RESUMEN
Silencing Zonula occludens 2 (ZO-2), a tight junctions (TJ) scaffold protein, in epithelial cells (MDCK ZO-2 KD) triggers: 1) Decreased cell to substratum attachment, accompanied by reduced expression of claudin-7 and integrin ß1, and increased vinculin recruitment to focal adhesions and stress fibers formation; 2) Lowered cell-cell aggregation and appearance of wider intercellular spaces; 3) Increased RhoA/ROCK activity, mediated by GEF-HI recruitment to cell borders by cingulin; 4) Increased Cdc42 activity, mitotic spindle disorientation and the appearance of cysts with multiple lumens; 5) Increased Rac and cofilin activity, multiple lamellipodia formation and random cell migration but increased wound closure; 6) Diminished cingulin phosphorylation and disappearance of planar network of microtubules at the TJ region; and 7) Increased transepithelial electrical resistance at steady state, coupled to an increased expression of ZO-1 and claudin-4 and a decreased expression of claudin-2 and paracingulin. Hence, ZO-2 is a crucial regulator of Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.
Asunto(s)
Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-2/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genética , Animales , Claudina-2/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Perros , Células Epiteliales/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Fosforilación , Uniones Estrechas/genética , TransfecciónRESUMEN
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.
Asunto(s)
Endocitosis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ouabaína/farmacología , Proteolisis/efectos de los fármacos , Proteínas de Uniones Estrechas/metabolismo , Animales , Células Cultivadas , Perros , Células de Riñón Canino Madin DarbyRESUMEN
Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Ouabaína/farmacología , Uniones Estrechas/efectos de los fármacos , Animales , Proteína Tirosina Quinasa CSK , Dextranos/química , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Iones , Modelos Biológicos , Potasio/química , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src QuinasasRESUMEN
The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na(+),K(+)-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na(+),K(+)-ATPase polarity depends on interactions between the beta subunits of Na(+),K(+)-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these beta subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.
Asunto(s)
Polaridad Celular , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Línea Celular , Cricetinae , Cricetulus , Perros , Células Epiteliales/citología , Células Epiteliales/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
The space between neighboring epithelial cells is sealed by the tight junction (TJ). When this seal is leaky, such as in the proximal tubule of the kidney or the gallbladder, substances may cross the epithelium between the cells (paracellular pathway). Yet, when TJs are really hermetic, as is the case in the epithelium of the urinary bladder or the colon, substances can mainly cross the epithelium through the transcellular pathway. The structure of the TJ involves (so far) some 50-odd protein species. Failure of any of these components causes a variety of diseases, some of them so serious that fetuses are not viable. A fast-growing number of diseases are recognized to depend or involve alterations in the TJ. These include autoimmune diseases, in which intestinal TJs allow the passage of antigens from the intestinal flora, challenging the immune system to produce antibodies that may cross react with proteins in the brain, thyroid gland or pancreas. TJs are also involved in cancer development, infections, allergies, etc. The present article does not catalogue all TJ diseases known so far, but describes one of each type as illustration. It also depicts the efforts being made to find pharmaceutical agents that would seal faulty TJs or release their grip to allow for the passage of large molecules through the upper respiratory and digestive tracts, such as insulin, thyroid, appetite-regulatory peptide, etc.