Ouabain Enhances Cell-Cell Adhesion Mediated by β1 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 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.

A caveolin binding motif in Na/K-ATPase is required for stem cell differentiation and organogenesis in mammals and C. elegans

“…S3, we observed no change in fetal survival after administration of pNaKtide to female mice before mating and continued until the end of pregnancy. It is important to mention that pNaKtide has been proven to be specific and effective in blocking the NKA/Src receptor signaling in vivo (22)(23)(24)(25)(26), and our control experiments showed that pNaKtide could cross the placental barrier. Moreover, this lack of pNaKtide effect on mouse embryogenesis appears to be consistent with a previous report demonstrating that neutralization of endogenous ouabain by injection of an anti-ouabain antibody did affect the kidney development of neonatal mice but did not affect their overall survival (20).…”

“…These findings have substantially increased our understanding of 1 NKA/Src interaction in cell biology and animal physiology. It is important to mention that several groups not associated with us have successfully used pNaKtide to block ouabain and NKA signaling in vitro and in vivo (23)(24)(25)(26)51). While our group and others continue to characterize the molecular basis and biological function of the NKA/Src receptor complex, we propound that the question of NKA/caveolin-1 interaction is a more pressing one in the context of this study.…”

A Model for the Homotypic Interaction between Na+,K+-ATPase β1 Subunits Reveals the Role of Extracellular Residues 221–229 in Its Ig-Like Domain

“…As previously shown, transfection of canine NaK β 1 subunit in CHO fibroblast increases cell adhesion [10,35]. We performed Dispase assays to assess cell-cell adhesion in the stably transfected cell lines.…”

Twenty years of exceptional success: The molecular education and research consortium in undergraduate computational chemistry (MERCURY)

“…The faculty published 2 peer‐reviewed books, [ 238,239 ] 9 peer‐reviewed book chapters, [ 240–248 ] and 115 peer‐reviewed research papers. [ 249–363 ] This comes to 1.6 peer‐reviewed products/faculty/year during the 3‐year grant period, which is 3.2 times the rate of publication for natural science faculty at PUIs. [ 46 ]…”