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The Journal of Physiological Sciences

, Volume 66, Issue 1, pp 53–65 | Cite as

Effects of ezrin knockdown on the structure of gastric glandular epithelia

  • Saori Yoshida
  • Hiroto Yamamoto
  • Takahito Tetsui
  • Yuka Kobayakawa
  • Ryo Hatano
  • Ken-ichi Mukaisho
  • Takanori Hattori
  • Hiroyuki Sugihara
  • Shinji AsanoEmail author
Original Paper

Abstract

Ezrin, an adaptor protein that cross-links plasma membrane-associated proteins with the actin cytoskeleton, is concentrated on apical surfaces of epithelial cells, especially in microvilli of the small intestine and stomach. In the stomach, ezrin is predominantly expressed on the apical canalicular membrane of parietal cells. Transgenic ezrin knockdown mice in which the expression level of ezrin was reduced to <7 % compared with the wild-type suffered from achlorhydria because of impairment of membrane fusion between tubulovesicles and apical membranes. We observed, for the first time, hypergastrinemia and foveolar hyperplasia in the gastric fundic region of the knockdown mice. Dilation of fundic glands was observed, the percentage of parietal and chief cells was reduced, and that of mucous-secreting cells was increased. The parietal cells of knockdown mice contained dilated tubulovesicles and abnormal mitochondria, and subsets of these cells contained abnormal vacuoles and multilamellar structures. Therefore, lack of ezrin not only causes achlorhydria and hypergastrinemia but also changes the structure of gastric glands, with severe perturbation of the secretory membranes of parietal cells.

Keywords

Ezrin Parietal cells Epithelium Secretory membrane 

Notes

Acknowledgments

We thank Professor Tsukita for giving us the Vil2 kd/kd mice. We thank Dr Yosuke Matsumoto, Mr Hiroki Murakami, Ms Karin Ikeda, and Ms Kaori Akiyama for their help with breeding and genotyping of mice and for technical support. This research was supported in part by Grants-in-Aid for Scientific Research (21590082 and 24590104) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.A., and a High-Tech Research Center Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.A.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical approval

All work with animals was performed with the approval of the Animal Ethics Committees of Ritsumeikan University.

Supplementary material

12576_2015_393_MOESM1_ESM.pptm (49 kb)
Supplementary Fig. 1 mRNA expression levels of ezrin in the gastric corpus and pyloric antrum segments were compared between the wild-type and Vil2 kd/kd mice. The mRNA expression levels were normalized by the expression level of GAPDH, and the values were shown as the percentage to the expression levels in the wild-type. All data represent mean ± S.E. N=3 for wild-type and Vil2 kd/kd mice. **P < 0.01 versus wild-type. (PPTM 48 kb) (PPTM 48 kb)
12576_2015_393_MOESM2_ESM.pptm (570 kb)
Supplementary Fig. 2 Con A staining of the sections of gastric corpus of wild-type and Vil2 kd/kd mice. Sections of the gastric corpus of wild-type (a, c) and Vil2 kd/kd mice (b, d) were stained with H.E. (a, b) and Con A (c, d), respectively. Scale bar 100 µm. (PPTM 570 kb) (PPTM 570 kb)
12576_2015_393_MOESM3_ESM.pptm (501 kb)
Supplementary Fig. 3 Expression of ezrin and pepsin C in the gastric corpus segments. Immunofluoresence observation of the gastric corpus of wild-type and Vil2 kd/kd mice with the anti-ezrin (green) and anti-pepsin C antibodies (red), respectively at low (a) and high (b) magnifications. Immunofluoresence of the bottom part of gastric corpus was shown in (b). Scale bars 100 µm (b, c). (PPTM 501 kb) (PPTM 501 kb)
12576_2015_393_MOESM4_ESM.pptm (310 kb)
Supplementary Fig. 4 Expression of moesin in the gastric corpus segments of wild-type (a) and Vil2 kd/kd mice (b). The expression pattern of moesin was different from that of ezrin in the gastric corpus. In addition, moesin did not compensate the loss of ezrin in the Vil2 kd/kd parietal cells. Scale bar 100 µm. (PPTM 309 kb) (PPTM 309 kb)

References

  1. 1.
    Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3:586–599CrossRefPubMedGoogle Scholar
  2. 2.
    Fehon RG, McClatchey AI, Bretscher A (2010) Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol 11(4):276–287PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Tsukita S, Yonemura S (1999) Cortical actin organization: lessons from ERM (ezrin/radixin/moesin) proteins. J Biol Chem 274:34507–34510CrossRefPubMedGoogle Scholar
  4. 4.
    Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1992) A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J Cell Sci 103:131–143PubMedGoogle Scholar
  5. 5.
    Andréoli C, Martin M, Le Borgne R, Reggio H, Mangeat P (1994) Ezrin has properties to self-associate at the plasma membrane. J Cell Biol 107:2509–2521Google Scholar
  6. 6.
    Gary R, Bretscher A (1995) Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol Biol Cell 6:1061–1075PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Berryman M, Franck Z, Bretascher A (1993) Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci 105:1025–1043PubMedGoogle Scholar
  8. 8.
    Hanzel DK, Urushidani T, Usinger WR, Smolka A, Forte JG (1989) Immunolocalization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells. Am J Physiol (Gastrointest Liver Physiol) 256:G1082–G1089Google Scholar
  9. 9.
    Hanzel D, Reggio H, Bretscher A, Forte JG, Mangeat P (1991) The secretion-stimulated 80 K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli. EMBO J 10(9):2363–2373PubMedCentralPubMedGoogle Scholar
  10. 10.
    Zhou R, Cao X, Watson C, Miao Y, Guo Z, Forte JG, Yao X (2003) Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation. J Biol Chem 278(37):35651–35659CrossRefPubMedGoogle Scholar
  11. 11.
    Saotome I, Curto M, McClatchey AI (2004) Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine. Dev Cell 6:855–864CrossRefPubMedGoogle Scholar
  12. 12.
    Casaletto JB, Saotome I, Curto M, McClatchey AI (2011) Ezrin-mediated apical integrity is required for intestinal homeostasis. Proc Natl Acad Sci 108(29):11924–11929PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Tamura A, Kikuchi S, Hata M, Katsuno T, Matsui T, Hayashi H, Suzuki Y, Noda T, Tsukita S, Tsukita S (2005) Achlorhydria by ezrin knockdown: defects in the formation/expansion of apical canaliculi in gastric parietal cells. J Cell Biol 169(1):21–28PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Hatano R, Fujii E, Segawa H, Mukaisho K, Matsubara M, Miyamoto K, Hattori T, Sugihara H, Asano S (2013) Ezrin, a membrane cytoskeletal cross-linker, is essential for the regulation of phosphate and calcium homeostasis. Kidney Int 83:41–49CrossRefPubMedGoogle Scholar
  15. 15.
    Hatano R, Akiyama K, Tamura A, Hosogi S, Marunaka Y, Caplan MJ, Ueno Y, Tsukita S, Asano S (2015) Knockdown of ezrin causes intrahepatic cholestasis by the dysregulation of bile fluidity in the bile duct epithelium. Hepatology 61(5):1660–1671CrossRefPubMedGoogle Scholar
  16. 16.
    Dockray GJ, Varro A, Dimaline R, Wang T (2001) The gastrins: their production and biological activities. Annu Rev Physiol 63:119–139CrossRefPubMedGoogle Scholar
  17. 17.
    Pagliocca A, Hegyi P, Venglovecz V, Rackstraw SA, Khan Z, Burdyga G, Wang TC, Dimaline R, Verro A, Dockray GJ (2008) Identification of ezrin as a target of gastrin in immature mouse gastric parietal cells. Exp Physiol 93:1174–1189CrossRefPubMedGoogle Scholar
  18. 18.
    Schultheis PJ, Clarke LL, Meneton P, Harline M, Boivin GP, Sternmermann G, Duffy JJ, Doetschman T, Miller ML, Shull GE (1998) Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion. J Clin Invest 101(6):1243–1253PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Scarff KL, Judd LM, Toh B-H, Gleeson PA, van Driel IR (1999) Gastric H+, K+-adenosine triphosphatase β subunit is required for normal function, development, and membrane structure of mouse parietal cells. Gastroenterology 117:605–618CrossRefPubMedGoogle Scholar
  20. 20.
    Spicer Z, Miller ML, Andringa A, Riddle TM, Duffy JJ, Doetschman T, Shull GE (2000) Stomachs of mice lacking the gastric H, K-ATPase α-subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia. J Biol Chem 275(28):21555–21565CrossRefPubMedGoogle Scholar
  21. 21.
    Roepke TK, Anantharam A, Kirchhoff P, Busque SM, Young JB, Geibel JP, Lerner DJ, Abbott GW (2006) The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion. J Biol Chem 281(33):23740–23747CrossRefPubMedGoogle Scholar
  22. 22.
    Judd LM, Andringa A, Rubio CA, Spicer Z, Shull GE, Miller ML (2005) Gastric achlorhydria in H/K-ATPase-deficient (Atp4a(−/−)) mice causes severe hyperplasia, mucocystic metaplasia and upregulation of growth factors. J Gastroenterol Hepatol 20:1266–1278CrossRefPubMedGoogle Scholar
  23. 23.
    Karam SM, Straiton T, Hassan WM, Leblond CP (2003) Defining epithelial cell progenitors in the human oxyntic mucosa. Stem Cells 21:322–336CrossRefPubMedGoogle Scholar
  24. 24.
    Falk P, Roth KA, Gordon JI (1994) Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cell lineages. Am J Physiol Gastrointest Liver Physiol 266(29):G987–G1003Google Scholar
  25. 25.
    Zhu L, Hatakeyama J, Zhang B, Makdisi J, Ender C, Forte JG (2009) Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin. Am J Physiol (Gastrointest Liver Physiol) 296:G185–G195CrossRefGoogle Scholar
  26. 26.
    Gawenis LR, Ledoussal C, Judd LM, Prasad V, Alper SL, Stuart-Tilley A, Woo AL, Grisham C, Sanford LP, Doetschman T, Miller ML, Shull GE (2004) Mice with a targeted disruption of the AE2 Cl/HCO3 exchanger are achlorhydric. J Biol Chem 279(29):30531–30539CrossRefPubMedGoogle Scholar
  27. 27.
    Goldenring JR, Nomura S (2006) Differentiation of the gastric mucosa III. Animal models of oxytic atrophy and metaplasia. Am J Physiol (Gastrointest Liver Physiol) 291:G999–G1004CrossRefGoogle Scholar
  28. 28.
    Hattori T (1986) Development of adenocarcinomas in the stomach. Cancer 57:1528–1534CrossRefPubMedGoogle Scholar
  29. 29.
    Longman RJ, Douthwaite J, Sylvester PA, Poulsom R, Corfield AP, Thomas MG, Wright NA (2000) Coordinated localization of mucins and trefoil peptides in the ulcer associated cell lineage and the gastrointestinal mucosa. Gut 47:792–800PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Ding X, Deng H, Wang D, Zhou J, Huang Y, Zhao X, Yu X, Wang M, Wang F, Ward T, Aikhionbare F, Yao X (2010) Phospho-regulated ACAP4-ezrin interaction is essential for histamine-stimulated parietal cell secretion. J Biol Chem 285:18769–18780PubMedCentralPubMedGoogle Scholar
  31. 31.
    Fujisawa S, Romin Y, Barlas A, Petrovic LM, Turkekul M, Fan N, Xu K, Garcia AR, Monette S, Klimstra DS, Erinjeri JP, Solomon SB, Manova-Todorova K, Sofocleous CT (2014) Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastasis. Cytotechnology 66:259–273PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Kakei N, Ichinose M, Tatematsu M, Shimizu M, Oka M, Yahagi N, Matsushima M, Kurokawa K, Yonezawa S, Furihata C, Shiokawa K, Kageyama T, Miki K, Fukamachi H (1995) Effects of long-term omeprazole treatment on adult rat gastric mucosa—enhancement of the epithelial cell proliferation and suppression of its differentiation. Biochem Biophys Res Commun 214:861–868CrossRefPubMedGoogle Scholar
  33. 33.
    Nomura S, Yamaguchi H, Ogawa M, Wang TC, Lee JR, Goldenring JR (2005) Alterations in gastric mucosal lineages induced by acute oxyntic atrophy in wild-type and gastrin-deficient mice. Am J Physiol (Gastrointest Liver Physiol) 288:G362–G375CrossRefGoogle Scholar
  34. 34.
    Jain RN, Al-Menhali AA, Keeley TM, Ren J, El-Zaatari M, Chen X, Merchant JL, Ross TS, Chew CS, Samuelson LC (2008) Hip1r is expressed in gastric parietal cells and is required for tubulovesicle formation and cell survival in mice. J Clin Invest 118:2459–2470PubMedCentralPubMedGoogle Scholar
  35. 35.
    Wang TC, Goldenring JR, Dangler C, Ito S, Mueller A, Jeon WK, Koh TJ, Fox JG (1998) Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection. Gastroenterology 114:675–689CrossRefPubMedGoogle Scholar
  36. 36.
    Lopez-Diaz L, Hinkle KL, Jain RN, Zavros Y, Brunkan CS, Keeley T, Eaton KA, Merchant JL, Chew CS, Samuelson LC (2006) Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice. Am J Physiol (Gastrointest Liver Physiol) 290:G970–G979CrossRefGoogle Scholar
  37. 37.
    Goldenring JR, Ray GS, Coffey RJ, Meunier PC, Haley PJ, Barnes TB (2000) Car BD (2000) Reversible drug-induced oxyntic atrophy in rats. Gastroenterology 118:1080–1093CrossRefPubMedGoogle Scholar
  38. 38.
    Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Klionsky DJ (2007) Autophagy: form phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 8:931–937CrossRefPubMedGoogle Scholar
  40. 40.
    Nardacci R, Sartori C, Stefanini S (2000) Selective autophagy of clofibrate-induced rat liver peroxisomes. Cytochemistry and immunocytochemistry on tissue specimens and on fractions obtained by nycodenz density gradient centrifugation. Cell Mol Biol 46:1277–1290PubMedGoogle Scholar

Copyright information

© The Physiological Society of Japan and Springer Japan 2015

Authors and Affiliations

  • Saori Yoshida
    • 1
  • Hiroto Yamamoto
    • 1
    • 2
  • Takahito Tetsui
    • 1
  • Yuka Kobayakawa
    • 1
  • Ryo Hatano
    • 1
  • Ken-ichi Mukaisho
    • 2
  • Takanori Hattori
    • 2
  • Hiroyuki Sugihara
    • 2
  • Shinji Asano
    • 1
    Email author
  1. 1.Department of Molecular Physiology, College of Pharmaceutical SciencesRitsumeikan UniversityKusatsuJapan
  2. 2.Department of PathologyShiga University of Medical SciencesOtsuJapan

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