Journal of Gastroenterology

, Volume 48, Issue 2, pp 193–202 | Cite as

Hepatocyte growth factor stimulates the migration of gastric epithelial cells by altering the subcellular localization of the tight junction protein ZO-1

  • Yuichiro Nasu
  • Akio IdoEmail author
  • Shirou Tanoue
  • Shinichi Hashimoto
  • Fumisato Sasaki
  • Shuji Kanmura
  • Hitoshi Setoyama
  • Masatsugu Numata
  • Keita Funakawa
  • Akihiro Moriuchi
  • Hiroshi Fujita
  • Toshio Sakiyama
  • Hirofumi Uto
  • Makoto Oketani
  • Hirohito Tsubouchi
Original Article—Alimentary Tract



Hepatocyte growth factor (HGF) is essential for epithelial restitution, a process in which epithelial cells rapidly migrate to cover desquamated epithelium after mucosal injury in the gastrointestinal tract. In this study, we aimed to elucidate the molecular mechanisms of the HGF-mediated reconstitution of gastric epithelial structures by analyzing the expression and subcellular dynamics of tight junction proteins.


We treated human gastric epithelial MKN74 cells with HGF, and examined the effects of HGF on cell migration and proliferation, and the expression and subcellular dynamics of tight junction proteins; as well, we investigated the effect of HGF on paracellular permeability to macromolecules (using fluorescein isothiocyanate [FITC]-dextran).


HGF significantly stimulated the migration of MKN74 cells, but not their proliferation, in a dose-dependent manner. HGF did not affect the expression of tight junction proteins, including claudin-1, -3, -4 and -7; occludin; and zonula occludens (ZO)-1. However, fluorescence immunostaining revealed that, in the cell membrane, the levels of ZO-1, but not those of occludin or claudin-4, were transiently decreased 1 h after HGF treatment. The results were further confirmed by western blotting: HGF reduced the amount of ZO-1 protein in the cell membrane fraction concomitantly with an increase in cytoplasmic ZO-1. Furthermore, HGF reduced the interaction between ZO-1 and occludin, and induced the tyrosine phosphorylation of occludin, whereas the phosphorylation status of ZO-1 was not affected by exposure to HGF. Despite a decrease in the ZO-1/occludin interaction, HGF did not affect paracellular permeability to macromolecules.


HGF alters the subcellular localization of ZO-1, probably through the tyrosine phosphorylation of occludin, which may induce cell dispersion during epithelial restitution.


HGF Migration Epithelial restitution Mucosal injury Tight junction 



The authors thank Ms. Yuko Morinaga-Nakamura for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and by Grants-in-Aid from the Ministry of Health, Labour and Welfare of Japan.

Conflicts of interest

The authors declare no conflicts of interest.


  1. 1.
    Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama O, Takahashi K, et al. Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J Clin Invest. 1988;81:414–9.PubMedCrossRefGoogle Scholar
  2. 2.
    Igawa T, Kanda S, Kanetake H, Saitoh Y, Ichihara A, Tomita Y, et al. Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells. Biochem Biophys Res Commun. 1991;174:831–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Joplin R, Hishida T, Tsubouchi H, Daikuhara Y, Ayres R, Neuberger JM, et al. Human intrahepatic biliary epithelial cells proliferate in vitro in response to human hepatocyte growth factor. J Clin Invest. 1992;90:1284–9.PubMedCrossRefGoogle Scholar
  4. 4.
    Takahashi M, Ota S, Terano A, Yoshiura K, Matsumura M, Niwa Y, et al. Hepatocyte growth factor induces mitogenic reaction to the rabbit gastric epithelial cells in primary culture. Biochem Biophys Res Commun. 1993;191:528–34.PubMedCrossRefGoogle Scholar
  5. 5.
    Takahashi M, Ota S, Ogura K, Nakamura T, Omata M. Hepatocyte growth factor stimulates wound repair of the rabbit esophageal epithelial cells in primary culture. Biochem Biophys Res Commun. 1995;216:298–305.PubMedCrossRefGoogle Scholar
  6. 6.
    Hori T, Ido A, Uto H, Hasuike S, Moriuchi A, Hayashi K, et al. Activation of hepatocyte growth factor in monkey stomach following gastric mucosal injury. J Gastroenterol. 2004;39:133–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Takahashi M, Ota S, Shimada T, Hamada E, Kawabe T, Okudaira T, et al. Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture. J Clin Invest. 1995;95:1994–2003.PubMedCrossRefGoogle Scholar
  8. 8.
    Ido A, Numata M, Kodama M, Tsubouchi H. Mucosal repair and growth factors: recombinant human hepatocyte growth factor as an innovative therapy for inflammatory bowel disease. J Gastroenterol. 2005;40:925–31.PubMedCrossRefGoogle Scholar
  9. 9.
    Nusrat A, Delp C, Madara J. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest. 1992;89:1501–11.PubMedCrossRefGoogle Scholar
  10. 10.
    Dignass A, Lynch-Devaney K, Podolsky D. Hepatocyte growth factor/scatter factor modulates intestinal epithelial cell proliferation and migration. Biochem Biophys Res Commun. 1994;202:701–9.PubMedCrossRefGoogle Scholar
  11. 11.
    Itoh H, Naganuma S, Takeda N, Miyata S, Uchinokura S, Fukushima T, et al. Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice. Gastroenterology. 2004;127:1423–35.PubMedCrossRefGoogle Scholar
  12. 12.
    Anderson JM, Van Itallie CM, Fanning AS. Setting up a selective barrier at the apical junction complex. Curr Opin Cell Biol. 2004;16:140–5.PubMedCrossRefGoogle Scholar
  13. 13.
    Matter K, Balda MS. Functional analysis of tight junctions. Methods. 2003;30:228–34.PubMedCrossRefGoogle Scholar
  14. 14.
    D’Atri F, Citi S. Molecular complexity of vertebrate tight junctions. Mol Membr Biol. 2002;19:103–12.PubMedCrossRefGoogle Scholar
  15. 15.
    Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE. Tight junction proteins. Prog Biophys Mol Biol. 2003;81:1–44.PubMedCrossRefGoogle Scholar
  16. 16.
    Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Physiol. 2004;286:C1213–28.PubMedCrossRefGoogle Scholar
  17. 17.
    Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;123:1777–88.PubMedCrossRefGoogle Scholar
  18. 18.
    Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141:1539–50.PubMedCrossRefGoogle Scholar
  19. 19.
    Martìn-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, et al. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998;142:117–27.PubMedCrossRefGoogle Scholar
  20. 20.
    Feldman GJ, Mullin JM, Ryan MP. Occludin: structure, function and regulation. Adv Drug Deliv Rev. 2005;57:883–917.PubMedCrossRefGoogle Scholar
  21. 21.
    Seth A, Sheth P, Elias BC, Rao R. Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer. J Biol Chem. 2007;282:11487–98.PubMedCrossRefGoogle Scholar
  22. 22.
    Sheth P, Basuroy S, Li C, Naren AP, Rao RK. Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem. 2003;278:49239–45.PubMedCrossRefGoogle Scholar
  23. 23.
    Mineta K, Yamamoto Y, Yamazaki Y, Tanaka H, Tada Y, Saito K, et al. Predicted expansion of the claudin multigene family. FEBS Lett. 2011;585:606–12.PubMedCrossRefGoogle Scholar
  24. 24.
    Matter K, Aijaz S, Tsapara A, Balda MS. Mammalian tight junctions in the regulation of epithelial differentiation and proliferation. Curr Opin Cell Biol. 2005;17:453–8.PubMedCrossRefGoogle Scholar
  25. 25.
    Gonzalez-Mariscal L, Betanzos A, Avila-Flores A. MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol. 2000;11:315–24.PubMedCrossRefGoogle Scholar
  26. 26.
    Ide N, Hata Y, Nishioka H, Hirao K, Yao I, Deguchi M, et al. Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells. Oncogene. 1999;18:7810–5.PubMedCrossRefGoogle Scholar
  27. 27.
    Stevenson B, Siliciano J, Mooseker M, Goodenough D. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol. 1986;103:755–66.PubMedCrossRefGoogle Scholar
  28. 28.
    Jesaitis LA, Goodenough DA. Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J Cell Biol. 1994;124:949–61.PubMedCrossRefGoogle Scholar
  29. 29.
    Haskins J, Gu L, Wittchen ES, Hibbard J, Stevenson BR. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol. 1998;141:199–208.PubMedCrossRefGoogle Scholar
  30. 30.
    Fanning A, Jameson B, Jesaitis L, Anderson J. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem. 1998;273:29745–53.PubMedCrossRefGoogle Scholar
  31. 31.
    Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol. 1999;147:1351–63.PubMedCrossRefGoogle Scholar
  32. 32.
    Huo L, Wen W, Wang R, Kam C, Xia J, Feng W, et al. Cdc42-dependent formation of the ZO-1/MRCKbeta complex at the leading edge controls cell migration. EMBO J. 2011;30:665–78.PubMedCrossRefGoogle Scholar
  33. 33.
    Tuomi S, Mai A, Nevo J, Laine JO, Vilkki V, Ohman TJ et al. PKCepsilon regulation of an alpha5 integrin-ZO-1 complex controls lamellae formation in migrating cancer cells. Sci Signal. 2009;2:ra32.Google Scholar
  34. 34.
    Du D 29, Xu F, Yu L, Zhang C, Lu X, Yuan H et al. The tight junction protein, occludin, regulates the directional migration of epithelial cells. Dev Cell. 2010;18:52–63.Google Scholar
  35. 35.
    Agarwal R, Mori Y, Cheng Y, Jin Z, Olaru AV, Hamilton JP, et al. Silencing of claudin-11 is associated with increased invasiveness of gastric cancer cells. PLoS ONE. 2009;4:e8002.PubMedCrossRefGoogle Scholar
  36. 36.
    Ikari A, Sato T, Takiguchi A, Atomi K, Yamazaki Y, Sugatani J. Caudin-2 knockdown decreases matrix metalloproteinase-9 activity and cell migration via suppression of nuclear Sp1 in A549 cells. Life Sci. 2011;88:628–33.PubMedCrossRefGoogle Scholar
  37. 37.
    Lapointe TK, Buret AG. Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of occludin, without altering epithelial permeability. Am J Physiol Gastrointest Liver Physiol. 2012;302:G343–51.PubMedCrossRefGoogle Scholar
  38. 38.
    Chen HC. Boyden chamber assay. Methods Mol Biol. 2005;294:15–22.PubMedGoogle Scholar
  39. 39.
    Hewitt K, Agarwal R, Morin PJ. The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer. 2006;6:186.PubMedCrossRefGoogle Scholar
  40. 40.
    Rao RK, Basuroy S, Rao VU, Karnaky KJ, Gupta A. Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress. Biochem J. 2002;369:471–81.CrossRefGoogle Scholar
  41. 41.
    Singh AB, Harris RC. Epidermal growth factor receptor activation differentially regulates Claudin expression and enhances transcriptional resistance in Madin–Darby Canine Kidney cells. J Biol Chem. 2004;279:3543–52.PubMedCrossRefGoogle Scholar
  42. 42.
    Jin M, Barron E, He S, Ryan S, Hinton D. Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor. Invest Ophthalmol Vis Sci. 2002;43:2782–90.PubMedGoogle Scholar
  43. 43.
    Schmidt A, Utepbergenov D, Mueller S, Beyermann M, Schneider-Mergener J, Krause G, et al. Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation. Cell Mol Life Sci. 2004;61:1354–65.PubMedCrossRefGoogle Scholar
  44. 44.
    Jiang W, Martin T, Matsumoto K, Nakamura T, Mansel R. Hepatocyte growth factor/scatter factor decreases the expression of occludin and transendothelial resistance (TER) and increases paracellular permeability in human vascular endothelial cells. J Cell Physiol. 1999;181:319–29.PubMedCrossRefGoogle Scholar
  45. 45.
    Sanada Y, Oue N, Mitani Y, Yoshida K, Nakayama H, Yasui W. Down-regulation of the claudin-18 gene, identified through serial analysis of gene expression data analysis, in gastric cancer with an intestinal phenotype. J Pathol. 2006;208:633–42.PubMedCrossRefGoogle Scholar
  46. 46.
    Tsukita S, Katsuno T, Yamazaki Y, Umeda K, Tamura A. Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function. Ann N Y Acad Sci. 2009;1165:44–52.PubMedCrossRefGoogle Scholar
  47. 47.
    Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol. 2001;2:285–93.PubMedCrossRefGoogle Scholar
  48. 48.
    Katsuno T, Umeda K, Matsui T, Hata M, Tamura A, Itoh M, et al. Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells. Mol Biol Cell. 2008;19:2465–75.PubMedCrossRefGoogle Scholar
  49. 49.
    Krueger S, Hundermark T, Kuester D, Kalinski T, Peitz U, Roessner A. Helicobacter pylori alters the distribution of ZO-1 and p120ctn in primary human gastric epithelial cells. Pathol Res Pract. 2007;203:433–44.PubMedCrossRefGoogle Scholar
  50. 50.
    Royal I, Lamarche-Vane N, Lamorte L, Kaibuchi K, Park M. Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol Biol Cell. 2000;11:1709–25.PubMedGoogle Scholar
  51. 51.
    Osanai M, Murata M, Nishikiori N, Chiba H, Kojima T, Sawada N. Occludin-mediated premature senescence is a fail-safe mechanism against tumorigenesis in breast carcinoma cells. Cancer Sci. 2007;98:1027–34.PubMedCrossRefGoogle Scholar
  52. 52.
    Elias BC, Suzuki T, Seth A, Giorgianni F, Kale G, Shen L, et al. Phosphorylation of Tyr-398 and Tyr-402 in occludin prevents its interaction with ZO-1 and destabilizes its assembly at the tight junctions. J Biol Chem. 2009;284:1559–69.PubMedCrossRefGoogle Scholar
  53. 53.
    Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol. 1997;137:1393–401.PubMedCrossRefGoogle Scholar
  54. 54.
    Basuroy S, Sheth P, Kuppuswamy D, Balasubramanian S, Ray RM, Rao RK. Expression of kinase-inactive c-Src delays oxidative stress-induced disassembly and accelerates calcium-mediated reassembly of tight junctions in the Caco-2 cell monolayer. J Biol Chem. 2003;278:11916–24.PubMedCrossRefGoogle Scholar
  55. 55.
    Kale G, Naren A, Sheth P, Rao R. Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3. Biochem Biophys Res Commun. 2003;302:324–9.PubMedCrossRefGoogle Scholar
  56. 56.
    Grisendi S, Arpin M, Crepaldi T. Effect of hepatocyte growth factor on assembly of zonula occludens-1 protein at the plasma membrane. J Cell Physiol. 1998;176:465–71.PubMedCrossRefGoogle Scholar
  57. 57.
    Hollande F, Blanc E, Bali J, Whitehead R, Pelegrin A, Baldwin G, et al. HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line. Am J Physiol Gastrointest Liver Physiol. 2001;280:G910–21.PubMedGoogle Scholar
  58. 58.
    Royal I, Fournier T, Park M. Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis. J Cell Physiol. 1997;173:196–201.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2012

Authors and Affiliations

  • Yuichiro Nasu
    • 1
  • Akio Ido
    • 1
    Email author
  • Shirou Tanoue
    • 1
  • Shinichi Hashimoto
    • 1
  • Fumisato Sasaki
    • 1
  • Shuji Kanmura
    • 1
  • Hitoshi Setoyama
    • 1
  • Masatsugu Numata
    • 1
  • Keita Funakawa
    • 1
  • Akihiro Moriuchi
    • 1
  • Hiroshi Fujita
    • 1
  • Toshio Sakiyama
    • 1
  • Hirofumi Uto
    • 1
  • Makoto Oketani
    • 1
  • Hirohito Tsubouchi
    • 1
  1. 1.Digestive Disease and Life-style Related DiseaseKagoshima University Graduate School of Medical and Dental SciencesKagoshimaJapan

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