FXYD5 (dysadherin) may mediate metastatic progression through regulation of the ß-Na+-K+-ATPase subunit in the 4T1 mouse breast cancer model.

FXYD5 is a Na+-K+-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5-specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5-related morphological changes are mediated through its interaction with Na+-K+-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression may downregulate the ß1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5-expressing cells may correlate with matrix metalloproteinase 9 secretion and adhesion changes in 4T1 wild-type cells.

FXYD5 Protein Has a Pro-inflammatory Role in Epithelial Cells.

The FXYD proteins are a family of small membrane proteins that share an invariant four amino acid signature motif F-X-Y-D and act as tissue-specific regulatory subunits of the Na,K-ATPase. FXYD5 (also termed dysadherin or RIC) is a structurally and functionally unique member of the FXYD family. As other FXYD proteins, FXYD5 specifically interacts with the Na,K-ATPase and alters its kinetics by increasing Vmax However, unlike other family members FXYD5 appears to have additional functions, which cannot be readily explained by modulation of transport kinetics. Knockdown of FXYD5 in MDA-MB-231 breast cancer cells largely decreases expression and secretion of the chemokine CCL2 (MCP-1). A related effect has also been observed in renal cell carcinoma cells. The current study aims to further characterize the relationship between the expression of FXYD5 and CCL2 secretion. We demonstrate that transfection of M1 epithelial cell line with FXYD5 largely increases lipopolysaccharide (LPS) stimulated CCL2 mRNA and secretion of the translated protein. We have completed a detailed analysis of the molecular events leading to the above response. Our key findings indicate that FXYD5 generates a late response by increasing the surface expression of the TNFα receptor, without affecting its total protein level, or mRNA transcription. LPS administration to mice demonstrates induced secretion of CCL2 and TNFα in FXYD5-expressing lung peripheral tissue, which suggests a possible role for FXYD5 in normal epithelia during inflammation.

Cardiac glycosides induced toxicity in human cells expressing α1-, α2-, or α3-isoforms of Na-K-ATPase.

The Na+-K+-ATPase is specifically inhibited by cardiac glycosides, some of which may also function as endogenous mammalian hormones. Previous studies using Xenopus oocytes, yeast cells, or purified isoforms demonstrated that affinities of various cardiac glycosides for three isoforms of the Na+-K+-ATPase (α1-α3ß1) may differ, a finding with potential clinical implication. The present study investigates isoform selectivity and effects of cardiac glycosides on cultured mammalian cells under more physiological conditions. H1299 cells (non-small cell lung carcinoma) were engineered to express only one α-isoform (α1, α2, or α3) by combining stable transfection of isoforms and silencing endogenous α1. Cardiac glycoside binding was measured by displacement of bound 3H-ouabain. The experiments confirm moderate α1/α3:α2 selectivity of ouabain, moderate α2:α1 selectivity of digoxin, and enhanced α2:α1 selectivity of synthetic derivatives (Katz A, Tal DM, Heller D, Haviv H, Rabah B, Barkana Y, Marcovich AL, Karlish SJD. J Biol Chem 289: 21153-21162, 2014). Relative α2:α1 selectivity of digoxin vs. ouabain was also manifested by enhanced internalization of α2 in response to digoxin. Cellular proliferation assays of H1299 cells confirmed the patterns of α2:α1 selectivity for ouabain, digoxin, and a synthetic derivative and reveal a crucial role of surface pump density on sensitivity to cardiac glycosides. Because cardiac glycosides are being considered as drugs for treatment of cancer, effects of ouabain on proliferation of 12 cancer and noncancer cell lines, with variable plasma membrane expression of α1, have been tested. These demonstrated that sensitivity to ouabain indeed depends linearly on the plasma membrane surface density of Na+-K+-ATPase irrespective of status, malignant or nonmalignant.

Ouabain-induced internalization and lysosomal degradation of the Na+/K+-ATPase.

Internalization of the Na(+)/K(+)-ATPase (the Na(+) pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na(+)/K(+)-ATPase molecule or more generally by the disruption of cation homeostasis (Na(+), K(+), Ca(2+)) due to the partial inhibition of active Na(+) and K(+) transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K(+)-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na(+)/K(+)-ATPase complex.

Modulation of cell polarization by the Na+-K+-ATPase-associated protein FXYD5 (dysadherin).

FXYD5 (dysadherin or also called a related to ion channel, RIC) is a transmembrane auxiliary subunit of the Na(+)-K(+)-ATPase shown to increase its maximal velocity (Vmax). FXYD5 has also been identified as a cancer-associated protein whose expression in tumor-derived cell lines impairs cytoskeletal organization and increases cell motility. Previously, we have demonstrated that the expression of FXYD5 in M1 cells derived from mouse kidney collecting duct impairs the formation of tight and adherence junctions. The current study aimed to further explore effects of FXYD5 at a single cell level. It was found that in M1, as well as three other cell lines, FXYD5 inhibits transformation of adhered single cells from the initial radial shape to a flattened, elongated shape in the first stage of monolayer formation. This is also correlated to less ordered actin cables and fewer focal points. Structure-function analysis has demonstrated that the transmembrane domain of FXYD5, and not its unique extracellular segment, mediates the inhibition of change in cell shape. This domain has been shown before to be involved in the association of FXYD5 with the Na(+)-K(+)-ATPase, which leads to the increase in Vmax. Furthermore, specific transmembrane point mutations in FXYD5 that either increase or decrease its effect on cell elongation had a corresponding effect on the coimmunoprecipitation of FXYD5 with α Na(+)-K(+)-ATPase. These findings lend support to the possibility that FXYD5 affects cell polarization through its transmembrane domain interaction with the Na(+)-K(+)-ATPase. Yet interaction of FXYD5 with other proteins cannot be excluded.

Intracellular trafficking of FXYD1 (phospholemman) and FXYD7 proteins in Xenopus oocytes and mammalian cells.

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1ß1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions.

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1ß1) to form α1ß1FXYD complexes. Compared with the control (α1ß1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C(12)E(8)), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E(1) ↔ E(2) conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1ß1 or α1ß1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.

Phospholemman (FXYD1) raises the affinity of the human α1ß1 isoform of Na,K-ATPase for Na ions.

The human α(1)/His(10)-ß(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-ß-maltoside, and purified by metal chelate chromatography. The α(1)ß(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-ß(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-ß(1) and α(1)/His(10)-ß(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ∼30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)ß(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.

FXYD5 (dysadherin) regulates the paracellular permeability in cultured kidney collecting duct cells.

FXYD5 (dysadherin or RIC) is a member of the FXYD family of single-span transmembrane proteins associated with the Na(+)-K(+)-ATPase. Several studies have demonstrated enhanced expression of FXYD5 during metastasis and effects on cell adhesion and motility. The current study examines effects of FXYD5 on the paracellular permeability in the mouse kidney collecting duct cell line M1. Expressing FXYD5 in these cells leads to a large decrease in amiloride-insensitive transepithelial electrical resistance as well as increased permeability to 4-kDa dextran. Impairment of cell-cell contact was also demonstrated by staining cells for the tight and adherence junction markers zonula occludens-1 and ß-catenin, respectively. This is further supported by large expansions of the interstitial spaces, visualized in electron microscope images. Expressing FXYD5 in M1 cells resulted in a decrease in N-glycosylation of ß1 Na(+)-K(+)-ATPase, while silencing it in H1299 cells had an opposite effect. This may provide a mechanism for the above effects, since normal glycosylation of ß1 plays an important role in cell-cell contact formation (Vagin O, Tokhtaeva E, Sachs G. J Biol Chem 281: 39573-39587, 2006).

Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance.

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na(+) channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 microM range.

FXYD proteins: tissue-specific regulators of the Na,K-ATPase.

Work in several laboratories has led to the identification of a family of short single-span transmembrane proteins named after the invariant extracellular motif: FXYD. Four members of this group have been shown to interact with the Na,K-adenosine triphosphatase (ATPase) and alter the pump kinetics. Thus, it is assumed that FXYD proteins are tissue-specific regulatory subunits, which adjust the kinetic properties of the pump to the specific needs of the relevant tissue, cell type, or physiologic state, without affecting it elsewhere. A number of studies have provided evidence for additional and possibly unrelated functions of the FXYD proteins. This review summarizes current knowledge on the structure, function, and cellular distribution of FXYD proteins with special emphasis on their role in kidney electrolyte homeostasis.

A specific functional interaction between CHIF and Na,K-ATPase: role of FXYD proteins in the cellular regulation of the pump.

CHIF (corticosteroid hormone-induced factor) is a member of the FXYD family that shares approximately 50% homology with the gamma subunit of Na,K-ATPase. It is expressed in renal collecting duct and distal colon, and is upregulated by Na(+) deprivation and high K(+) diet. Both CHIF and gamma are coimmunoprecipitated by an anti-alpha subunit antibody, and alpha is immunoprecipitated by anti-gamma and anti-CHIF antibodies. (86)Rb(+) flux experiments in CHIF-transfected HeLa cells demonstrate that CHIF increases the affinity for cytoplasmic Na(+), but does not affect the affinity for extracellular K(Rb). A physiological role of CHIF in kidney function is further elucidated by the phenotypic analysis of CHIF knockout mice. Taken together with data by others, it appears that FXYD proteins are tissue-specific subunits or regulators of the Na,K-ATPase whose function is to adjust the pump kinetics to particular physiological needs.

FXYD proteins: new tissue- and isoform-specific regulators of Na,K-ATPase.

The recently defined FXYD protein family contains seven members that are small, single-span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the gamma subunit of Na,K-ATPase, which was shown to be a specific regulator of renal alpha1-beta1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the gamma subunit and have found that CHIF (corticosteroid hormone-induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K-ATPase and preferentially with alpha1-beta isozymes in native tissues, and produce distinct effects on the transport properties of Na,K-ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K-ATPase by FXYD proteins that involves a tissue-specific expression of an auxiliary subunit of distinct Na,K-ATPase isozymes.

FXYD7, the first brain- and isoform-specific regulator of Na,K-ATPase: biosynthesis and function of its posttranslational modifications.

The FXYD protein family has recently been defined as a result of the search for homologues of the Na,K-ATPase gamma subunit, CHIF, and phospholemman in EST and gene data banks. FXYD7 has been seen to have a role as a brain- and isozyme-specific regulator of Na/K-ATPase. In this study, the biosynthesis, membrane topology, nature, and role of the processing of FXYD7 are investigated.

Immunocytochemical localization of Na,K-ATPase gamma subunit and CHIF in inner medulla of rat kidney.

The gamma subunit of Na,K-ATPase and CHIF both belong to the FXYD single-membrane-spanning protein family and have been suggested to have regulatory functions in kidney tubules. CHIF is known to be present in the collecting duct, and gamma has been demonstrated in several segments of the rat kidney tubule, but never clearly in the inner medullary collecting duct (IMCD). Here, we demonstrate the cellular and subcellular localization of the gamma subunit and CHIF in the IMCD in inner medulla by using Western blotting, laser-scanning confocal immunofluorescence, and immunoelectron microscopy. In the initial quarter of the IMCD (next to the outer medulla), antibodies against the C-terminal of gamma as well as splice variant gammaa labeled the basolateral surface of intercalated cells (ICs), while principal cells (PCs) remained unlabeled. In the middle segment of the IMCD, all PCs exhibited distinct basolateral staining for the gammaC-terminal as well as gammaa and CHIF. Immunoelectron microscopy showed that the gammaC-terminal and CHIF were associated with the inner leaflet of the basolateral plasma membrane in the labeled cells. Immunoblotting demonstrated the presence of both the gammaC-terminal and gammaa in inner medullary tissue. However, splice variant gammab was not detected in inner medulla by immunocytochemistry or immunoblotting. The present observations demonstrate that the Na,K-ATPase gamma subunit and CHIF are strategically located in the inner medulla to participate in the fine-tuning of urine ion composition through the regulation of the Na,K-ATPase activity in the IMCD.

Induction of FKBP51 by aldosterone in intestinal epithelium.

Screening female rat distal colon preparations for aldosterone-induced genes identified the Hsp90-binding immunophilin FKBP51 as a major aldosterone-induced mRNA and protein. Limited induction of FKBP51 was observed also in other aldosterone-responsive tissues such as kidney medulla and heart. Ex vivo measurements in colonic tissue have characterized time course, dose response and receptor specificity of the induction of FKBP51. FKBP51 mRNA and protein were strongly up regulated by physiological concentrations of aldosterone in a late (greater than 2.5h) response to the hormone. Maximal increase in FKBP51 mRNA requires aldosterone concentrations that are higher than those needed to fully occupy the mineralocorticoid receptor (MR). Yet, the response is fully inhibited by the MR antagonist spironolactone and not inhibited and even stimulated by the glucocorticoid receptor (GR) antagonist RU486. These and related findings cannot be explained by a simple activation and dimerization of either MR or GR but are in agreement with response mediated by an MR-GR heterodimer. Overexpression or silencing FKBP51 in the kidney collecting duct cell line M1 had little or no effect on the aldosterone-induced increase in transepithelial Na(+) transport.

Structural and functional interactions between FXYD5 and the Na+-K+-ATPase.

FXYD5 is a member of a family of tissue-specific regulators of the Na(+)-K(+)-ATPase expressed in kidney tubules. Previously, we have shown that FXYD5 interacts with the alphabeta-subunits of the Na(+)-K(+)-ATPase and increases its V(max) (Lubarski I, Pihakaski-Maunsbach K, Karlish SJ, Maunsbach AB, Garty H. J Biol Chem 280: 37717-37724, 2005). The current study further characterizes structural interaction and structure-function relationships of FXYD5. FXYD5/FXYD4 chimeras expressed in Xenopus laevis oocytes have been used to demonstrate that both the high-affinity association with the pump and the increase in V(max) are mediated by the transmembrane domain of FXYD5. Several amino acids that participate in the high-affinity interaction between FXYD5 and the alpha-subunit of the Na(+)-K(+)-ATPase have been identified. The data suggest that different FXYD proteins interact similarly with the Na(+)-K(+)-ATPase and their transmembrane domains play a key role in both the structural interactions and functional effects. Other experiments have identified at least one splice variant of FXYD5 with 10 additional amino acids at the COOH terminus, suggesting the possibility of other functional effects not mediated by the transmembrane domain. FXYD5 could be specifically bound to wheat germ agglutinin beads, indicating that it is glycosylated. However, unlike previous findings in metastatic cells, such glycosylation does not evoke a large increase in the size of the protein expressed in native epithelia and X. laevis oocytes.