Ion dependence of Na-K-ATPase-mediated epithelial cell adhesion and migration

the role of Na-K-ATPase in the regulation of epithelial cell polarity, adhesion, and migration has been demonstrated. Some of these processes, such as tight junction (TJ) formation, require ion transport function of Na-K-ATPase by its catalytic subunit Na-K-α. Other functions, such as motility suppression, cell adhesion, and contact inhibition, are mediated by the regulatory subunit Na-K-β, typically in association with Na-K-α but independent of the pump activity. Our recent study (5) showed that Na-K-β controls cell migration and phosphatidylinositol 3-kinase (PI3K)/ERK signaling via Na/Ca exchanger 1 (NCX1)-mediated regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i), highlighting the ion dependency of Na-K-β-mediated suppression of cell migration. Na-K-β also regulates membrane localization of other ion transporters, such as the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel and Na-K-2Cl cotransporter isoform 2 (NKCC2). This leads us to speculate that these ion transporters might also be involved in facilitating additional functions of Na-K-β. In summary, we have come full circle: we started with the notion that the sole function of Na-K-ATPase is regulation of ion homeostasis, progressed to acceptance of the view that its subunit components can mediate diverse cellular processes by activating specific signaling pathways, independent of Na-K-ATPase activity, and, finally, closed the loop with recent studies that indicate that Na-K-β not only regulates membrane localization and activity of Na-K-α but also of other ion transporters such as NCX1, BK_Ca, and NKCC2, thereby governing ion homeostasis in numerous ways. Here, we propose that ion homeostasis may be crucial for the multiple cell functions mediated by Na-K-β.

Na-K-ATPase Subunit Components and Their Function in Ion Homeostasis

Na-K-ATPase is a hetero-oligomeric protein with a catalytic α-subunit (Na-K-α), a regulatory β-subunit (Na-K-β), and an auxiliary γ-subunit (reviewed in Refs. 17 and 55). Na-K-α is a polytopic membrane protein that possesses binding sites for Na⁺, K⁺, and ATP. Using energy gained from ATP hydrolysis, Na-K-α mediates efflux of three Na⁺ and influx of two K⁺ against the electrochemical gradient. Na-K-α is also the receptor for cardiac glycosides, such as digitalis and ouabain, which inhibit Na-K-ATPase activity. Na-K-β is a single-transmembrane glycoprotein with a large extracellular domain and a short cytoplasmic tail. The conventional role of Na-K-β is enhancement of the rate of synthesis, half-life, trafficking, and membrane stabilization of Na-K-α and regulation of its enzymatic activity (1, 12, 18, 41). There are four isoforms of Na-K-α and three isoforms of Na-K-β, which are expressed in a tissue-specific manner. Na-K-α₁ and Na-K-β₁ are ubiquitously expressed and, in this Viewpoint, are referred to as Na-K-α and Na-K-β, respectively, unless otherwise specified. The γ-subunit, belonging to the FXYD family of proteins, which is named based on the presence of an invariant amino acid signature sequence containing the FXYD motif (53), regulates Na-K-ATPase pump activity by binding to Na-K-α (57).

The regulation of ion homeostasis by the Na⁺ pump and its physiological role have been well studied since its discovery in 1957 (51). The pump activity is pivotal in maintaining the osmotic equilibrium and membrane potential, providing gradient for other Na⁺-dependent ion transporters and ancillary solute transport. Na-K-ATPase is functionally coupled to other ion transporters, such as NCX1 (4, 45), ATP-sensitive K⁺ channels (32, 38), glutamate transporters (46), and the BK_Ca channel (54), to execute housekeeping and specialized functions related to ion homeostasis.

Na-K-ATPase Functions in Addition to Ion Homeostasis

Recognition of the adhesion molecule on glia as Na-K-β₂, with 40% identity to Na-K-β₁ (21), indicates that other isoforms of Na-K-β may function similarly and that Na-K-ATPase subunits may have additional functions unrelated to pumping ions. Several decades after its discovery, additional functions of Na-K-ATPase, such as regulation of TJ, epithelial polarity, cell attachment, adhesion, migration, and proliferation, and signal transduction, were identified (6, 10, 13, 40, 58, 61). Epithelial cells mediate contact between adjacent cells and basement membrane via membrane protein complexes that form TJ, adherens junctions (AJ), desmosomes, gap junctions, and hemidesmosomes (15). TJ and septate junctions (the TJ equivalent in invertebrates) regulate paracellular ion transport by forming zones of apparent membrane fusions, or the “kissing points” (60), between adjacent cells and polarize the epithelial cell membrane into apical and basolateral membrane domains (11, 22, 56). Na-K-ATPase, initially used as a marker for basolateral membrane domain in epithelial cells, was later established as a regulator of TJ and epithelial polarity (13, 24, 33, 42, 64). Complete inhibition of Na-K-ATPase pump activity with ouabain for shorter duration retained cell viability but prevented TJ formation, increased paracellular permeability, and disrupted existing TJ in epithelial cells (19, 42, 64). The molecular mechanism by which Na-K-ATPase pump inhibition disrupted TJ formation involved 1) inhibition of RhoA activity, mediating rearrangement of TJ proteins and desmosomes (42), 2) reduction of protein phosphatase 2A (PP2A) activity, inducing hyperphosphorylation of the TJ protein occludin and disassembly of TJ strands (39), 3) regulation of Src family kinases (20), and 4) activation of ERK, leading to upregulation of claudin 3 and disruption of the apical junctional complex (14). These findings demonstrate the role of Na-K-ATPase pump activity in TJ biogenesis.

Increasing evidence suggests the presence of Na-K-ATPase in apical junctions (31, 39, 65). In agreement with this localization, Na-K-ATPase and its enzymatic activity are required for apicobasal polarity and vectorial transport of ions to facilitate lumen formation during organogenesis, as demonstrated in three-dimensional cultures of Madin-Darby canine kidney (MDCK) cells in collagen, zebrafish gut, Drosophila trachea, and mouse blastocysts (3, 36, 64). From these studies, we gain a new appreciation for the role of Na-K-ATPase-mediated ion homeostasis in TJ formation and organogenesis.

Na-K-ATPase Functions Independent of Ion Transport

As these additional functions of Na-K-ATPase were uncovered, it was necessary to clarify whether these functions are dependent on the pump activity or on the protein-protein interactions mediated by its constituent subunits. Pharmacological inhibition of Na-K-ATPase pump activity by ouabain at higher concentrations was used to ascertain the involvement of the ion transport activity in mediating these functions. Another approach was utilized following the identification of specific amino acid residues on opposite faces of the transmembrane domain of Na-K-β that mediate 1) homo-oligomerization and cell adhesion via the glycine zipper motif on one face and 2) hetero-oligomerization with Na-K-α via the heptad repeat motif on the other face of the transmembrane domain, thereby regulating pump activity (7). Cells expressing mutant Na-K-β that fail to hetero-oligomerize with Na-K-α were used to determine the contribution of ion transport activity in additional cell functions governed by Na-K-ATPase.

AJ mediate the formation of adhesive contacts between adjacent cells and chiefly comprise E-cadherin, a classical cell adhesion molecule, coupled to the actin cytoskeleton via interacting proteins (63, 68). Earlier studies revealed that Na-K-β functions synergistically with E-cadherin in restoring the epithelial phenotype of virally transformed cells (43). Further reports show that Na-K-β by itself can mediate intercellular adhesion by cis-homo-oligomerization, i.e., interaction between the transmembrane regions of two Na-K-β molecules within the same cell via the glycine zipper motif, in addition to Na-K-β trans-homo-oligomerization between adjacent cells facilitated by N-glycans and the immunoglobulin-like fold (resembling cell adhesion molecules) within the extracellular domain (2, 7, 50, 59, 62). Cell adhesion was unaltered in the heptad repeat motif mutant of Na-K-β, which had reduced Na-K-ATPase pump activity, confirming that Na-K-β-induced cell adhesion was independent of Na-K-ATPase pump activity (7). This characteristic of Na-K-β was shown to be K⁺-dependent (28), consistent with the presence of two K⁺-binding sites in extracellular and cytoplasmic domains of Na-K-β (12).

Knockdown of Na-K-β activated PI3K/ERK signaling associated with increased proliferation, reduced contact inhibition, and disrupted polarized phenotype in epithelial cells (8). Na-K-ATPase pump activity was not involved in these functions, since the heptad repeat mutant of Na-K-β with compromised Na-K-ATPase function did not activate PI3K/ERK signaling or disrupt epithelial polarity. Na-K-ATPase also functions as a signaling scaffold for Src, p85 subunit of PI3K, PKC, PP2A, PLC, and inositol 1,4,5-trisphosphate receptor proteins (16, 23, 37, 67, 69). Activation of these signaling pathways in the presence of ligand (cardiac glycosides) at concentrations that do not inhibit Na-K-ATPase activity resulted in proliferation or differentiation in a cell-type-specific manner (27, 30, 34, 67). Thus, regulation of signal transduction downstream of Na-K-ATPase is independent of pump activity.

Overexpression of Na-K-β in invasive MSV-MDCK cells suppressed motility and invasion (6, 43), while knockdown of Na-K-β increased migration and reduced contact inhibition (5, 8, 25), confirming the role of Na-K-β in the regulation of cell migration. This migration was dependent on protein-protein interactions of Na-K-ATPase subunits with PI3K and annexin II but independent of pump function (6). Thus the role of Na-K-β as a motility suppressor independent of ion transport function was consolidated.

Our recent study showed that activation of PI3K/ERK signaling and increased migration of Na-K-β knockdown cells were Ca²⁺-dependent (5). The increase in [Ca²⁺]_i was mediated by the reduction of functional NCX1, a major regulator of Ca²⁺ in renal epithelial cells. Although Na-K-β-mediated suppression of PI3K/ERK signaling and motility is independent of Na-K-ATPase activity, it requires NCX1 function and is Ca²⁺-dependent. Similarly, suppression of proliferation and contact inhibition may also be Ca²⁺-dependent.

Na-K-β Regulates Other Ion Transporters

We showed recently that Na-K-β interacts with NCX1 and regulates its membrane localization, ascertaining the role of Na-K-β as a molecular chaperone responsible for the membrane trafficking of proteins in addition to Na-K-α (5). Similarly, other studies have shown that Na-K-β interacts with and targets the transport of BK_Ca channels to specific regions of plasma membrane, thereby regulating [Ca²⁺]_i (26). The reduction of BK_Ca channel protein in the absence of Na-K-β led to significantly reduced membrane potential (26). Changes in membrane potential due to altered BK_Ca channel activity increased cancer cell migration and invasion (66), indicating that the BK_Ca channel may function downstream of Na-K-β in regulation of migration. Na-K-β also controls exocytosis and membrane expression of the NKCC2 channel, which is an important player in the regulation of Na⁺ and Cl⁻ reabsorption (9). We propose that Na-K-β acts as a molecular chaperone for multiple ion transporters, resulting in regulation of ion homeostasis circuitously.

Many studies have shown the involvement of specific ions and their transporters in the regulation of cell functions (29, 47, 49), for example, homeostasis of intracellular Na⁺ concentration in maintenance of the epithelial phenotype (44), [Ca²⁺]_i in regulation of cellular migration machinery and biogenesis of TJ formation (35, 52), and K⁺ balance in control of cell volume, thereby regulating sperm motility (48). Our study identified NCX1 and [Ca²⁺]_i as downstream mediators in suppression of motility by Na-K-β. It is tempting to speculate that, similar to NCX1, other ion transporters regulated by Na-K-β are also responsible for governing multiple and overlapping pathways in which Na-K-ATPase subunits regulate diverse cellular processes.

Conclusions

Na-K-ATPase, composed of evolutionarily conserved Na-K-α and Na-K-β subunits, performs numerous cell functions. Na-K-ATPase-regulating TJ biogenesis is dependent on pump activity, while Na-K-β-regulating cell migration independent of pump function was shown by Na-K-ATPase inhibition (6). Use of cells expressing heptad repeat motif mutants of Na-K-β (7) enabled the determination of a minimal contribution of Na-K-ATPase pump activity in cell adhesion and contact inhibition (8). Although Na-K-ATPase pump activity was not involved, our recent study showed that restoration of the function of another ion transporter, NCX1, in Na-K-β knockdown cells reverted [Ca²⁺]_i dependent PI3K/ERK signaling and suppressed migration (5). Moreover, Na-K-β has been shown to regulate other ion transporters, such as BK_Ca and NKCC2, apart from Na-K-α. Based on these studies, we infer that Na-K-β facilitates its moonlighting functions by regulating cell surface expression of ion transporters and, thereby, controlling ion homeostasis (Fig. 1). Thus, in disease conditions associated with loss of Na-K-β, the membrane localization and function of Na-K-α, as well as other ion transporters, are likely to be affected, leading to synchronous deterioration of ion homeostasis.

Fig. 1.

Role of Na-K-ATPase in ion-dependent regulation of epithelial cell functions. A: since its discovery by Jens Skou in 1957 (51), the role of Na-K-ATPase (composed of Na-K-α and Na-K-β subunits) in the regulation of ion homeostasis has been studied extensively. B: studies in the early 2000s elucidated other functions of Na-K-ATPase in the formation and maintenance of tight junctions (TJ) and preservation of epithelial polarity. A pictorial representation shows Na-K-ATPase localized to TJ regulating paracellular permeability and the formation of fluid-filled lumen. C: in the mid to late 2000s, various groups studied the pump-independent roles of Na-K-ATPase, specifically, the role of protein-protein interactions in the regulation of cell-cell adhesion and in suppression of motility and proliferation. The cartoon depicts protein-protein interactions mediated by Na-K-ATPase subunits. The glycine zipper motif (shown in green in the homodimerization cartoon) and N-glycan domains (shown as 3 red circles in the extracellular region) of Na-K-β mediate the interaction of Na-K-β with Na-K-β involved in cell adhesion. The heptad repeat motif of Na-K-β that mediates the interaction of Na-K-α with Na-K-β is shown in orange in the heterodimerization (Na-K-α/β) cartoon. Na-K-α interaction with Src, inositol 1,4,5-trisphosphate receptor (IP3R), and p85 subunit of phosphatidylinositol 3-kinase (PI3K) is shown, along with Na-K-β association with annexin II (A II) and protein phosphatase 2A (PP2A). D: additional functions of Na-K-β in the regulation of membrane localization of other ion transporters [such as Na/Ca exchanger 1 (NCX1), large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel, and Na-K-2Cl cotransporter isoform 2 (NKCC2)], apart from its cognate partner Na-K-α, have come to light in recent years. Recently, we showed that Na-K-β-mediated migration is Ca²⁺-dependent and the loss of Na-K-β mediates reduction in NCX1, thereby increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activating PI3K/ERK. In conclusion, ions are the mediators of Na-K-ATPase-dependent cellular processes governing formation and maintenance of the epithelial phenotype. The cartoon represents the increase in the rate of migration of epithelial cells with Na-K-β knockdown, mediated by a reduction of NCX1 and an increase in intracellular Ca²⁺.

GRANTS

This study was supported by American Heart Association Grant 10SDG260011, National Institute of General Medical Sciences Delaware CTR ACCEL Grant U54 GM-104941, National Institute of General Medical Sciences Delaware IDeA Networks of Biomedical Research Excellence Grant P20 GM-103446, and the Nemours Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.L.B. and S.P.B. prepared the figure; S.L.B. and S.P.B. drafted the manuscript; S.L.B., A.G., and S.P.B. edited and revised the manuscript; S.L.B., A.G., and S.P.B. approved the final version of the manuscript; A.G. and S.P.B. developed the concept and designed the research.