The Journal of Membrane Biology

, Volume 247, Issue 1, pp 23–33 | Cite as

Ouabain-Induced Alterations of the Apical Junctional Complex Involve α1 and β1 Na,K-ATPase Downregulation and ERK1/2 Activation Independent of Caveolae in Colorectal Cancer Cells

  • Waldemir Fernandes de Souza
  • Leandro Augusto Barbosa
  • Lijun Liu
  • Wallace Martins de Araujo
  • Julio Cesar Madureira de-Freitas-Junior
  • Natalia Fortunato-Miranda
  • Carlos Frederico Leite Fontes
  • José Andrés Morgado-DíazEmail author


Studies have reported that Na,K-ATPase interacts with E-cadherin to stabilize (AJs) and regulate the expression of claudins, the main proteins present in the tight junction (TJ) in epithelial cells containing caveolae. However, the role of this ATPase in the regulation of the AJ and TJ proteins in colorectal cancer cells as well as the molecular events underlying this event in a caveolae-independent system remain undefined. In the present study, we used ouabain, a classic drug known to inhibit Na,K-ATPase, and Caco-2 cells, which are a well-established human colorectal cancer model that does not exhibit caveolae. We demonstrated that ouabain treatment resulted in a reduction of the β1 Na,K-ATPase protein and cell redistribution of the AJ proteins E-cadherin and β-catenin, as well as the α1 Na,K-ATPase subunit. Furthermore, ouabain increased claudin-3 protein levels, impaired the TJ barrier function and increased cell viability and proliferation during the early stages of treatment. Additionally, the observed ouabain-induced events were dependent on the activation of ERK1/2 signaling; but in contrast to previous studies, this signaling cascade was caveolae-independent. In conclusion, our findings strongly suggest that α1 and β1 Na,K-ATPase downregulation and ERK1/2 activation induced by ouabain are interlinked events that play an important role during cell–cell adhesion loss, which is an important step during the tumor progression of colorectal carcinomas.


Colorectal cancer Apical junctional complex Na,K-ATPase Ouabain 



This research was supported by Ministério da Saúde (Brazil), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Grant E26/170.026/2008), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant 573806/2008-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. The text was reviewed by American journal experts.

Supplementary material

232_2013_9607_MOESM1_ESM.tiff (629 kb)
Fig. S1 Effect of low dose of ouabain and ionic imbalance on cell distribution of E-cadherin.Monolayers of Caco-2 cells were grown on glass coverslips. Cells were treated with a low concentration of ouabain (100 nM) for 6 h or with a molar excess of NaCl (250 mM) for 2.5 h. After, cells were processed for immunofluorescence analysis of E-cadherin distribution. Bar = 10 μm (TIFF 629 kb)
232_2013_9607_MOESM2_ESM.tif (59 kb)
Fig. S2 Impact of ERK1/2 pathway inhibition on the ouabain-induced effects in β1 Na,K-ATPase levels. Monolayers of Caco-2 cells were grown, pretreated with an inhibitor of the ERK1/2 pathway (PD98059) for 1 h (when indicated) and incubated with ouabain for 6 h. After treatment, total cell lysates were harvested and the expression of β1 Na,K-ATPase was visualized by immunoblotting. Ponceau staining (Ponc.) was used as a loading control. Numbers represent the ratio of the optical density of treated to untreated cells (TIFF 59 kb)


  1. Bagrov AY, Shapiro JI, Fedorova OV (2009) Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets. Am J Hypertens 22(5):559–563PubMedCentralCrossRefPubMedGoogle Scholar
  2. Barbosa LA, Goto-Silva L, Redondo PA et al (2003) TPA-induced signal transduction: a link between PKC and EGFR signaling modulates the assembly of intercellular junctions in Caco-2 cells. Cell Tissue Res 312:319–331CrossRefPubMedGoogle Scholar
  3. Breuza L, Corby S, Arsanto JP et al (2002) The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells. J Cell Sci 115(Pt 23):4457–4467CrossRefPubMedGoogle Scholar
  4. Cereijido M, Contreras RG, Shoshani L, Larre I (2012) The Na+-K+-ATPase as self-adhesion molecule and hormone receptor. Am J Physiol Cell Physiol 302:C473–C481CrossRefPubMedGoogle Scholar
  5. Contreras RG, Flores-Maldonado C, Lazaro A et al (2004) Ouabain binding to Na+, K+-ATPase relaxes cell attachment and sends a specific signal (NACos) to the nucleus. J Membr Biol 198:147–158CrossRefPubMedGoogle Scholar
  6. de Freitas JC Jr, Silva Bdu R, de Souza WF et al (2011) Inhibition of N-linked glycosylation by tunicamycin induces E-cadherin-mediated cell–cell adhesion and inhibits cell proliferation in undifferentiated human colon cancer cells. Cancer Chemother Pharmacol 68(1):227–238CrossRefGoogle Scholar
  7. de Rezende Corrêa G, Araujo dos Santos A, Frederico Leite Fontes C, Giestal de Araujo E (2005) Ouabain induces an increase of retinal ganglion cell survival in vitro: the involvement of protein kinase C. Brain Res 1049(1):89–94CrossRefPubMedGoogle Scholar
  8. Esteves MB, Marques-Santos LF, Affonso-Mitidieri OR, Rumjanek VM (2005) Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes. An Acad Bras Cienc 77(2):281–292CrossRefPubMedGoogle Scholar
  9. Felth J, Rickardson L, Rosén J et al (2009) Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs. J Nat Prod 72:1969–1974CrossRefPubMedGoogle Scholar
  10. Ikari A, Takiguchi A, Atomi K, Sugatani J (2011) Epidermal growth factor increases clathrin-dependent endocytosis and degradation of claudin-2 protein in MDCK II cells. J Cell Physiol 226:2448–2456CrossRefPubMedGoogle Scholar
  11. Inge LJ, Rajasekaran SA, Yoshimoto K et al (2008) Evidence for a potential tumor suppressor role for the Na, K-ATPase beta1-subunit. Histol Histopathol 23:459–467PubMedCentralPubMedGoogle Scholar
  12. Kometiani P, Liu L, Askari A (2005) Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells. Mol Pharmacol 67:929–936CrossRefPubMedGoogle Scholar
  13. Krug SM, Günzel D, Conrad MP et al (2012) Charge-selective claudin channels. Ann N Y Acad Sci 1257:20–28CrossRefPubMedGoogle Scholar
  14. Larre I, Lazaro A, Contreras RG et al (2010) Ouabain modulates epithelial cell tight junction. Proc Natl Acad Sci USA 107(25):11387–11392PubMedCentralCrossRefPubMedGoogle Scholar
  15. Larre I, Contreras RG, Cereijido M (2011) Ouabain modulates cell contacts as well as functions that depend on cell adhesion. Methods Mol Biol 763:155–168CrossRefPubMedGoogle Scholar
  16. Li Q, Mattingly RR (2008) Restoration of E-cadherin cell–cell junctions requires both expression of E-cadherin and suppression of ERK MAP kinase activation in Ras-transformed breast epithelial cells. Neoplasia 10:1444–1458PubMedCentralCrossRefPubMedGoogle Scholar
  17. Li Z, Xie Z (2009) The Na/K-ATPase/Src complex and cardiotonic steroid-activated protein kinase cascades. Pflugers Arch 457:635–644CrossRefPubMedGoogle Scholar
  18. Liang M, Tian J, Liu L et al (2007) Identification of a pool of non-pumping Na/K-ATPase. J Biol Chem 282(14):10585–10593CrossRefPubMedGoogle Scholar
  19. Liu J, Shapiro JI (2007) Regulation of sodium pump endocytosis by cardiotonic steroids: molecular mechanisms and physiological implications. Pathophysiology 14:171–181PubMedCentralCrossRefPubMedGoogle Scholar
  20. Liu L, Mohammadi K, Aynafshar B et al (2003) Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase. Am J Physiol Cell Physiol 284:C1550–C1560CrossRefPubMedGoogle Scholar
  21. Liu L, Ivanov AV, Gable ME et al (2011) Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase. Biochemistry 50(40):8664–8673PubMedCentralCrossRefPubMedGoogle Scholar
  22. Mijatovic T, Van Quaquebeke E, Delest B et al (2007) Cardiotonic steroids on the road to anti-cancer therapy. Biochim Biophys Acta 1776:32–57PubMedGoogle Scholar
  23. Mineta K, Yamamoto Y, Yamazaki Y et al (2011) Predicted expansion of the claudin multigene family. FEBS Lett 585(4):606–612CrossRefPubMedGoogle Scholar
  24. Mitchell LA, Overgaard CE, Ward C et al (2011) Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function. Am J Physiol Lung Cell Mol Physiol 301:L40–L49PubMedCentralCrossRefPubMedGoogle Scholar
  25. Miyoshi J, Takai Y (2008) Structural and functional associations of apical junctions with cytoskeleton. Biochim Biophys Acta 1778:670–691CrossRefPubMedGoogle Scholar
  26. Mohammadi K, Kometiani P, Xie Z, Askari A (2001) Role of protein kinase C in the signal pathways that link Na+/K+-ATPase to ERK1/2. J Biol Chem 276(45):42050–42056CrossRefPubMedGoogle Scholar
  27. Morth JP, Poulsen H, Toustrup-Jensen MS et al (2009) The structure of the Na+, K+-ATPase and mapping of isoform differences and disease-related mutations. Philos Trans R Soc Lond B 364(1514):217–227CrossRefGoogle Scholar
  28. Oliveira SS, Oliveira IM, Souza W, Morgado-Díaz JA (2005) Claudins upregulation in human colorectal cancer. FEBS Lett 579:6179–6185CrossRefPubMedGoogle Scholar
  29. Rajasekaran SA, Ball WJ Jr, Bander NH et al (1999) Reduced expression of β-subunit of Na, K-ATPase in human clear-cell renal cell carcinoma. J Urol 162:574–580CrossRefPubMedGoogle Scholar
  30. Rajasekaran SA, Palmer LG, Quan K et al (2001) Na, K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility. Mol Biol Cell 12:279–295PubMedCentralCrossRefPubMedGoogle Scholar
  31. Rajasekaran SA, Barwe SP, Rajasekaran AK (2005) Multiple functions of Na, K-ATPase in epithelial cells. Semin Nephrol 25:328–334CrossRefPubMedGoogle Scholar
  32. Rajasekaran SA, Huynh TP, Wolle DG et al (2010) Na, K-ATPase subunits as markers for epithelial–mesenchymal transition in cancer and fibrosis. Mol Cancer Ther 9:1515–1524PubMedCentralCrossRefPubMedGoogle Scholar
  33. Singh AB, Harris RC (2004) Epidermal growth factor receptor activation differentially regulates claudin expression and enhances transepithelial resistance in Madin-Darby canine kidney cells. J Biol Chem 279(5):3543–3552CrossRefPubMedGoogle Scholar
  34. Takizawa Y, Kishimoto H, Kitazato T et al (2012) Changes in protein and mRNA expression levels of claudin family after mucosal lesion by intestinal ischemia/reperfusion. Int J Pharm 426(1–2):82–89CrossRefPubMedGoogle Scholar
  35. Tian J, Li X, Liang M et al (2009) Changes in sodium pump expression dictate the effects of ouabain on cell growth. J Biol Chem 284:14921–14929PubMedCentralCrossRefPubMedGoogle Scholar
  36. Vagin O, Sachs G, Tokhtaeva E (2007) The roles of the Na, K-ATPase beta 1 subunit in pump sorting and epithelial integrity. J Bioenerg Biomembr 39:367–372CrossRefPubMedGoogle Scholar
  37. Wang H, Haas M, Liang M et al (2004) Ouabain assembles signaling cascades through the caveolar Na+/K+-ATPase. J Biol Chem 279(17):17250–17259CrossRefPubMedGoogle Scholar
  38. Wang Z, Zheng M, Li Z et al (2009) Cardiac glycosides inhibit p53 synthesis by a mechanism relieved by Src or MAPK inhibition. Cancer Res 69:6556–6564PubMedCentralCrossRefPubMedGoogle Scholar
  39. Weber CR (2012) Dynamic properties of the tight junction barrier. Ann N Y Acad Sci 1257:77–84PubMedCentralCrossRefPubMedGoogle Scholar
  40. Xie Z, Askari A (2002) Na+/K+-ATPase as a signal transducer. Eur J Biochem 269(10):2434–2439CrossRefPubMedGoogle Scholar
  41. Yap AS, Crampton MS, Hardin J (2007) Making and breaking contacts: the cellular biology of cadherin regulation. Curr Opin Cell Biol 19:508–514PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Waldemir Fernandes de Souza
    • 1
  • Leandro Augusto Barbosa
    • 2
  • Lijun Liu
    • 3
  • Wallace Martins de Araujo
    • 1
  • Julio Cesar Madureira de-Freitas-Junior
    • 1
  • Natalia Fortunato-Miranda
    • 1
  • Carlos Frederico Leite Fontes
    • 4
  • José Andrés Morgado-Díaz
    • 1
    Email author
  1. 1.Programa de Biologia Celular, Centro de PesquisasInstituto Nacional de CâncerRio de JaneiroBrazil
  2. 2.Laboratório de Bioquímica Celular, Campus do Centro Oeste Dona LindúUniversidade Federal de São João Del-ReiDivinópolisBrazil
  3. 3.Department of Physiology and PharmacologyUniversity of ToledoToledoUSA
  4. 4.Laboratório de Estrutura e Regulação de Proteínas e ATPases, Departamento de Bioquímica MédicaUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil

Personalised recommendations