
  9. Gomes-Marcondes, M. C. and Tisdale, M. J. 2002. Induction of protein catabolism and the ubiquitin-proteasome pathway by mild oxidative stress. Cancer Lett. 180, 69-74. https://doi.org/10.1016/S0304-3835(02)00006-X
  10. Hyatt, J. P., Roy, R. R., Baldwin, K. M. and Edgerton, V. R. 2003. Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei. Am. J. Physiol. Cell. Physiol. 285, C1161-1173. https://doi.org/10.1152/ajpcell.00128.2003
  11. Jagoe, R. T. and Goldberg, A. L. 2001. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy? Curr. Opin. Clin. Nutr. Metab. Care 4, 183-190. https://doi.org/10.1097/00075197-200105000-00003
  12. Jesinkey, S. R., Korrapati, M. C., Rasbach, K. A., Beeson, C. C. and Schnellmann, R. G. 2014. Atomoxetine prevents dexamethasone-induced skeletal muscle atrophy in mice. J. Pharmacol. Exp. Ther. 351, 663-673. https://doi.org/10.1124/jpet.114.217380
  13. Kandarian, S. C. and Jackman, R. W. 2006. Intracellular signaling during skeletal muscle atrophy. Muscle Nerve 33, 155-165. https://doi.org/10.1002/mus.20442
  14. Kiess, M., Gill, R. M. and Hamel, P. A. 1995. Expression of the positive regulator of cell cycle progression, cyclin D3, is induced during differentiation of myoblasts into quiescent myotubes. Oncogene 10. 159-166.
  15. Ko, J. A., Kimura, Y., Matsuura, K., Yamamoto, H., Gondo, T. and Inui, M. 2006. PDZRN3 (LNX3, SEMCAP3) is required for the differentiation of C2C12 myoblasts into myotubes. J. Cell. Sci. 119, 5106-5113. https://doi.org/10.1242/jcs.03290
  16. Laviano, A., Meguid, M. M., Preziosa, I. and Rossi Fanelli, F. 2007. Oxidative stress and wasting in cancer. Curr. Opin. Clin. Nutr. Metab. Care 10, 449-456. https://doi.org/10.1097/MCO.0b013e328122db94
  17. Lawler, J. M., Song, W. and Demaree, S. R. 2003. Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free Radic. Biol. Med. 35, 9-16. https://doi.org/10.1016/S0891-5849(03)00186-2
  18. Li, X., Moody, M. R., Engel, D., Walker, S., Clubb, F. J. Jr., Sivasubramanian, N., Mann, D. L. and Reid, M. B. 2000. Cardiac-specific overexpression of tumor necrosis factor-alpha causes oxidative stress and contractile dysfunction in mouse diaphragm. Circulation 102, 1690-1696. https://doi.org/10.1161/01.CIR.102.14.1690
  19. Li, Y. P., Chen, Y., Li, A. S. and Reid, M. B. 2003. Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes. Am. J. Physiol. Cell. Physiol. 285, C806-812. https://doi.org/10.1152/ajpcell.00129.2003
  20. Marinovic, A. C., Zheng, B., Mitch, W. E. and Price, S. R. 2007. Tissue-specific regulation of ubiquitin (UbC) transcription by glucocorticoids: in vivo and in vitro analyses. Am. J. Physiol. Renal. Physiol. 292, F660-666. https://doi.org/10.1152/ajprenal.00178.2006
  21. Mastrocola, R., Reffo, P., Penna, F., Tomasinelli, C. E., Boccuzzi, G., Baccino, F. M., Aragno, M. and Costelli, P. 2008. Muscle wasting in diabetic and in tumor-bearing rats: role of oxidative stress. Free Radic. Biol. Med. 44, 584-593. https://doi.org/10.1016/j.freeradbiomed.2007.10.047
  22. Menconi, M., Gonnella, P., Petkova, V., Lecker, S. and Hasselgren, P. O. 2008. Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes. J. Cell. Biochem. 105, 353-364. https://doi.org/10.1002/jcb.21833
  23. Murton, A. J., Constantin, D. and Greenhaff, P. L. 2008. The involvement of the ubiquitin proteasome system in human skeletal muscle remodelling and atrophy. Biochim. Biophys. Acta 1782, 730-743. https://doi.org/10.1016/j.bbadis.2008.10.011
  24. Otis, J. S., Ashikhmin, Y. I., Brown, L. A. and Guidot, D. M. 2008. Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats. AIDS Res. Ther. 5, 8. https://doi.org/10.1186/1742-6405-5-8
  25. Passmore, L. A. and Barford, D. 2004. Getting into position: the catalytic mechanisms of protein ubiquitylation. Biochem. J. 379, 513-525. https://doi.org/10.1042/bj20040198
  26. Pickart, C. M. and Eddins, M. J. 2004. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55-72. https://doi.org/10.1016/j.bbamcr.2004.09.019
  27. Qin, J., Du, R., Yang, Y. Q., Zhang, H. Q., Li, Q., Liu, L., Guan, H., Hou, J. and An, X. R. 2013. Dexamethasone-induced skeletal muscle atrophy was associated with upregulation of myostatin promoter activity. Res. Vet. Sci. 94, 84-89. https://doi.org/10.1016/j.rvsc.2012.07.018
  28. Stitt, T. N., Drujan, D., Clarke, B. A., Panaro, F., Timofeyva, Y., Kline, W. O., Gonzalez, M., Yancopoulos, G. D. and Glass, D. J. 2004. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol. Cell 14, 395-403. https://doi.org/10.1016/S1097-2765(04)00211-4
  29. Talarmin, H., Derbre, F., Lefeuvre-Orfila, L., Leon, K., Droguet, M., Pennec, J. P. and Giroux-Metges, M. A. 2017. The diaphragm is better protected from oxidative stress than hindlimb skeletal muscle during CLP-induced sepsis. Redox Rep. 22, 218-226. https://doi.org/10.1080/13510002.2016.1223793
  30. Tintignac, L. A., Lagirand, J., Batonnet, S., Sirri, V., Leibovitch, M. P. and Leibovitch, S. A. 2005. Degradation of MyoD mediated by the SCF (MAFbx) ubiquitin ligase. J. Biol. Chem. 280, 2847-2856. https://doi.org/10.1074/jbc.M411346200
  31. Wing, S. S. 2005. Control of ubiquitination in skeletal muscle wasting. Int. J. Biochem. Cell. Biol. 37, 2075-2087. https://doi.org/10.1016/j.biocel.2004.11.011
  32. Yamamoto, D., Ikeshita, N., Matsubara, T., Tasaki, H., Herningtyas, E. H., Toda, K., Iida, K., Takahashi, Y., Kaji, H., Chihara, K. and Okimura, Y. 2008. GHRP-2, a GHS-R agonist, directly acts on myocytes to attenuate the dexamethasone-induced expressions of muscle-specific ubiquitin ligases, Atrogin-1 and MuRF1. Life Sci. 82, 460-466. https://doi.org/10.1016/j.lfs.2007.11.019