Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Clostridium difficile Toxin A Inhibits Wnt Signaling Pathway in Gut Epithelial Cells

대장상피세포 속 Wnt 신호 경로에 대한 C. difficile 톡신A의 영향

  • Yoon, I Na (Division of Life Science and Chemistry, College of Natural Science, Daejin University) ;
  • Kim, Ho (Division of Life Science and Chemistry, College of Natural Science, Daejin University)
  • 윤이나 (대진대학교 과학기술대학 생명화학부 생명과학전공) ;
  • 김호 (대진대학교 과학기술대학 생명화학부 생명과학전공)
  • Received : 2018.06.05
  • Accepted : 2018.08.28
  • Published : 2018.09.30
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Abstract

Clostridium difficile toxin A causes pseudomembranous colitis. The pathogenesis of toxin A-induced colonic inflammation includes toxin A-dependent epithelial cell apoptosis, resulting in the loss of barrier function provided by epithelial cells against luminal pathogens. Toxin A-dependent epithelial cell apoptosis has been linked to toxin A-induced production of reaction oxygen species and subsequent p38MAPK activation; $p21^{CIP1/WAF1}$ upregulation-dependent cell cycle arrest; cytoskeletal disaggregation; and/or the induction of Fas ligand on epithelial cells. However, the molecular mechanisms underlying toxin A-induced apoptosis remain poorly understood. This study tested whether toxin A could block the Wnt signaling pathway, which is involved in gut epithelial cell proliferation, differentiation and antiapoptotic progression. Toxin A treatment of nontransformed human colonocytes (NCM460) rapidly reduced ${\beta}$-catenin protein, an essential component of the Wnt signaling pathway. Exposure of mouse ileum to toxin A also significantly reduced ${\beta}$-catenin protein levels. MG132 inhibition of proteasome-dependent protein degradation resulted in the recovery of toxin A-mediated reduction of ${\beta}$-catenin, indicating that toxin A may activate intracellular processes, such as $GSK3{\beta}$, to promote degradation of ${\beta}$-catenin. Immunoblot analysis showed that toxin A increased active phosphorylation of $GSK3{\beta}$. Because the Wnt signaling pathway is essential for gut epithelial cell proliferation and anti-apoptotic processes, our results suggest that toxin A-mediated inhibition of the Wnt signaling pathway may be required for maximal toxin A-induced apoptosis of gut epithelial cells.

C. difficile 톡신A에 의한 대장상피세포 자살과정은 위막성대장염(Pseudomembranous colitis)의 주요 원인으로 고려되고 있다. 톡신A는 활성산소 를 증가시켜 세포자살 신호를 유도한다. 또한 톡신A는 미세섬유나 미세소관과 같은 세포골격계 형성을 저해함으로써 자살을 유도한다고 알려져 있다. 하지만 톡신A가 야기하는 소화기 상피세포 자살경로는 아직 불분명하다. 본 연구에서는 소화관 상피세포의 성장과 분화 그리고 기능에 중요하다고 알려져 온 Wnt 신호경로에 대한 톡신A의 영향을 확인해보았다. 이를 위해 비암화-인간대장세포주(NCM460)에 톡신A를 처치하고 Wnt 신호 분자들의 변화를 추적하였다. 또한 톡신A를 주입한 생쥐의 회장 상피세포 속 Wnt 신호경로 변화도 평가하였다. 인간 대장상피세포에서 톡신A는 Wnt 경로의 핵심 신호분자인 ${\beta}$-catenin 단백질의 양을 빠르게 감소시켰다. 이 현상은 생쥐 회장 상피세포에서도 동일하게 확인되었다. 연구자 등은 톡신A가 $GSK3{\beta}$ 활성형 인산화(Thr390)를 증가시킴도 확인하였다. 이는 톡신A가 $GSK3{\beta}$의 활성을 높여서 ${\beta}$-catenin의 인산화시키고 이를 통해 단백질 분해 과정이 촉진되었음을 보여준다. 이 결과들을 종합하면, 톡신A에 의한 소화관 상피세포 자살과정이 상피세포의 성장과 자살을 조절하는 Wnt 신호경로 차단과 밀접하게 연관되어 있음을 보여준다.

Keywords

References

  1. Brunt, L. H., Begg, K., Kague, E., Cross, S. and Hammond, C. L. 2017. Wnt signalling controls the response to mechanical loading during zebrafish joint development. Development 144, 2798-2809. https://doi.org/10.1242/dev.153528
  2. Clevers, H. 2006. Wnt/beta-catenin signaling in development and disease. Cell 127, 469-480. https://doi.org/10.1016/j.cell.2006.10.018
  3. Fevr, T., Robine, S., Louvard, D. and Huelsken, J. 2007. Wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells. Mol. Cell. Biol. 27, 7551-7559. https://doi.org/10.1128/MCB.01034-07
  4. Han, G., Casson, R. J., Chidlow, G. and Wood, J. P. 2014. The mitochondrial complex I inhibitor rotenone induces endoplasmic reticulum stress and activation of GSK-3beta in cultured rat retinal cells. Invest. Ophthalmol. Vis. Sci. 55, 5616-5628. https://doi.org/10.1167/iovs.14-14371
  5. He, D., Hagen, S. J., Pothoulakis, C., Chen, M., Medina, N. D., Warny, M. and LaMont, J. T. 2000. Clostridium difficile toxin A causes early damage to mitochondria in cultured cells. Gastroenterology 119, 139-150. https://doi.org/10.1053/gast.2000.8526
  6. He, D., Sougioultzis, S., Hagen, S., Liu, J., Keates, S., Keates, A. C., Pothoulakis, C. and Lamont, J. T. 2002. Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation. Gastroenterology 122, 1048-1057. https://doi.org/10.1053/gast.2002.32386
  7. Hoffman, J., Kuhnert, F., Davis, C. R. and Kuo, C. J. 2004. Wnts as essential growth factors for the adult small intestine and colon. Cell Cycle 3, 554-557.
  8. Inoue, S., Nakase, H., Matsuura, M., Mikami, S., Ueno, S., Uza, N. and Chiba, T. 2009. The effect of proteasome inhibitor MG132 on experimental inflammatory bowel disease. Clin. Exp. Immunol. 156, 172-182. https://doi.org/10.1111/j.1365-2249.2008.03872.x
  9. Jeffers, M., McDonald, W. F., Chillakuru, R. A., Yang, M., Nakase, H., Deegler, L. L., Sylander, E. D., Rittman, B., Bendele, A., Sartor, R. B. and Lichenstein, H. S. 2002. A novel human fibroblast growth factor treats experimental intestinal inflammation. Gastroenterology 123, 1151-1162. https://doi.org/10.1053/gast.2002.36041
  10. Just, I., Fritz, G., Aktories, K., Giry, M., Popoff, M. R., Boquet, P., Hegenbarth, S. and von Eichel-Streiber, C. 1994. Clostridium difficile toxin B acts on the GTP-binding protein Rho. J. Biol. Chem. 269, 10706-10712.
  11. Just, I., Selzer, J., von Eichel-Streiber, C. and Aktories, K. 1995. The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile. J. Clin. Invest. 95, 1026-1031. https://doi.org/10.1172/JCI117747
  12. Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M. and Aktories, K. 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500-503. https://doi.org/10.1038/375500a0
  13. Just, I., Wilm, M., Selzer, J., Rex, G., von Eichel-Streiber, C., Mann, M. and Aktories, K. 1995. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J. Biol. Chem. 270, 13932-13936. https://doi.org/10.1074/jbc.270.23.13932
  14. Kanazawa, S., Tsunoda, T., Onuma, E., Majima, T., Kagiyama, M. and Kikuchi, K. 2001. VEGF, basic-FGF, and TGF-beta in Crohn's disease and ulcerative colitis: a novel mechanism of chronic intestinal inflammation. Am. J. Gastroenterol. 96, 822-828.
  15. Kelly, C. P. and LaMont, J. T. 1998. Clostridium difficile infection. Annu. Rev. Med. 49, 375-390. https://doi.org/10.1146/annurev.med.49.1.375
  16. Kelly, C. P., Pothoulakis, C. and LaMont, J. T. 1994. Clostridium difficile colitis. N. Engl. J. Med. 330, 257-262. https://doi.org/10.1056/NEJM199401273300406
  17. Kim, D. H., Hwang, J. S., Lee, I. H., Nam, S. T., Hong, J., Zhang, P., Lu, L. F., Lee, J., Seok, H., Pothoulakis, C., Lamont, J. T. and Kim, H. 2016. The insect peptide CopA3 increases colonic epithelial cell proliferation and mucosal barrier function to prevent inflammatory responses in the gut. J. Biol. Chem. 291, 3209-3223. https://doi.org/10.1074/jbc.M115.682856
  18. Kim, H., Kokkotou, E., Na, X., Rhee, S. H., Moyer, M. P., Pothoulakis, C. and Lamont, J. T. 2005. Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase. Gastroenterology 129, 1875-1888. https://doi.org/10.1053/j.gastro.2005.09.011
  19. Kim, H., Rhee, S. H., Kokkotou, E., Na, X., Savidge, T., Moyer, M. P., Pothoulakis, C. and LaMont, J. T. 2005. Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J. Biol. Chem. 280, 21237-21245. https://doi.org/10.1074/jbc.M413842200
  20. Kim, H., Rhee, S. H., Pothoulakis, C. and Lamont, J. T. 2009. Clostridium difficile toxin A binds colonocyte Src causing dephosphorylation of focal adhesion kinase and paxillin. Exp. Cell. Res. 315, 3336-3344 https://doi.org/10.1016/j.yexcr.2009.05.020
  21. Kim, H., Rhee, S. H., Pothoulakis, C. and Lamont, J. T. 2007. Inflammation and apoptosis in Clostridium difficile enteritis is mediated by PGE2 up-regulation of Fas ligand. Gastroenterology 133, 875-886. https://doi.org/10.1053/j.gastro.2007.06.063
  22. Kim, Y., Kugler, M. C., Wei, Y., Kim, K. K., Li, X., Brumwell, A. N. and Chapman, H. A. 2009. Integrin alpha3beta1-dependent beta-catenin phosphorylation links epithelial Smad signaling to cell contacts. J. Cell. Biol. 184, 309-322. https://doi.org/10.1083/jcb.200806067
  23. Liu, X., Wu, S., Xia, Y., Li, X. E., Zhou, Z. D. and Sun, J. 2011. Wingless homolog Wnt11 suppresses bacterial invasion and inflammation in intestinal epithelial cells. Am. J. Physiol. Gastrointest Liver Physiol. 301, G992-G1003. https://doi.org/10.1152/ajpgi.00080.2011
  24. Na, X., Zhao, D., Koon, H. W., Kim, H., Husmark, J., Moyer, M. P., Pothoulakis, C. and LaMont, J. T. 2005. Clostridium difficile toxin B activates the EGF receptor and the ERK/ MAP kinase pathway in human colonocytes. Gastroenterology 128, 1002-1011. https://doi.org/10.1053/j.gastro.2005.01.053
  25. Nam, H. J., Kang, J. K., Kim, S. K., Ahn, K. J., Seok, H., Park, S. J., Chang, J. S., Pothoulakis, C., Lamont, J. T. and Kim, H. 2010. Clostridium difficile toxin A decreases acetylation of tubulin, leading to microtubule depolymerization through activation of histone deacetylase 6, and this mediates acute inflammation. J. Biol. Chem. 285, 32888-32896. https://doi.org/10.1074/jbc.M110.162743
  26. Resar, L., Chia, L. and Xian, L. 2018. Lessons from the Crypt: HMGA1-Amping up Wnt for Stem Cells and Tumor Progression. Cancer Res. 78, 1890-1897. https://doi.org/10.1158/0008-5472.CAN-17-3045
  27. Riegler, M., Sedivy, R., Sogukoglu, T., Castagliuolo, I., Pothoulakis, C., Cosentini, E., Bischof, G., Hamilton, G., Teleky, B., Feil, W., Lamont, J. T. and Wenzl, E. 1997. Epidermal growth factor attenuates Clostridium difficile toxin A- and B-induced damage of human colonic mucosa. Am. J. Physiol. 273, G1014-G1022.
  28. Uchiyama, K., Sakiyama, T., Hasebe, T., Musch, M. W., Miyoshi, H., Nakagawa, Y., He, T. C., Lichtenstein, L., Naito, Y., Itoh, Y., Yoshikawa, T., Jabri, B., Stappenbeck, T. and Chang, E. B. 2016. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development. Sci. Rep. 6, 32094. https://doi.org/10.1038/srep32094
  29. Wang, C. Y., Yang, T. T., Chen, C. L., Lin, W. C. and Lin, C. F. 2014. Reactive oxygen species-regulated glycogen synthase kinase-3beta activation contributes to all-trans retinoic acid-induced apoptosis in granulocyte-differentiated HL60 cells. Biochem. Pharmacol. 88, 86-94. https://doi.org/10.1016/j.bcp.2013.12.021
  30. Zhang, X., Yang, M., Shi, H., Hu, J., Wang, Y., Sun, Z. and Xu, S. 2017. Reduced E-cadherin facilitates renal cell carcinoma progression by WNT/beta-catenin signaling activation. Oncotarget 8, 19566-19576.