Ouabain Enhances Cell-Cell Adhesion Mediated by β1 Subunits of the Na+,K+-ATPase in CHO Fibroblasts.

Claudia Andrea Vilchis-Nestor,María Luisa Roldán,Teresita Padilla-Benavides,Juan G Navea,Liora Shoshani,Angelina Leonardi

doi:10.3390/ijms20092111

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 β1 subunit become adhesive, and those homotypic interactions amongst β1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α 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 α subunit. In this study, we investigated whether the adhesion between β1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β1 subunit 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 β1 cells. 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 examined the effect of ouabain on the activation of signaling pathways in CHO β1 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 β1 cells. Collectively, our findings suggest that the β1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.

Highlights

The Na+,K+-ATPase or sodium pump is an ubiquitous plasma membrane transporter that creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across cellular membranes [1,2]
Cell-Cell Adhesion of chinese hamster ovary (CHO) Fibroblasts Expressing Canine β1 Subunit of Na+,K+-ATPase is Mediated by β1 Homotypic Interactions in-trans
Confocal microscopy analyses showed that CHO cells transfected with the plasmid encoding the canine β1 subunit express the protein in the plasma membrane, thereby showing a distribution resembling that observed in epithelial MDCK cells (Figure 1A, middle and right panels)

Summary

Introduction

The Na+,K+-ATPase or sodium pump is an ubiquitous plasma membrane transporter that creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across cellular membranes [1,2]. The catalytic α subunit consists of 10 transmembrane domains (TM), and exchanges 3 Na+ ions from the cytosol for 2 K+ ions from the extracellular milieu using the energy released from. The γ subunit is a small, single span TM protein belonging to the FXYD family, which is differentially expressed in tissues and modulates the pump’s function [6,7]. The β subunit of the sodium pump has different functions that depend on the isoform expressed (β1, β2 or β3), and on the accompanying α subunit isoform (α1–α4) [9]. The main function of the β subunit is to act as a chaperone for the α subunit, by contributing to the assembly and delivery of the pump to the plasma membrane [10,11]. Different β isoforms have been associated with different K+ affinities [13]

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Discussion

Conclusion