Journal of Gastroenterology

, Volume 46, Issue 4, pp 456–468 | Cite as

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis

  • Toshiro FukuiEmail author
  • Masanobu Kishimoto
  • Atsushi Nakajima
  • Masao Yamashina
  • Shinji Nakayama
  • Takeo Kusuda
  • Yutaku Sakaguchi
  • Katsunori Yoshida
  • Kazushige Uchida
  • Akiyoshi Nishio
  • Koichi Matsuzaki
  • Kazuichi Okazaki
Original Article—Alimentary Tract



The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells.


Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin–eosin and observed these cells under a light microscope.


In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist.


We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.


Linker phosphorylation Smad2/3 Helicobacter-associated gastritis Epithelial stem cells 



This study was supported by Grants-in-Aid for Scientific Research (20790516 and 22790676) from the Japan Society for the Promotion of Science (JSPS) and by Intractable Diseases Health and Labor Sciences Research Grants (to K.O.) from the Ministry of Labor and Welfare of Japan.


  1. 1.
    Karam SM, Leblond CP. Identifying and counting epithelial cell types in the “corpus” of the mouse stomach. Anat Rec. 1992;232:231–46.PubMedCrossRefGoogle Scholar
  2. 2.
    Lee ER, Trasler J, Dwivedi S, Leblond CP. Division of the mouse gastric mucosa into zymogenic and mucous regions on the basis of gland features. Am J Anat. 1982;164:187–207.PubMedCrossRefGoogle Scholar
  3. 3.
    Bjerknes M, Cheng H. Multipotential stem cells in adult mouse gastric epithelium. Am J Physiol Gastrointest Liver Physiol. 2002;283:G767–77.PubMedGoogle Scholar
  4. 4.
    Brittan M, Wright NA. The gastrointestinal stem cell. Cell Prolif. 2004;37:35–53.PubMedCrossRefGoogle Scholar
  5. 5.
    Hattori T. On cell proliferation and differentiation of the fundic mucosa of the golden hamster. Fractographic study combined with microscopy and 3H-thymidine autoradiography. Cell Tissue Res. 1974;148:213–26.PubMedCrossRefGoogle Scholar
  6. 6.
    Karam SM, Hassan WM, John R. Expression of retinoid receptors in multiple cell lineages in the gastric mucosae of mice and humans. J Gastroenterol Hepatol. 2005;20:1892–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Lee ER, Leblond CP. Dynamic histology of the antral epithelium in the mouse stomach: II. Ultrastructure and renewal of isthmal cells. Am J Anat. 1985;172:205–24.PubMedCrossRefGoogle Scholar
  8. 8.
    Karam SM, Leblond CP. Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cell. Anat Rec. 1993;236:259–79.PubMedCrossRefGoogle Scholar
  9. 9.
    Watt FM. Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci. 1998;353:831–7.PubMedCrossRefGoogle Scholar
  10. 10.
    Visser JW, Van Bekkum DW. Purification of pluripotent hemopoietic stem cells: past and present. Exp Hematol. 1990;18:248–56.PubMedGoogle Scholar
  11. 11.
    Chen S, Takahara M, Kido M, Takeuchi S, Uchi H, Tu Y, et al. Increased expression of an epidermal stem cell marker, cytokeratin 19, in cutaneous squamous cell carcinoma. Br J Dermatol. 2008;159:952–5.PubMedCrossRefGoogle Scholar
  12. 12.
    Hall PA, Coates PJ, Ansari B, Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci. 1994;107(Pt 12):3569–77.PubMedGoogle Scholar
  13. 13.
    Brenes F, Ruiz B, Correa P, Hunter F, Rhamakrishnan T, Fontham E, et al. Helicobacter pylori causes hyperproliferation of the gastric epithelium: pre- and post-eradication indices of proliferating cell nuclear antigen. Am J Gastroenterol. 1993;88:1870–5.PubMedGoogle Scholar
  14. 14.
    Cahill RJ, Xia H, Kilgallen C, Beattie S, Hamilton H, O’Morain C. Effect of eradication of Helicobacter pylori infection on gastric epithelial cell proliferation. Dig Dis Sci. 1995;40:1627–31.PubMedCrossRefGoogle Scholar
  15. 15.
    Fan XG, Kelleher D, Fan XJ, Xia HX, Keeling PW. Helicobacter pylori increases proliferation of gastric epithelial cells. Gut. 1996;38:19–22.PubMedCrossRefGoogle Scholar
  16. 16.
    Fox JG, Blanco M, Murphy JC, Taylor NS, Lee A, Kabok Z, et al. Local and systemic immune responses in murine Helicobacter felis active chronic gastritis. Infect Immun. 1993;61:2309–15.PubMedGoogle Scholar
  17. 17.
    Fraser AG, Sim R, Sankey EA, Dhillon AP, Pounder RE. Effect of eradication of Helicobacter pylori on gastric epithelial cell proliferation. Aliment Pharmacol Ther. 1994;8:167–73.PubMedCrossRefGoogle Scholar
  18. 18.
    Fukui T, Nishio A, Okazaki K, Kasahara K, Saga K, Tanaka J, et al. Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells. Helicobacter. 2007;12:486–97.PubMedCrossRefGoogle Scholar
  19. 19.
    Hibi K, Mitomi H, Koizumi W, Tanabe S, Saigenji K, Okayasu I. Enhanced cellular proliferation and p53 accumulation in gastric mucosa chronically infected with Helicobacter pylori. Am J Clin Pathol. 1997;108:26–34.PubMedGoogle Scholar
  20. 20.
    Lynch DA, Mapstone NP, Clarke AM, Sobala GM, Jackson P, Morrison L, et al. Cell proliferation in Helicobacter pylori associated gastritis and the effect of eradication therapy. Gut. 1995;36:346–50.PubMedCrossRefGoogle Scholar
  21. 21.
    Panella C, Ierardi E, Polimeno L, Balzano T, Ingrosso M, Amoruso A, et al. Proliferative activity of gastric epithelium in progressive stages of Helicobacter pylori infection. Dig Dis Sci. 1996;41:1132–8.PubMedCrossRefGoogle Scholar
  22. 22.
    Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261–91.PubMedCrossRefGoogle Scholar
  23. 23.
    Satyanarayana A, Kaldis P. Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene. 2009;28:2925–39.PubMedCrossRefGoogle Scholar
  24. 24.
    Sherr CJ, Roberts JM. Living with or without cyclins and cyclin-dependent kinases. Genes Dev. 2004;18:2699–711.PubMedCrossRefGoogle Scholar
  25. 25.
    Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases. Trends Biochem Sci. 2005;30:630–41.PubMedCrossRefGoogle Scholar
  26. 26.
    Susaki E, Nakayama K, Nakayama KI. Cyclin D2 translocates p27 out of the nucleus and promotes its degradation at the G0–G1 transition. Mol Cell Biol. 2007;27:4626–40.PubMedCrossRefGoogle Scholar
  27. 27.
    Matsuura I, Denissova NG, Wang G, He D, Long J, Liu F. Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature. 2004;430:226–31.PubMedCrossRefGoogle Scholar
  28. 28.
    Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–91.PubMedCrossRefGoogle Scholar
  29. 29.
    Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997;390:465–71.PubMedCrossRefGoogle Scholar
  30. 30.
    Wrana JL. Crossing Smads. Sci STKE. 2000;2000:re1.Google Scholar
  31. 31.
    Mori S, Matsuzaki K, Yoshida K, Furukawa F, Tahashi Y, Yamagata H, et al. TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions. Oncogene. 2004;23:7416–29.PubMedCrossRefGoogle Scholar
  32. 32.
    Kretzschmar M, Doody J, Timokhina I, Massague J. A mechanism of repression of TGFbeta/Smad signaling by oncogenic Ras. Genes Dev. 1999;13:804–16.PubMedCrossRefGoogle Scholar
  33. 33.
    Sapkota G, Knockaert M, Alarcon C, Montalvo E, Brivanlou AH, Massague J. Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways. J Biol Chem. 2006;281:40412–9.PubMedCrossRefGoogle Scholar
  34. 34.
    Matsuzaki K, Kitano C, Murata M, Sekimoto G, Yoshida K, Uemura Y, et al. Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer. Cancer Res. 2009;69:5321–30.PubMedCrossRefGoogle Scholar
  35. 35.
    Matsuzaki K. Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis. Histol Histopathol. 2006;21:645–62.PubMedGoogle Scholar
  36. 36.
    Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–84.PubMedCrossRefGoogle Scholar
  37. 37.
    Sekimoto G, Matsuzaki K, Yoshida K, Mori S, Murata M, Seki T, et al. Reversible Smad-dependent signaling between tumor suppression and oncogenesis. Cancer Res. 2007;67:5090–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Murata M, Matsuzaki K, Yoshida K, Sekimoto G, Tahashi Y, Mori S, et al. Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B. Hepatology. 2009;49:1203–17.PubMedCrossRefGoogle Scholar
  39. 39.
    Furukawa F, Matsuzaki K, Mori S, Tahashi Y, Yoshida K, Sugano Y, et al. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology. 2003;38:879–89.PubMedGoogle Scholar
  40. 40.
    Zhang Y, Huang X. Investigation of doublecortin and calcium/calmodulin-dependent protein kinase-like-1-expressing cells in the mouse stomach. J Gastroenterol Hepatol. 2010;25:576–82.PubMedCrossRefGoogle Scholar
  41. 41.
    Okumura T, Ericksen RE, Takaishi S, Wang SS, Dubeykovskiy Z, Shibata W, et al. K-ras mutation targeted to gastric tissue progenitor cells results in chronic inflammation, an altered microenvironment, and progression to intraepithelial neoplasia. Cancer Res. 2010;70:8435–45.PubMedCrossRefGoogle Scholar
  42. 42.
    Kikuchi M, Nagata H, Watanabe N, Watanabe H, Tatemichi M, Hibi T. Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia. BMC Gastroenterol. 2010;10:65.PubMedGoogle Scholar
  43. 43.
    Giannakis M, Stappenbeck TS, Mills JC, Leip DG, Lovett M, Clifton SW, et al. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches. J Biol Chem. 2006;281:11292–300.PubMedCrossRefGoogle Scholar
  44. 44.
    Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459:262–5.PubMedCrossRefGoogle Scholar
  45. 45.
    Karam SM. Lineage commitment and maturation of epithelial cells in the gut. Front Biosci. 1999;4:D286–98.PubMedCrossRefGoogle Scholar
  46. 46.
    Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–7.PubMedCrossRefGoogle Scholar
  47. 47.
    Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet. 2008;40:915–20.PubMedCrossRefGoogle Scholar
  48. 48.
    Karam SM, Straiton T, Hassan WM, Leblond CP. Defining epithelial cell progenitors in the human oxyntic mucosa. Stem Cells. 2003;21:322–36.PubMedCrossRefGoogle Scholar
  49. 49.
    Qiao XT, Ziel JW, McKimpson W, Madison BB, Todisco A, Merchant JL, et al. Prospective identification of a multilineage progenitor in murine stomach epithelium. Gastroenterology. 2007;133:1989–98.PubMedCrossRefGoogle Scholar
  50. 50.
    Weidner N, Moore DH 2nd, Vartanian R. Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel “paraffin”-reactive MIB1 antibody. Hum Pathol. 1994;25:337–42.PubMedCrossRefGoogle Scholar
  51. 51.
    Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell. 1991;65:701–13.PubMedCrossRefGoogle Scholar
  52. 52.
    Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993;7:812–21.PubMedCrossRefGoogle Scholar
  53. 53.
    Furukawa Y, Kikuchi J, Nakamura M, Iwase S, Yamada H, Matsuda M. Lineage-specific regulation of cell cycle control gene expression during haematopoietic cell differentiation. Br J Haematol. 2000;110:663–73.PubMedCrossRefGoogle Scholar
  54. 54.
    Snippert HJ, van Es JH, van den Born M, Begthel H, Stange DE, Barker N, et al. Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine. Gastroenterology. 2009;136:2187–94. e1.Google Scholar

Copyright information

© Springer 2010

Authors and Affiliations

  • Toshiro Fukui
    • 1
    Email author
  • Masanobu Kishimoto
    • 1
  • Atsushi Nakajima
    • 1
  • Masao Yamashina
    • 1
  • Shinji Nakayama
    • 1
  • Takeo Kusuda
    • 1
  • Yutaku Sakaguchi
    • 1
  • Katsunori Yoshida
    • 1
  • Kazushige Uchida
    • 1
  • Akiyoshi Nishio
    • 1
  • Koichi Matsuzaki
    • 1
  • Kazuichi Okazaki
    • 1
  1. 1.The Third Department of Internal Medicine, Division of Gastroenterology and HepatologyKansai Medical UniversityMoriguchiJapan

Personalised recommendations