Involvement of eIF2α in halofuginone-driven inhibition of TGF-β1-induced EMT

Abstract

Halofuginone (HF) is an extract from the widely used traditional Chinese medicine (TCM) Dichroa febrifuga that facilitates the recovery of wounds and attenuates hepatic fibrosis. However, the role of HF in the epithelial-mesenchymal transition (EMT) of IPEC-J2 cells remains unclear. The current study explored the anti-EMT effect of HF in IPEC-J2 cells and illustrates its molecular mechanism. Transforming growth factor β1 (TGF-β1), as a recognized profibrogenic cytokine, decreased the level of the epithelial marker E-cadherin and increased the level of the mesenchymal markers, such as N-cadherin, fibronectin (FN), vimentin (Vim), and α-smooth muscle actin (α-SMA), in IPEC-J2 cells depending on the exposure time and dose. HF markedly prevented the EMT induced by TGF-β1. Dissection of the mechanism revealed that HF inhibited IPEC-J2 cell EMT via modulating the phosphorylation of SMAD2/3 and the SMAD2/3-SMAD4 complex nuclear translocation. Furthermore, HF could promote the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which modulates the SMAD signaling pathway. These results suggested that HF inhibits TGF-β1-induced EMT in IPEC-J2 cells through the eIF2α/SMAD signaling pathway. Our findings suggest that HF can serve as a potential anti-EMT agent in intestinal fibrosis therapy.

This is a preview of subscription content, access via your institution.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

References

  1. Agrawal M and Colombel JF 2019 Treat-to-target in inflammatory bowel diseases, what is the target and how do we treat? Gastrointest. Endoscopy Clin. 29 421–436

    Google Scholar 

  2. Batlle E, Sancho E, Franci C, Domínguez D, Monfar M, Josep Baulida J and de Herreros AG 2000 The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat. Cell Biol. 2 84–89

    CAS  PubMed  Google Scholar 

  3. Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D and Yuan J 2005 A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307 935–939

    CAS  PubMed  Google Scholar 

  4. Chen H, Zhang J and Liu W 2009 Experimental study of curcumin against schistosomiasis liver fibrosis and its mechanism. Chin. Traditional Herbal Drugs 08 1274–1277

    Google Scholar 

  5. Choi ET, Callow AD, Sehgal NL, Brown DM and Ryan US 1995 Halofuginone, a specific collagen type I inhibitor, reduces anastomotic intimal hyperplasia. Arch. Surg. 130 257–261

    CAS  PubMed  Google Scholar 

  6. Chu H, Khosravi A, Kusumawardhani IP, Kwon AH, Vasconcelos AC, Cunha LD, Mayer AE, Shen Y, et al. 2016 Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease. Science 352 1116–1120

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Cook JA, Choudhuri R, Degraff W, Gamson J and Mitchell JB 2010 Halofuginone enhances the radiation sensitivity of human tumor cell lines. Cancer Lett. 289 119–126

    CAS  PubMed  Google Scholar 

  8. Cuffari C 2006 Inflammatory bowel disease in children: a pediatrician’s perspective. Minerva Pediatr. 58 139–157

    CAS  PubMed  Google Scholar 

  9. Dabak H, Karlidag T, Akpolat N, Keles E, Alpay HC, Serin M, Kaygusuz I, Yalcin S and Isik O 2010 The effects of methylprednisolone and halofuginone on preventing esophageal and hypopharyngeal fibrosis in delivered radiotherapy. Eur. Arch. Otorhinolaryngol. 267 1429–1435

    PubMed  Google Scholar 

  10. Di Sabatino A, Jackson CL, Pickard KM, Buckley M, Rovedatti L, Leakey NA, Picariello L, Cazzola P, Monteleone G, Tonelli F, Corazza GR, MacDonald TT and Pender SL 2009 Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn’s disease strictures. Gut 58 777–789

    PubMed  Google Scholar 

  11. Fausther M and Dranoff JA 2015 Beyond scar formation: portal myofibroblast-mediated angiogenesis in the fibrotic liver. Hepatology 61 766–768

    PubMed  PubMed Central  Google Scholar 

  12. Fichtner-Feigl S, Young CA, Kitani A, Geissler EK, Schlitt HJ and Strober W 2008 IL-13 signaling via IL-13R alpha2 induces major downstream fibrogenic factors mediating fibrosis in chronic TNBS colitis. Gastroenterology 135 2003–2013

    CAS  PubMed  Google Scholar 

  13. Ge Y, Sun M, Wu W, Ma C, Zhang C, He C, Li J, Cong Y, Zhang D and Liu Z 2019 MicroRNA-125a suppresses intestinal mucosal inflammation through targeting ETS-1 in patients with inflammatory bowel diseases. J. Autoimmun. 101 109–120

    CAS  PubMed  Google Scholar 

  14. Geremia A, Biancheri P, Allan P, Corazza GR and Di Sabatino A 2014 Innate and adaptive immunity in inflammatory bowel disease. Autoimmun. Rev. 13 3–10

    CAS  PubMed  Google Scholar 

  15. Halevy O, Genin O, Barzilai-Tutsch H, Pima Y, Levi O, Moshe I and Pines M 2013 Inhibition of muscle fibrosis and improvement of muscle histopathology in dysferlin knock-out mice treated with halofuginone. Histol Histopathol. 28 211–226

    CAS  PubMed  Google Scholar 

  16. Hasegawa M, Matsushita Y, Horikawa M, Higashi K, Tomigahara Y., Kaneko H, Shirasaki F, Fujimoto M, Takehara K and Sato S 2009 A novel inhibitor of SMAD-dependent transcriptional activation suppresses tissue fibrosis in mouse models of systemic sclerosis. Arthritis Rheum. 60 3465–3475

    CAS  PubMed  Google Scholar 

  17. Ince MN and Elliott DE 2007 Immunologic and molecular mechanisms in inflammatory bowel disease. Surg. Clin. N. Am. 87 681–696

    PubMed  Google Scholar 

  18. Ishii H, Choudhuri R, Mathias A, Sowers AL, Flanders KC, Cook JA and Mitchell JB 2009 Halofuginone mediated protection against radiation-induced leg contracture. Int. J. Oncol. 35 315–319

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Juarez P, Mohammad KS, Yin JJ, Fournier PG, McKenna RC, Davis HW, Peng XH, Niewolna M, Javelaud D, Chirgwin JM, Mauviel A and Guise TA 2012 Halofuginone inhibits the establishment and progression of melanoma bone metastases. Cancer Res. 72 6247–6256

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kaimori A, Potter J, Kaimori J, Wang C, Mezey E and Koteish A 2007 Transforming growth factor-beta1 induces an epithelial-to-mesenchymal transition state in mouse hepatocytes in vitro. J. Biol. Chem. 282 22089–22101

    CAS  PubMed  Google Scholar 

  21. Keller TL, Zocco D, Sundrud MS, Hendrick M, Edenius M, Yum J, Kim YJ, Lee HK, Cortese JF, Wirth DF, Dignam JD, Rao A, Yeo CY, Mazitschek R and Whitman M 2012 Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat. Chem. Biol. 8 311–317

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Koohestani F, Qiang W, MacNeill A L, Druschitz SA, Serna VA, Adur M, Kurita T and Nowak RA 2016 Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model. Hum. Reprod. 31 1540–1551

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Krstic J, Trivanovic D, Mojsilovic S and Santibanez JF 2015 Transforming growth factor-beta and oxidative stress interplay implications in tumorigenesis and cancer progression. Oxid. Med. Cell Longev. 2015 654594

    PubMed  PubMed Central  Google Scholar 

  24. Latella G, Rogler G, Bamias G, Breynaert C, Florholmen J, Pellino G, Reif S, Speca S and Lawrance IC 2014 Results of the 4th scientific workshop of the ECCO (I): pathophysiology of intestinal fibrosis in IBD. J. Crohns Colitis 8 1147–1165

    PubMed  Google Scholar 

  25. Liang J, Zhang B, Shen RW, Liu JB, Gao MH, Li Y, Li YY and Zhang W 2013 Preventive effect of halofuginone on concanavalin A-induced liver fibrosis. PLoS One 8 e82232

    PubMed  PubMed Central  Google Scholar 

  26. Liu L, Zhu Y, Noë M, Li Q and Pasricha PJ 2018 Neuronal transforming growth factor beta signaling via SMAD3 contributes to pain in animal models of chronic pancreatitis. Gastroenterology 154 2252–2265

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Malik TA 2015 Inflammatory bowel disease: historical perspective, epidemiology, and risk factors. Surg. Clin. N. Am. 95 1105–1122

    PubMed  Google Scholar 

  28. McGonigle S, Beall MJ and Pearce EJ 2002 Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response. Biochemistry 41 579–587

    CAS  PubMed  Google Scholar 

  29. Mejlvang J, Kriajevska M, Berditchevski F, Bronstein I, Lukanidin EM, Pringle JH, Mellon JK and Tulchinsky EM 2007 Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition. Exp. Cell Res. 313 380–393

    CAS  PubMed  Google Scholar 

  30. Nawroth I, Alsner J, Behlke MA, Besenbacher F, Overgaard J, Howard KA and Kjems J 2010 Intraperitoneal administration of chitosan/DsiRNA nanoparticles targeting TNFalpha prevents radiation-induced fibrosis. Radiother. Oncol. 97 143–148

    CAS  PubMed  Google Scholar 

  31. Nelson EF, Huang CW, Ewel JM, Chang AA and Yuan C 2012 Halofuginone down-regulates SMAD3 expression and inhibits the TGFbeta-induced expression of fibrotic markers in human corneal fibroblasts. Mol. Vis. 18 479–487

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Nevo Y, Halevy O, Genin O, Moshe I, Turgeman T, Harel M, Biton E, Reif S and Pines M 2010 Fibrosis inhibition and muscle histopathology improvement in laminin-alpha2-deficient mice. Muscle Nerve 42 218–229

    PubMed  Google Scholar 

  33. Nimmons D and Limdi JK 2016 Elderly patients and inflammatory bowel disease. World J. Gastrointest. Pharmacol. Ther. 7 51–65

    PubMed  PubMed Central  Google Scholar 

  34. Opstelten JL, de Vries JHM, Wools A, Siersema PD, Oldenburg B and Witteman BJM 2019 Dietary intake of patients with inflammatory bowel disease: A comparison with individuals from a general population and associations with relapse. Clin. Nutr. 38 1892–1898

    PubMed  Google Scholar 

  35. Ravikumara M and Sandhu BK 2006 Epidemiology of inflammatory bowel diseases in childhood. Indian J. Pediatr. 73 717–721

    CAS  PubMed  Google Scholar 

  36. Robertson H, Kirby JA, Yip WW, Jones DE and Burt AD 2007 Biliary epithelial-mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis. Hepatology 45 977–981

    CAS  PubMed  Google Scholar 

  37. Rodriguez-Garcia A, Samso P, Fontova P, Simon-Molas H, Manzano A, Castaño E, Rosa JL, Martinez-Outshoorn U, Ventura F, Navarro-Sabaté À and Bartrons R 2017 TGF-beta1 targets SMAD, p38 MAPK, and PI3K/Akt signaling pathways to induce PFKFB3 gene expression and glycolysis in glioblastoma cells. FEBS J. 284 3437–3454

    CAS  PubMed  Google Scholar 

  38. Shimbori C, Bellaye PS, Xia J, Gauldie J, Ask K, Ramos C, Becerril C, Pardo A, Selman M and Kolb M 2016 Fibroblast growth factor-1 attenuates TGF-beta1-induced lung fibrosis. J. Pathol. 240 197–210

    CAS  PubMed  Google Scholar 

  39. Song D G, Kim D, Jung J W, Nam SH, Kim JE, Kim H, Kim JH, Lee S, Pan C, Kim S and Lee JW 2018 Glutamyl-prolyl-tRNA synthetase induces fibrotic extracellular matrix via both transcriptional and translational mechanisms. FASEB J. 33 4341–4354

    PubMed  Google Scholar 

  40. Song S, Gong Z and Zhang Q 2004 Therapeutic effect and mechanism of traditional Chinese compound decoction of Radix Curcumae, Rhizoma Sparganii, Rhizoma Zedoariae on fibrotic liver in rats. Chin. Traditional Herbal Drugs 03 59–62

    Google Scholar 

  41. Spector I, Honig H, Kawada N, Nagler A, Genin O and Pines M 2010 Inhibition of pancreatic stellate cell activation by halofuginone prevents pancreatic xenograft tumor development. Pancreas 39 1008–1015

    CAS  PubMed  Google Scholar 

  42. Spilka R, Ernst C, Mehta AK and Haybaeck J 2013 Eukaryotic translation initiation factors in cancer development and progression. Cancer Lett. 340 9–21

    CAS  PubMed  Google Scholar 

  43. Van Limbergen J, Geddes K, Henderson P, Russell RK, Drummond HE, Satsangi J, Griffiths AM, Philpott DJ and Wilson DC 2015 Paneth cell marker CD24 in NOD2 knockout organoids and in inflammatory bowel disease (IBD). Gut 64 353–354

    PubMed  Google Scholar 

  44. Wang P, Liu T, Cong M, Wu X, Bai Y, Yin C, An W, Wang B, Jia J and You H 2009 Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta? Liver Int. 29 575–584

    CAS  PubMed  Google Scholar 

  45. Wellner G, Mordechay S, Evans P, Genin O, Pines M and Halevy O 2019 Hydroxy group requirement for halofuginone-dependent inhibition of muscle fibrosis and improvement of histopathology in the mdx mouse model for Duchenne muscular dystrophy. Histol. Histopathol. 18083–18083

  46. Witte D, Otterbein H, Forster M, Giehl K, Zeiser R, Lehnert H and Ungefroren H 2017 Negative regulation of TGF-beta1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b. Sci. Rep. 7 17313

    PubMed  PubMed Central  Google Scholar 

  47. Yoon YH, Rha KS, Kim DH, Kim EH, Kim JM and Koo BS 2011 Is there any synergic effect for coadministration of mitomycin C and halofuginone on the skin wound healing? Am. J. Otolaryngol. 32 130–134

    CAS  PubMed  Google Scholar 

  48. Zavadil J, Bitzer M, Liang D, Yang YC, Massimi A, Kneitz S, Piek E and Bottinger EP 2001 Genetic programs of epithelial cell plasticity directed by transforming growth factor-beta. Proc. Natl. Acad. Sci. USA 98 6686–6691

    CAS  PubMed  Google Scholar 

  49. Zhou C, Ai L, Wang M and Wang X 2011 Protection of Eucommia ulmoides polysaccharide on liver fibrosis. Chin. Traditional Herbal Drugs 02 324–329

    Google Scholar 

Download references

Acknowledgements

This article was funded by the National Natural Science Foundation of China (No. 31702263, 31972715), the Innovative Project of Bai Nong Ying Cai for College Students of Henan University of Science and Technology in 2018 (No. BNYC2018-2-06) and the Key Specialized Research and Development Breakthrough program in Henan province (No. 202102110101, 182102110347).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Xiaojing Xia or Jianhe Hu.

Additional information

Corresponding editor: Kundan Sengupta

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Duan, M., Wei, X., Cheng, Z. et al. Involvement of eIF2α in halofuginone-driven inhibition of TGF-β1-induced EMT. J Biosci 45, 71 (2020). https://doi.org/10.1007/s12038-020-00042-5

Download citation

Keywords

  • Halofuginone
  • TGF-β1
  • EMT
  • eIF2α
  • IBD