Ouabain Enhances Cell-Cell Adhesion Mediated by ?₁ Subunits of the Na⁺,K⁺-ATPase in CHO Fibroblasts.

ABSTRACT: Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na⁺,K⁺-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ?₁ subunit become adhesive, and those homotypic interactions amongst ?₁ subunits of the Na⁺,K⁺-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the ? subunit of the Na⁺,K⁺-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the ? subunit. In this study, we investigated whether the adhesion between ?₁ subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ?₁ subunit of the Na⁺,K⁺-ATPase (CHO ?₁), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ?₁ cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ?₁ subunit of the Na⁺,K⁺-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ?₁ cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ?₁ cells. Collectively, our findings suggest that the ?₁ subunit adhesion is modulated by the expression levels of the Na⁺,K⁺-ATPase at the plasma membrane, which is regulated by ouabain.

SUBMITTER: Vilchis-Nestor CA 

PROVIDER: S-EPMC6539428 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature