Ouabain enhances cell-cell adhesion mediated by β1-subunits of the Na+,K+-ATPase in CHO fibroblasts

Abstract

AbstractAdhesion is an important characteristic of epithelial cells to provide a crucial barrier to pathogens and substances. In polarized epithelial cells, cell-adhesion depends on tight junctions, adherent junctions and the Na+,K+-ATPase. All these are located in the basolateral membrane of the cells. The hormone ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, and inhibits the pump activity when used at above μM concentrations. At physiological nM concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through activation of signaling pathways associated to the α subunit. Our group showed that non-adherent CHO cells transfected with the canine β1subunit become adhesive, and that homotypic interactions between β1subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Therefore, in this study we investigated whether the adhesion between β1subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β1subunit of the Na+,K+-ATPase (CHO-β1) and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO-β1cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β1 subunit of the Na+,K+-ATPase at the cell membrane. We also screened the effect of ouabain on activation of signaling pathways in CHO-β1cells, and their effect on cell adhesion. We found that c-Src, is activated by ouabain and is therefore likely to regulate the adhesive properties of CHO-β1cells. Collectively, our findings suggest that the β1subunits adhesion is modulated by the levels of expression and activation of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.