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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

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Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Programa de Biologia Celular, Centro de Pesquisas, Instituto Nacional de Câncer, Rua André Cavalcanti, 37, 5° andar, Rio de Janeiro, RJ, 20231-050, Brazil

    Waldemir Fernandes de Souza, Wallace Martins de Araujo, Julio Cesar Madureira de-Freitas-Junior, Natalia Fortunato-Miranda & José Andrés Morgado-Díaz

  2. Laboratório de Bioquímica Celular, Campus do Centro Oeste Dona Lindú, Universidade Federal de São João Del-Rei, Av Sebastião Gonçalves Coelho 400, Chanadour, Divinópolis, MG, 35501-296, Brazil

    Leandro Augusto Barbosa

  3. Department of Physiology and Pharmacology, University of Toledo, 3035 Arlington Avenue, Toledo, OH, 43614-5804, USA

    Lijun Liu

  4. Laboratório de Estrutura e Regulação de Proteínas e ATPases, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Alameda Bauhínia 400, Rio de Janeiro, 21941-590, Brazil

    Carlos Frederico Leite Fontes

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  1. Waldemir Fernandes de Souza
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  2. Leandro Augusto Barbosa
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  4. Wallace Martins de Araujo
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  8. José Andrés Morgado-Díaz
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Correspondence to José Andrés Morgado-Díaz.

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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)

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)

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de Souza, W.F., Barbosa, L.A., Liu, L. et al. 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. J Membrane Biol 247, 23–33 (2014). https://doi.org/10.1007/s00232-013-9607-y

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