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TPA-induced signal transduction: a link between PKC and EGFR signaling modulates the assembly of intercellular junctions in Caco-2 cells

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Abstract

Recent studies suggest that signal transduction may have an important role in the development and regulation of the metastatic phenotype. Here, we investigated the role of the epidermal growth factor receptor (EGFR), and protein kinase C (PKC), in the process of reassembly of cadherin-dependent cell-cell adhesion of Caco-2 cells. We used chemical activation of PKC and EGFR with 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent, pretreatment with protein kinase inhibitors and subcellular fractionation to analyze the effect of the phorbol ester on the redistribution of junctional proteins. Transepithelial resistance (TER), electron microscopy and immunofluorescence analyses were also carried out. Activation with TPA resulted in disassembly of adherens junctions (AJs), but the tight junction (TJ) structure and function remained unaltered. TPA affected E-cadherin levels. In Caco-2 cells at day 2 of culture, when most E-cadherin is not associated with the cytoskeleton, a decrease in the level of this protein was observed as soon as 6 h after TPA addition. However, at day 5 of culture, the major effect observed after 6 h of treatment was a translocation of the protein from the Triton-insoluble to the -soluble fraction. On the other hand, TPA did not significantly affect the E-cadherin-associated proteins α and β-catenins. Potent specific EGFR inhibitors, such as PD153035 and Tyrphostin 25, as well as Calphostin C, an inhibitor of PKC, significantly blocked the effect of TPA on AJs. Furthermore, inhibition of the TPA effect by the PD98059 MAPK inhibitor suggests that activation of this kinase was the final event in the modulation of cadherin-dependent cell-cell adhesion. Pretreatment of cell monolayers with Calphostin C before EGF treatment, one of the ligands of EGFR, blocked the redistribution of E-cadherin caused by EGF. Based on these results, we conclude that both EGFR and PKC activation are involved in TPA-induced cell signaling for modulation of cadherin-dependent cell-cell adhesion and cell shape in Caco-2 cells.

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Acknowledgements

We thank Dr. Marcello Barcinski for critically reading the manuscript, Dr. Marcia Attias for assistance with the high-resolution scanning electron microscope, and Dr. Carlos Frederico Leite Fontes and Vanessa Honorato de Oliveira for help with the biochemical assays.

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  1. Leandro A. Barbosa

    Present address: Laboratório de Membranas Transportadoras, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de Janeiro, Brazil

Authors and Affiliations

  1. Divisão de Biologia Celular, Coordenação de Pesquisa, Instituto Nacional de Câncer, Praça da Cruz Vermelha 23, 6º Andar, 20230-130, Rio de Janeiro, Brazil

    Leandro A. Barbosa, Livia Goto-Silva, Patricia A. Redondo, Silvia Oliveira, Giovani Montesano, Wanderley de Souza & Jose A. Morgado-Díaz

  2. Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha de Fundão, 21944-900, Rio de Janeiro, Brazil

    Wanderley de Souza

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  1. Leandro A. Barbosa
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Correspondence to Jose A. Morgado-Díaz.

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This work was supported by Fundação Ary Frauzino para Pesquisa e Controle do Câncer (FAF), Instituto Nacional de Câncer de Rio de Janeiro (INCa), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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Barbosa, L.A., Goto-Silva, L., Redondo, P.A. et al. 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, 319–331 (2003). https://doi.org/10.1007/s00441-003-0727-z

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