Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Abstract

SummaryNa,K-ATPase is a hetero-oligomer of an a-and a b-subunit. The a-subunit (Na,K-a) possesses the catalytic function, whereas the bsubunit (Na,K-b) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-b 1 transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-b 1 and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-a 1 . We now provide evidence that knockdown of Na,K-b 1 prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-b 1 or heptad repeat motif mutant of Na,K-b 1 , but not by the glycine zipper motif mutant that abrogates Na,K-b 1 cis homo-oligomerization. These studies suggest that Na,K-b 1 cis homo-oligomerization rather than hetero-oligomerization with Na,K-a 1 is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

Carcinoma cells induce lumen filling and EMT in epithelial cells by soluble E-cadherin-mediated activation of EGFR

“…This novel 3D assay allowed us to demonstrate that carcinoma cells disrupt epithelial architecture by inducing preneoplastic lumen filling. Interference with the key regulators of the apico-basal polarity and tight junction assembly, such as the Crumbs3 and PAR complexes, β1 integrin, RhoA and the Na + /K + -ATPase β-subunit generates multiple lumina or no lumen in epithelial cysts (Barwe et al, 2013;Martin-Belmonte et al, 2007;Schluter et al, 2009;Shin et al, 2005;Straight et al, 2004;Yu et al, 2008). However, it is not well known how these markers are affected during carcinogenesis.…”

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell–cell trans-dimerization of Na,K-ATPase β1 subunits

“…In the alveolar epithelium, these gradients are responsible for water reabsorption from alveolar spaces, which is crucial for normal gas exchange (Mutlu and Sznajder, 2005;Sznajder, 2001;Vádasz et al, 2007). The two Na,K-ATPase subunits, a catalytic α subunit and an N-glycosylated structural β subunit are required for normal maturation, membrane targeting and transport activity of the enzyme (Barwe et al, 2012;Geering, 2001;Rajasekaran et al, 2001;Tokhtaeva et al, 2009). Additional regulatory subunits that are not obligatory for activity belong to a family of proteins that share an extracellular FXYD sequence (Geering, 2006;Miller and Davis, 2008;Sweadner and Rael, 2000).…”

Sodium-Calcium Exchanger 1 Regulates Epithelial Cell Migration via Calcium-dependent Extracellular Signal-regulated Kinase Signaling

“…Na,K-ATPase has also been shown to function as a motility and tumor suppressor (13,14) and is involved in the maintenance of epithelial polarity (15) and cell adhesion (16,17). Moreover, we previously reported that knockdown of Na,K-ATPase ␤ 1 -subunit (Na,K-␤) in MDCK cells led to mesenchymal phenotype accompanied by increased cell proliferation via activation of phosphoinositide-3 kinase (PI3K)/Akt and extracellular-signal-regulated kinase (ERK1/2) pathways (18).…”

“…generated by introducing silent mutations within the shRNA recognition site in the Na,K-␤ cDNA, have been described previously (18). Immunoblot analysis confirmed 80% reduction in Na,K-␤ protein level in ␤-KD cells.…”

“…Cell Migration in ␤-KD Cells Requires Calcium-dependent ERK1/2 Activation-We previously demonstrated that ␤-KD cells have high ERK1/2 and Akt activation mediated by PI3K (18). These signaling pathways are known to play significant roles in inducing cell migration (27)(28)(29).…”