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Histochemistry and Cell Biology

, Volume 146, Issue 6, pp 757–768 | Cite as

P2X7R-dependent regulation of glycogen synthase kinase 3β and claudin-18 in alveolar epithelial type I cells of mice lung

  • K. Barth
  • R. Bläsche
  • A. Neißer
  • S. Bramke
  • J. A. Frank
  • M. KasperEmail author
Original Paper

Abstract

The purinergic receptor P2X7 represents an ATP-gated ionotropic receptor with a selective localization in alveolar epithelial type I cells of the lung. Despite the involvement of the receptor in inflammatory processes of the lung, it is not established whether this receptor plays a specific role in the alveolar epithelial cell biology. There is evidence that P2X7 receptor influences Wnt/β-catenin signalling pathways in alveolar epithelial cells under conditions of injury. Here, we investigated the expression of GSK-3β, a potent protein kinase involved in alveolar epithelial barrier functions, and of tight junction molecules occludin, claudin-4 and claudin-18 in wild-type and P2X7−/− mice. Western blot analysis, immunohistochemistry and quantitative real-time RT-PCR revealed a remarkable increase in claudin-18 mRNA and protein in lungs of P2X7−/− mice animals. Furthermore, alveolar epithelial cells from P2X7−/− animals showed decreased levels of GSK-3β protein and its inactive form GSK-3β (pS9). Conversely, claudin-18 knockout mice exhibited decreased P2X7 mRNA transcript abundance as measured by mRNA expression microarray and quantitative PCR. Our data are consistent with the hypothesis that P2X7R contributes to alveolar epithelial barrier function through effects on GSK-3β. Furthermore, these data suggest a potential reciprocal regulation of claudin-18 and P2X7R in the alveolar epithelium.

Keywords

Mouse lung Knockout P2X7 receptor GSK-3β β-catenin claudin-18 

References

  1. Barth K, Weinhold K, Guenther A, Young MT, Schnittler H, Kasper M (2007) Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells. FEBS J 274:3021–3033CrossRefPubMedGoogle Scholar
  2. Bazzoni G, Tonetti P, Manzi L, Cera MR, Balconi G, Dejana E (2005) Expression of junctional adhesion molecule—a prevents spontaneous and random motility. J Cell Sci 118:623–632CrossRefPubMedGoogle Scholar
  3. Bläsche R, Ebeling G, Perike S, Weinhold K, Kasper M, Barth K (2012) Activation of P2X7R and downstream effects in bleomycin treated lung epithelial cells. Int J Biochem Cell Biol 44:514–524CrossRefPubMedGoogle Scholar
  4. Brune K, Frank J, Schwingshackl A, Finigan J, Sidhaye VK (2015) Pulmonary epithelial barrier function: some new players and mechanisms. Am J Physiol Lung Cell Mol Physiol 308:L731–L745CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chen Z, Jin N, Narasaraju T, Chen J, McFarland LR, Scott M, Liu L (2004) Identification of two novel markers for alveolar epithelial type I and II cells. Biochem Biophys Res Commun 319:774–780CrossRefPubMedGoogle Scholar
  6. Dieckmann-Schuppert A, Schnittler HJ (1997) A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces. Cell Tissue Res 288:119–126CrossRefPubMedGoogle Scholar
  7. Eaton DC, Helms MN, Koval M, Bao HF, Jain L (2009) The contribution of epithelial sodium channels to alveolar function in health and disease. Annu Rev Physiol 71:403–423CrossRefPubMedGoogle Scholar
  8. Ebeling G, Blasche R, Hofmann F, Augstein A, Kasper M, Barth K (2014) Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells. PLoS ONE 9:e100282CrossRefPubMedPubMedCentralGoogle Scholar
  9. Frame S, Cohen P (2001) GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359:1–16CrossRefPubMedPubMedCentralGoogle Scholar
  10. Frank JA (2012) Claudins and alveolar epithelial barrier function in the lung. Ann N Y Acad Sci 1257:175–183CrossRefPubMedPubMedCentralGoogle Scholar
  11. Galam L, Rajan A, Failla A, Soundararajan R, Lockey RF, Kolliputi N (2016) Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression. Am J Physiol Lung Cell Mol Physiol 310:L572–L581CrossRefPubMedGoogle Scholar
  12. Gentile D, Natale M, Lazzerini PE, Capecchi PL, Laghi-Pasini F (2015) The role of P2X7 receptors in tissue fibrosis: a brief review. Purinergic Signal 11:435–440CrossRefPubMedPubMedCentralGoogle Scholar
  13. Guo Y, Mishra A, Weng T, Chintagari NR, Wang Y, Zhao C, Huang C, Liu L (2014) Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death. Cell Death Dis 5:e1286CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hart M, Concordet JP, Lassot I, Albert I, del los Santos R, Durand H, Perret C, Rubinfeld B, Margottin F, Benarous R, Polakis P (1999) The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell. Curr Biol 9:207–210CrossRefPubMedGoogle Scholar
  15. Herzog CR, Soloff EV, McDoniels AL, Tyson FL, Malkinson AM, Haugen-Strano A, Wiseman RW, Anderson MW, You M (1996) Homozygous codeletion and differential decreased expression of p15INK4b, p16INK4a-alpha and p16INK4a-beta in mouse lung tumor cells. Oncogene 13:1885–1891PubMedGoogle Scholar
  16. Hofmann F, Bläsche R, Kasper M, Barth K (2015) A co-culture system with an organotypic lung slice and an immortal alveolar macrophage cell line to quantify silica-induced inflammation. PLoS ONE 10:e0117056CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hollenhorst MI, Richter K, Fronius M (2011) Ion transport by pulmonary epithelia. J Biomed Biotechnol 2011:174306CrossRefPubMedPubMedCentralGoogle Scholar
  18. Johnson MD (2007) Ion transport in alveolar type I cells. Mol BioSyst 3:178–186CrossRefPubMedGoogle Scholar
  19. Jope RS, Johnson GV (2004) The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 29:95–102CrossRefPubMedGoogle Scholar
  20. Kasper M, Rudolf T, Haase M, Schuh D, Muller M (1993) Changes in cytokeratin, vimentin and desmoplakin distribution during the repair of irradiation-induced lung injury in adult rats. Virchows Arch B Cell Pathol Incl Mol Pathol 64:271–279CrossRefPubMedGoogle Scholar
  21. Koval M (2013) Claudin heterogeneity and control of lung tight junctions. Annu Rev Physiol 75:551–567CrossRefPubMedGoogle Scholar
  22. LaFemina MJ, Rokkam D, Chandrasena A, Pan J, Bajaj A, Johnson M, Frank JA (2010) Keratinocyte growth factor enhances barrier function without altering claudin expression in primary alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 299:L724–L734CrossRefPubMedPubMedCentralGoogle Scholar
  23. LaFemina MJ, Sutherland KM, Bentley T, Gonzales LW, Allen L, Chapin CJ, Rokkam D, Sweerus KA, Dobbs LG, Ballard PL, Frank JA (2014) Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice. Am J Respir Cell Mol Biol 51:550–558CrossRefPubMedPubMedCentralGoogle Scholar
  24. Linge A, Morishima N, Kasper M, Barth K (2007) Bleomycin induces caveolin-1 and -2 expression in epithelial lung cancer A549 cells. Anticancer Res 27:1343–1351PubMedGoogle Scholar
  25. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, He X (2002) Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108:837–847CrossRefPubMedGoogle Scholar
  26. Lucattelli M, Cicko S, Muller T, Lommatzsch M, De Cunto G, Cardini S, Sundas W, Grimm M, Zeiser R, Durk T, Zissel G, Sorichter S, Ferrari D, Di Virgilio F, Virchow JC, Lungarella G, Idzko M (2011) P2X7 receptor signaling in the pathogenesis of smoke-induced lung inflammation and emphysema. Am J Respir Cell Mol Biol 44:423–429CrossRefPubMedGoogle Scholar
  27. Mitchell LA, Ward C, Kwon M, Mitchell PO, Quintero DA, Nusrat A, Parkos CA, Koval M (2015) Junctional adhesion molecule A promotes epithelial tight junction assembly to augment lung barrier function. Am J Pathol 185:372–386CrossRefPubMedPubMedCentralGoogle Scholar
  28. Ohta H, Chiba S, Ebina M, Furuse M, Nukiwa T (2012) Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis. Am J Physiol Lung Cell Mol Physiol 302:L193–L205CrossRefPubMedGoogle Scholar
  29. Peifer M, Sweeton D, Casey M, Wieschaus E (1994) wingless signal and Zeste-white 3 kinase trigger opposing changes in the intracellular distribution of Armadillo. Development 120:369–380PubMedGoogle Scholar
  30. Pfleger C, Ebeling G, Blasche R, Patton M, Patel HH, Kasper M, Barth K (2012) Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro. Histochem Cell Biol 138:231–241CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ramirez SH, Fan S, Dykstra H, Rom S, Mercer A, Reichenbach NL, Gofman L, Persidsky Y (2013) Inhibition of glycogen synthase kinase 3beta promotes tight junction stability in brain endothelial cells by half-life extension of occludin and claudin-5. PLoS ONE 8:e55972CrossRefPubMedPubMedCentralGoogle Scholar
  32. Riteau N, Gasse P, Fauconnier L, Gombault A, Couegnat M, Fick L, Kanellopoulos J, Quesniaux VF, Marchand-Adam S, Crestani B, Ryffel B, Couillin I (2010) Extracellular ATP is a danger signal activating P2X7 receptor in lung inflammation and fibrosis. Am J Respir Crit Care Med 182:774–783CrossRefPubMedGoogle Scholar
  33. Severson EA, Kwon M, Hilgarth RS, Parkos CA, Nusrat A (2010) Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating occludin, claudin-1 and E-cadherin expression. Biochem Biophys Res Commun 397:592–597CrossRefPubMedPubMedCentralGoogle Scholar
  34. Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA (2001) Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 276:125–132CrossRefPubMedGoogle Scholar
  35. Wray C, Mao Y, Pan J, Chandrasena A, Piasta F, Frank JA (2009) Claudin-4 augments alveolar epithelial barrier function and is induced in acute lung injury. Am J Physiol Lung Cell Mol Physiol 297:L219–L227CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • K. Barth
    • 1
  • R. Bläsche
    • 1
  • A. Neißer
    • 1
  • S. Bramke
    • 1
  • J. A. Frank
    • 2
  • M. Kasper
    • 1
    Email author
  1. 1.Institute of Anatomy, Medical Faculty “Carl Gustav Carus”Technical University DresdenDresdenGermany
  2. 2.San Francisco VA Medical CenterUniversity of California, San FranciscoSan FranciscoUSA

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