title: Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.
abstract: In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.
authors.affiliation: Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (Cinvestav), Mexico City 07360, Mexico; Department of Pharmacology, Center for Research and Advanced Studies (Cinvestav), Mexico City 07360, Mexico.
authors.forename: Ruth
authors.initials: R
authors.lastname: Rincon-Heredia
authors.forename: David
authors.initials: D
authors.lastname: Flores-Benitez
authors.forename: Catalina
authors.initials: C
authors.lastname: Flores-Maldonado
authors.forename: José
authors.initials: J
authors.lastname: Bonilla-Delgado
authors.forename: Vicky
authors.initials: V
authors.lastname: García-Hernández
authors.forename: Odette
authors.initials: O
authors.lastname: Verdejo-Torres
authors.forename: Aida M
authors.initials: AM
authors.lastname: Castillo
authors.forename: Isabel
authors.initials: I
authors.lastname: Larré
authors.forename: Augusto C
authors.initials: AC
authors.lastname: Poot-Hernández
authors.forename: Martha
authors.initials: M
authors.lastname: Franco
authors.forename: Patricio
authors.initials: P
authors.lastname: Gariglio
authors.forename: José L
authors.initials: JL
authors.lastname: Reyes
authors.forename: Rubén G
authors.initials: RG
authors.lastname: Contreras
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D061985:Madin Darby Canine Kidney Cells; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology*; D059748:Proteolysis / Q000187:drug effects*; D062725:Tight Junction Proteins / Q000378:metabolism*
chemical_list: D062725:Tight Junction Proteins; D010042:Ouabain; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: Claudin; ERK1/2; Na(+), K(+)-ATPase; ZO-1; cSrc