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

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Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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References

  1. Karam SM, Leblond CP. Identifying and counting epithelial cell types in the “corpus” of the mouse stomach. Anat Rec. 1992;232:231–46.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  3. Bjerknes M, Cheng H. Multipotential stem cells in adult mouse gastric epithelium. Am J Physiol Gastrointest Liver Physiol. 2002;283:G767–77.

    PubMed  CAS  Google Scholar 

  4. Brittan M, Wright NA. The gastrointestinal stem cell. Cell Prolif. 2004;37:35–53.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  10. Visser JW, Van Bekkum DW. Purification of pluripotent hemopoietic stem cells: past and present. Exp Hematol. 1990;18:248–56.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  15. Fan XG, Kelleher D, Fan XJ, Xia HX, Keeling PW. Helicobacter pylori increases proliferation of gastric epithelial cells. Gut. 1996;38:19–22.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  22. Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261–91.

    Article  PubMed  CAS  Google Scholar 

  23. Satyanarayana A, Kaldis P. Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene. 2009;28:2925–39.

    Article  PubMed  CAS  Google Scholar 

  24. Sherr CJ, Roberts JM. Living with or without cyclins and cyclin-dependent kinases. Genes Dev. 2004;18:2699–711.

    Article  PubMed  CAS  Google Scholar 

  25. Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases. Trends Biochem Sci. 2005;30:630–41.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  28. Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–91.

    Article  PubMed  CAS  Google Scholar 

  29. Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997;390:465–71.

    Article  PubMed  CAS  Google Scholar 

  30. Wrana JL. Crossing Smads. Sci STKE. 2000;2000:re1.

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  35. Matsuzaki K. Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis. Histol Histopathol. 2006;21:645–62.

    PubMed  CAS  Google Scholar 

  36. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–84.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  45. Karam SM. Lineage commitment and maturation of epithelial cells in the gut. Front Biosci. 1999;4:D286–98.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  47. Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet. 2008;40:915–20.

    Article  PubMed  CAS  Google Scholar 

  48. Karam SM, Straiton T, Hassan WM, Leblond CP. Defining epithelial cell progenitors in the human oxyntic mucosa. Stem Cells. 2003;21:322–36.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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 

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Acknowledgments

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.

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Correspondence to Toshiro Fukui.

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Fukui, T., Kishimoto, M., Nakajima, A. et al. The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis. J Gastroenterol 46, 456–468 (2011). https://doi.org/10.1007/s00535-010-0364-8

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  • DOI: https://doi.org/10.1007/s00535-010-0364-8

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