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Am J Physiol Lung Cell Mol Physiol 294: L654-L664, 2008. First published February 8, 2008; doi:10.1152/ajplung.00430.2007
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FXYD5 modulates Na+ absorption and is increased in cystic fibrosis airway epithelia

Timothy J. Miller1,2 and Pamela B. Davis2

Departments of 1Pharmacology and 2Pediatrics, School of Medicine, Case Western Reserve University, Cleveland, Ohio

Submitted 17 October 2007 ; accepted in final form 3 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na+-K+-ATPase. We determined the kinetic effects of FXYD5 on Na+-K+-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na+ twofold and decreased the apparent affinity for K+ by 60% with a twofold increase in Vmax of K+, a pattern that would increase activity and Na+ removal from the cell. To test the effect of increased Na+ uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na+ channel (ENaC). Na+-K+-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na+ absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na+ in the airways, accompanied by increased Na+-K+-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-{kappa}B. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na+ hyperabsorption and surface liquid dehydration observed in CF airway epithelia.

Na+-K+-ATPase; sodium transport

THE FXYD protein family, typified by the {gamma}-subunit (FXYD2) of the Na+-K+-ATPase, is identified by a signature 35-residue domain containing an invariant, extracellular PFXYD sequence (7, 37). The mammalian family contains seven members, many of which have been shown to be tissue-specific subunits of the Na+-K+-ATPase responsible for fine tuning its kinetic behavior in response to extracellular signals (reviewed in Ref. 12,13). Although the primary function of Na+-K+-ATPase is to maintain intracellular sodium-potassium homeostasis, decreased expression of the {alpha}- and β-subunits has been correlated with neoplastic transformation (21, 28), and recent literature has implicated FXYD proteins in cancer progression (14, 17, 21, 25, 29–33).

Originally identified as a gene induced in NIH-3T3 cells transformed with the oncoprotein E2a-Pbx1, FXYD5 was subsequently cloned and characterized as a cancer-associated cell membrane glycoprotein (10, 14). FXYD5 encodes a 178-amino acid protein that includes a putative signal sequence, a single transmembrane domain, and a short cytoplasmic tail. FXYD5 is unique in the FXYD family, possessing a heavily O-glycosylated, extended extracellular domain (14, 40). Transfection of FXYD5, also known as dysadherin, into liver cells has led to decreased cell-cell adhesion correlated with diminished E-cadherin levels (14). Elevated FXYD5 expression in tumors from patients with thyroid, esophageal, colorectal, stomach, cervical, pancreatic, testicular, head/neck, or lung cancer correlates with a poor prognosis, suggesting that FXYD5 may be a critical determinant regulating the role of the Na+-K+-ATPase in determining cell adherence and polarity (1–3, 17, 29–32, 38, 41). Indeed, knockdown of FXYD5 expression correlates with decreased cell motility (33, 40), independent of E-cadherin expression, and recent evidence indicates that murine FXYD5 interacts with and may regulate the Na+-K+-ATPase in Xenopus laevis oocytes (18, 19). However, it is unclear how FXYD5 affects the functional properties of Na+-K+-ATPase pump activity.

FXYD5 is highly expressed in the basal layer of squamous epithelia, endothelia, and lymphocytes, as well as in ion transport tissues such as the kidney and lung (14, 30, 40, 41), where modulation of the Na+-K+-ATPase may be critical for specialized functions such as Na+ reabsorption. In lung epithelial cells, the Na+-K+-ATPase is critical for maintaining transepithelial Na+ transport and maintaining the alveolar fluid absorption necessary for efficient gas exchange. Thus we investigated whether FXYD5 expression is altered in epithelial cell and animal models that exhibit altered transepithelial Na+ transport.

Cystic fibrosis (CF) is a common genetic disease among Caucasians, caused by lesions in the CF transmembrane conductance regulator gene CFTR that result in the inability to transport Cl– through the apical membrane, particularly in epithelia lining the airway. A hallmark of CF is the hyperabsorption of Na+ through the epithelial Na+ channel (ENaC) (20). In the airway, increased Na+ uptake is postulated to dehydrate the periciliary layer, impair mucociliary clearance, cause mucous buildup in the lung, and lead to bacterial trapping. The increased bacterial burden causes infection, inflammation, and tissue damage characteristic of CF lungs (20). Because it has been shown that airway epithelia from CF patients have increased Na+-K+-ATPase pump number and activity compared with that shown in normal subjects (5, 26), we investigated whether FXYD5 is altered in the airway epithelia of CF mice and human systems and examined how FXYD5 may alter transepithelial Na+ transport along the ENaC-Na+-K+-ATPase axis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell Lines

The mouse lung epithelial cell line LA4, human embryonic kidney (HEK)-293 cells, and Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (Manassas, VA). LA4 cells were grown in Kaighn's modification of F12 medium (Mediatech, Herndon, VA), HEK-293 cells were grown in Earle's modification of MEM media (Mediatech), and MDCK cells were grown in 1:1 (vol/vol) DMEM-F-12 media (Mediatech). An MDCK cell line stably transfected with an inducible vector expressing the {alpha}-, β-, and {gamma}-subunits of the ENaC, a generous gift of Dr. Calvin Cotton [Case Western Reserve University (CWRU), Cleveland, OH], has been previously described (MDCK clone 29.1; Refs. 22, 34) and is referred to here as the MDCK-ENaC cell line. ENaC expression was induced by the addition of 1 µM dexamethasone and 2 mM sodium butyrate to serum-free MDCK culture medium and analyzed 36–48 h later. All media were supplemented with 10% heat-inactivated FBS, and all cells were grown in a 37°C, 5% CO2-95% O2 atmosphere.

Mouse Strains

Breeding pairs of heterozygote congenic mice (>N10) bearing the S489x mutation (B6.129P2-Cftrtm1unc; stock no. 2196) and C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Breeding pairs of heterozygote mice bearing the {Delta}F508 Cftr mutation in mixed genetic background were a kind gift from Dr. Kirk Thomas from the University of Utah and were backcrossed into the C57BL/6J background for at least 10 generations in the CF Animal Core Facility at CWRU before use. CF mice for these strains are indicated by their Cftr mutation and are referred to simply as "CF mice." Breeding pairs of heterozygote mice bearing the Na+ channel, nonvoltage-gated 1 β, Scnn1b, under control of the rat secretoglobin, family 1A, member 1, Scgb1a1 promoter [B6;C3H-Tg(Scgb1a1-Scnn1b)6608Bouc/J; stock no. 005315] were purchased from Jackson Laboratory. These mice are on the B6C3F1/J background and are a F1 hybrid cross of C57BL/6J and C3H/HeJ mice. All animal studies were performed under protocols approved by the CWRU Institutional Animal Care and Use Committee.

Human Nasal Scrapes

Nasal epithelia from CF patients and control individuals were obtained as previously described (16). Three or four scrapes from one CF female patient and three male CF patients (18–42 yr old) and six female and one male normal volunteers (26–53 yr old) were obtained and immediately stored at 4°C in RNAlater (Qiagen, Valencia, CA) for processing. Parallel scrapes were obtained and scored for percent epithelial cells present, which was used to normalize sample data. The protocol was approved by the Institutional Review Board. Human patients and volunteers gave informed consent under protocol approved by the Institutional Review Board, University Hospitals, Cleveland, OH.

Human Tracheal Epithelial Cells and Treatment With CFTR Inhibitor I-172

Human tracheal epithelial (HTE) cells were recovered from necropsy specimens as previously described (8) under an exempt Institutional Review Board protocol. Multiple donors were used for analysis. Briefly, HTE cells were grown in an air-liquid interface on collagen-coated, semi-permeable membranes (1 x 106 cells/1-cm2 filter, Transwell-clear polyester membrane; Costar, Corning, NY) as previously described (8, 27) and allowed to differentiate in serum-containing medium for 3 or 4 wk. Then, on day 0, cells were switched to submerged culture in serum-free medium and treated with either DMSO (1:1,000, vehicle control, normal cells; Sigma, St. Louis, MO) or 20 µM CFTR inhibitor I-172 (a kind gift from Alan Verkman, UCSF), prepared in DMSO, and diluted from a 1:1,000 stock. Similarly, the NF-{kappa}B inhibitor pyrrolidinecarbodithioate (PDTC; Sigma) was diluted in serum-free medium to a final concentration of 0.1 mM and added simultaneously with CFTR I-172 to both the basolateral and the apical media, which were replenished every 24 h. Cells were isolated for analysis after 3 days of drug treatment.

HTE were stimulated for 1 h at 37°C with 10 ng/ml TNF-{alpha} and 5 ng/ml IL-1β (Sigma) in 200 µl of HBSS (Invitrogen) added to the apical surface. NF-{kappa}B activation was inhibited by pretreating HTE with 20 µM BAY 11 7085 (a kind gift from John Mieyal, CWRU, Cleveland, OH) for 1 h. After stimulation, the apical surface was washed twice with HBSS, the air-liquid interface was reestablished, and the cells were isolated for RNA preparation 4 h later.

RNA Isolation

Total RNA was isolated from 6- to 8-wk-old mice and 10-cm dishes of LA4 cells, HEK-293 cells, MDCK cells, or HTE cultures using the RNAprotect kit according to the manufacturer's instructions (Qiagen) and stored at –80°C. RNA was quantified on a spectrophotometer and visualized by agarose gel electrophoresis to determine quality.

RT-PCR, Cloning, and Site-Directed Mutagenesis of FXYD5

The Superscript II one-step RT-PCR kit (Invitrogen) was used to reverse transcribe and amplify FXYD5 from HEK-293 cells. FXYD5 cDNA was isolated by RT-PCR using primers designed from accession numbers NM014164 (human) and NM008761 (mouse), which contained a HindIII and NotI restriction site on the 5' and 3' end, respectively. The following primers were used to generate FXYD5 clones: human 5'-AAGCTTGCTAGCGCCGCCACCATGTCGCCCTCTGGTCGCCTGTGTCT (forward) and 5'-AGTCGTCTAGATCACCTGCAACGATTCCGGCATAAC (reverse); mouse 5'-AAGCTTGCTAGCGCCGCCACCATGTCACTGTCCAGTCGCCTGTGTCT (forward) and 5'-AGTCGTCTAGATCACCTGTGGCGATTCAGGCAAATT (reverse). RT-PCR was performed as follows: reactions were incubated at 50°C for 30 min, followed by 2-min initial denaturation at 95°C and 40 cycles of 94°C, 1-min denaturation, 1-min 58°C primer annealing, and 45 s of primer extension. RT-PCR products were digested with HindIII and NotI restriction enzymes and agarose gel purified using the Qiaquick gel purification kit (Qiagen). cDNAs were subcloned in pBSK2 vector to create pBhF5k (human) and pBmF5k (murine). To generate an NH2-terminal Flag tag in human FXYD5 that did not alter the NH2-terminal signal sequence, codon Q22 was mutated using the Quickchange site-directed mutagenesis kit (Stratagene). A silent mutation was introduced (CAG mutated to CAA) to create a new restriction site (Acl1). The following sequence was then inserted in frame at the Acl1 site to produce pBhF5kQ22Flag: 5'-CGGATTACAAAGATGATGATGATAAGA-3'. The original and modified versions of FXYD5 were then subcloned into the pTracerCMV2 plasmid (Invitrogen) using the EcoRV/NotI restriction sites to create the pThF5kQ22Flag vector used for stable cell line expression. All cDNA sequences and mutations were verified by double-strand DNA sequencing.

Creation of MDCK-hF5Flag Stable Cell Line

MDCK cells were transfected in 10-cm dishes with vector (sham) or pThF5kQ22Flag using Fugene 6 (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions to create the MDCK-sham and MDCK-hF5Flag cell lines. Each line was selected and maintained in 400 µg/ml zeocin and analyzed after 3-wk incubation by fluorescence-activated cell sorting using the BD- FACSAria (BD Biosciences, San Jose, CA) at 488 nM to obtain multiple positive clones. Positive clones were identified by immunoblot and immunofluorescence analysis using antibodies directed against the Flag epitope [Sigma and Bethyl Labs (Montgomery, TX)] and the {alpha}1-Na+-K+-ATPase (clone 464.6; Millipore, Billerica, MA).

Quantitative RT-PCR

Total RNA (0.5 µg) was utilized to synthesize cDNA with the first-strand cDNA synthesis kit (Roche Molecular Biochemicals, Mannheim, Germany). cDNA synthesis was performed with oligo-p(dT)15 primer per kit instructions and stored at –20°C. Quantitative PCR was performed using the Lightcycler FastStart DNA master Sybr green kit (Roche). The mouse and canine sequences are conserved across the primer sequences used in this study. The primer sequences used for human and mouse FXYD5 are as follows: human 5'-ATGTCGCCCTCTGGTCGCCTG (forward) and 5'-TCACCTGCAACGATTCCGGCA (reverse); mouse 5'-ATGTCACTGTCCAGTCGCCTGTGTCTCCTCACT (forward) and 5'-TCACCTGTGGCGATTCAGGCAAAATTGAGACAA (reverse). DNA was amplified with a Roche Lightcycler with the following parameters: 95°C for 10-min initial denaturation followed by 35 cycles at 95°C for 10-s denaturation, 58°C for 7-s anneal, and 72°C for 10-s extension. Copy number was measured against a standard curve of linearized plasmid DNA and normalized to GAPDH. Second point derivatives were used for PCR analysis. Specificity of DNA amplification was assessed by melting point analysis and confirmed by agarose gel electrophoresis.

Preparation of Polyclonal FXYD5 Antibody

The sequence GKCRQLSQFCLNRHR was used by Covance (Princeton, NJ) to create rabbit polyclonal antibodies directed against FXYD5. Antisera were screened by ELISA and analyzed by immunoblot to identify successful immunization. Serum was collected, stored at –20°C, and affinity purified. FXYD5 562 polyclonal antisera recognized mouse FXYD5 as analyzed by immunohistochemistry of squamous epithelia (supplemental data) and immunoblot analysis (supplemental data). (Supplemental data for the article is available at the Am J Physiol Lung Cell Mol Physiol website.)

Immunohistochemistry of Mouse Tissue

Adult {Delta}F508 mice and their wild-type littermates were killed, and the lungs were fixed by perfusion using 2% paraformaldehyde in 1x PBS (pH 7.4) via the left ventricle. Lung tissue was immersed in the same fixative solution for 48 h at room temperature, dehydrated, and embedded in paraffin using standard histological procedures. Sections (5 µm) were mounted onto Fisher Superfrost slides coated with Vectabond (Fisher 12-550-15). After deparaffinization, the sections were treated with cold methanol for 15 min followed by 1% hydrogen peroxide in distilled water for 30 min at room temperature to block endogenous peroxidase activity, washed with PBS, and incubated with 1.5% normal goat serum-PBS (PK-6101; Vector Laboratories, Burlingame, CA) for 4 h at room temperature. The sections were then incubated with 6.8 µg/ml of affinity-purified 562 polyclonal rabbit antibody against FXYD5 overnight at 4°C. Blocking solution without primary antibody was used as a negative control. After sections were washed three times in 1x PBS, they were incubated with 1.5% biotinylated goat anti-rabbit IgG antibody for 1 h at room temperature. After washing three times with PBS, the sections were incubated with Vectastain ABC reagent for 30 min and washed again. The sections were developed with 3,3'-diaminobenzidine using 3,3'-diaminobenzidine substrate kit (Vector Laboratories) for 5 min. After they were washed two times with water, the sections were counterstained with hematoxylin (Thermo Electron, Waltham, MA).

Isolation of Crude Membranes

Crude membranes were prepared as previously described (19). Mouse tissue or cultured cells were isolated, washed with PBS, suspended in 25 mM imidazole, 1 mM EDTA, 250 mM sucrose, and protease inhibitor cocktail (Sigma), and manually homogenized with 35 strokes of a prechilled Dounce homogenizer. Nuclei, unbroken cells, and mitochondria were separated by centrifuging at 6,000 g for 5 min. The supernatant was collected and saved, whereas the pellet was homogenized again and centrifuged. The two supernatants were combined and centrifuged at 125,000 g to pellet crude microsomal membranes. The pellets were resuspended in 25 mM imidazole, 1 mM EDTA, and 10 mM RbCl and stored at 4°C.

Indirect Immunofluorescence

Cells were cultured in four-well chamber slides (Permanox), washed twice with PBS, and fixed for 5 min in 100% ice-cold methanol. Slides were then rinsed twice with PBS and blocked for nonspecific antibody binding by incubating the cells in 4% BSA for 1 h (Invitrogen). Primary rabbit anti-ECS (Flag) antibody (2 µg/ml; Bethyl Labs) or mouse anti-{alpha}1-subunit Na+-K+-ATPase antibody (1 µg/ml clone 464.6; Millipore) was diluted in 1% BSA-PBS, incubated on monolayers for 45 min, and aspirated. Monolayers were washed twice with PBS and then incubated in 1% BSA-PBS containing goat anti-mouse Alexa Fluor 488 or goat anti-rabbit IgG Alexa Fluor 568 (1:250; Invitrogen) for 45 min. Cells were then washed twice, and the nuclei were counterstained with Hoechst 33342 dye (Invitrogen) and mounted with Fluormount-G. Slides were allowed to dry overnight, and immunofluorescent localization was assessed on a Zeiss 200M Axiovert inverted microscope with a DG4 switchable fluorescent light source (Sutter Instrument, Novato, CA) and a 12-bit CoolSnap HQ camera (Roper Scientific, Tucson, AZ) under control of MetaMorph version 6.2 (Molecular Devices, Sunnyvale, CA). Images were obtained with a x63 numerical aperture 1.3 fluar lens using excitation and emission filter passbands of 260 ± 20 and 645 ± 30 nm, respectively. Typical exposure time for individual frames was 150 ms.

86Rb+ Uptake Transport Assay

Unidirectional Rb+ influxes into cells were measured and calculated as previously described, using 86Rb+ as a congener of K+ uptake, with minor modifications (23). Briefly, cells were grown to confluence in 24-well tissue culture plates (Costar) for 2–3 days. All solutions were maintained at 37°C. For Rb+ (K+)-dependent 86Rb+ uptake, cells were washed twice in the following assay buffer (in mM): 140 choline chloride, 10 NaCl, 10 HEPES (pH 7.4), 5 glucose, 1 MgCl2, 1 CaCl2, and 3 BaCl2, as well as 5 µM monensin and 50 µM bumetanide. For assays that varied intracellular Na+-dependent 86Rb+ uptake, the wash and incubation solutions were similar except that 2 mM RbCl, 10 µM monensin, and 2–70 mM NaCl were used with choline chloride added to maintain isotonicity. Cells were then preincubated in assay buffer in the presence or absence of 100 µM ouabain for 10 min at 37°C and washed twice. The assay was initiated by the addition of 25 µl (1–2 µCi 86RbCl) to 225 µl of assay buffer but with various concentrations of RbCl per well. The reaction was carried out for 6 min at 37°C during which the rate of 86Rb+ uptake remained constant (data not shown) (6), and the cells were washed in assay buffer without monensin. Cells were allowed to air dry for 1 h, solubilized in 500 µl of 2% SDS and 0.2 mM NaOH overnight, and sampled to determine 86Rb+ uptake in a scintillation counter. Multiple hF5Flag-positive clones were tested, with no significant difference in 86Rb+ uptake observed between clones. Ouabain-sensitive 86Rb+ uptake was calculated as the difference between total and ouabain-insensitive 86Rb+ accumulation, and samples were normalized to protein content using the DC protein assay (Bio-Rad).

Kinetic Analysis

The data for Na+- and K+-dependent 86Rb+ (K+) influxes were analyzed by nonlinear regression using Prism 4 (Graphpad Software). All data were analyzed with a Michaelis-Menten model for either a noncooperative two-site or three-site model that assumes identical noninteracting ligand binding sites as previously described (11, 15, 23, 39):

Formula 1(1)
where n is the number of sites and Ks is the apparent affinity for extracellular K+ or for intracellular Na+. Data were also analyzed according to a highly cooperative model:

Formula 2(2)
where K0.5 = K'1/n and n is the number of sites for extracellular K+ (n = 2) or cytoplasmic Na+ (n = 3). Experimental data points were fit to Eqs. 1 and 2 to obtain the values of Vmax and apparent K0.5. Specificity constants were obtained with the relationship

Formula 3(3)
where (S) represents the values obtained for Na+ or K+. Results from both Eqs. 1 and 2 are presented in Table 1, whereas Fig. 2 shows curves that have been fit using Eq. 1.


View this table:
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  		Table 1. Comparison of intracellular Na+ and extracellular K+ activation of Na+-K+-ATPase pump activity in FXYD5-transfected MDCK cells

 

Figure 2
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  		Fig. 2. Ouabain-sensitive 86Rb+ uptake in MDCK-hF5Flag cells. MDCK-hF5Flag ({blacktriangleup}) or MDCK-sham control ({blacksquare}) cells were preincubated 15 min with or without 100 µM ouabain as described in MATERIALS AND METHODS. 86Rb+ fluxes were measured over 6 min at constant external NaCl and varying external RbCl (0–2,000 µM; A) or constant external RbCl and varying external NaCl (0–70 mM; B). Inset: Scatchard analysis indicating noncooperativity of Rb+ binding (inset A) or negative cooperativity of Na+ binding (inset B). Initial ouabain-sensitive 86Rb+ (K+) influx rates were fit to Eq. 1 (noncooperative model) and represent 6 independent experiments ± SE. Kinetic constants are summarized in Table 1.

 
Statistics

Microsoft Excel was used for calculating Student's t-test. Sigma Stat (Systat Software, San Jose, CA) and Prism 4 (Graphpad Software) were used to calculate ANOVA statistics, using the Student-Neuman-Kuels regression analysis for pairwise comparisons. The Wilcoxon matched pairs test was used to calculate significance values for 1-substrate kinetic data obtained from 86Rb+ uptake experiments. P < 0.05 was used to declare statistical significance, unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
FXYD5 Modulates Na+-K+-ATPase Ion Transport

MDCK-hF5Flag cells stably express FXYD5 at the cell membrane. To develop an epithelial cell culture model system to assess the kinetic properties of FXYD5 on the Na+- K+-ATPase, MDCK cells were stably transfected with pThF5kFlag or empty (sham) vector and selected in zeocin. After 3 wk, positive clones were identified by fluorescence-activated cell sorting analysis (data not shown), subcultured, and assessed for immunofluorescence reactivity to anti-Flag antibodies. Cells were fixed in methanol and immunostained for FXYD5-Flag and the {alpha}1-subunit of the Na+-K+-ATPase. The Flag antibody reacted strongly to MDCK-hF5Flag cells and recognized membrane-localized mature human FXYD5 but not the empty vector (sham) control (Fig. 1, A and D). Immunofluorescent staining of the {alpha}1-subunit of the Na+-K+-ATPase demonstrated that membrane localization of pump subunits was unchanged in pThF5kFlag-transfected cells (Fig. 1, C and F) and that FXYD5-Flag colocalized with the Na+-K+-ATPase in the cell membrane (Fig. 1, B and E). Immunoblot analysis of crude membrane preparations of MDCK-hF5Flag cells demonstrated that mature human FXYD5-Flag protein migrated as an indistinct band of ~35 kDa, indicating heavy glycosylation (Fig. 1G), and that surface expression of the Na+-K+-ATPase was similar to vector-transfected cells (Fig. 1H). In separate experiments, FXYD5-Flag was coimmunoprecipitated with the {alpha}-subunit of the Na+-K+-ATPase, and this subunit was coimmunoprecipitated with antibody to Flag or to FXYD5 (data not shown). Thus we developed an appropriate epithelial cell model for testing the kinetic parameters of FXYD5 and its effects on Na+-K+-ATPase pump activity.


Figure 1
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  		Fig. 1. Madin-Darby canine kidney (MDCK)-hF5Flag cells stably express FXYD5-Flag at cell membrane. MDCK cells were stably transfected with pThF5kFlag or sham vector control and stained with anti-Flag antibody coupled to anti-rabbit Alexa Fluor 568 (red; A and D) and anti-{alpha}1-Na+-K+-ATPase antibody coupled to anti-mouse Alexa Fluor 488 (green; C and F) antibodies. Nuclei were counterstained with Hoechst dye (blue). Colocalization of FXYD5-Flag and Na+-K+-ATPase is observed (merge; E) compared with sham control (merge; B). G: immunoblot analyses of FXYD5-Flag, {alpha}1-subunit Na+-K+-ATPase, or actin loading controls demonstrating expression of FXYD5-Flag, but unchanged expression of Na+-K+-ATPase between sham and FXYD5-Flag cell lines. H: densitometric analysis of 4 blots of {alpha}1-Na+-K+-ATPase membrane preparations shown in G normalized to actin, demonstrating no significant (NS) change in {alpha}1-Na+-K+-ATPase.

 
FXYD5 alters Na+-K+-ATPase pump kinetics in MDCK-hF5Flag cells. The proteins of the FXYD family are believed to be responsible for fine tuning the kinetics of Na+-K+-ATPase pump activity in a tissue-specific manner. However, although previous studies have shown that FXYD5 associates with the Na+-K+-ATPase, it is unclear how FXYD5 modulates pump activity. To assess this question, Na+- and K+-dependent 86Rb+ (a congener of K+) uptake results were compared in MDCK-hF5Flag and vector control cells. Data were analyzed by fitting experimental data points to nonlinear regression models based on Michaelis-Menten kinetics as originally described by Garay and Garrahan (11) and are summarized in Table 1. Cells were incubated with 50 µM bumetanide and in the presence or absence of 100 µM ouabain to determine Na+-K+-ATPase-specific 86Rb+ uptake. Figure 2A shows that FXYD5 significantly increased the K0.5 for K+ by ~60% (K0.5 = 0.07 ± 0.01 mM for vector and 0.12 ± 0.01 mM for FXYD5 transfected; P < 0.03) and increased the K+-dependent maximal Na+-K+-pump uptake twofold compared with vector-transfected control MDCK cells (P < 0.01). In contrast, FXYD5 decreased the K0.5 for Na+ twofold (K0.5 = 9.24 ± 2.68 mM for vector and 4.53 ± 1.54 mM for FXYD5-transfected cells; P < 0.05) without significantly affecting the Na+-dependent maximum pump rate (Fig. 2B). The specificity constant (Vmax/K0.5) was calculated and used to compare Na+-K+-ATPase pump activity in the presence or absence of FXYD5 (Table 1) and to demonstrate that FXYD5 increases the overall pump efficiency for both Na+ and K+. These results indicate that, similar to FXYD4 (CHIF), FXYD5 increases Na+-K+-pump transport activity about fourfold at physiologically low intracellular Na+ concentrations (4) by increasing the apparent affinity for Na+ and increasing the maximal rate of K+ transport. Together, these data suggest that FXYD5 may be involved in the regulation of active Na+ reabsorption in ion transport tissues such as the lung and kidney.

FXYD5 is Decreased During Chronic Na+ Absorption

FXYD5 is downregulated after ENaC activation in MDCK-ENaC cells. To investigate whether increased Na+-K+- ATPase activity affects FXYD5 expression, we utilized a stable cell line that expresses high levels of the {alpha}-, β-, and {gamma}-ENaC subunits on induction in serum-free medium (34). Previous studies have shown that induction of ENaC expression generated a large increase in amiloride-sensitive short-circuit current, but the effect on Na+-K+-ATPase activity was not shown (22). As expected, induction of ENaC expression increased the amount of Na+-K+-ATPase immunostaining in the membranes of induced compared with control cells (Fig. 3, A–D). Similarly, a significant sixfold increase in ouabain- and bumetanide-sensitive 86Rb+ uptake was observed in MDCK-ENaC cells after ENaC induction (n = 10, P < 0.0001), indicating a large increase in Na+-K+-ATPase pump activity (Fig. 3E). Interestingly, quantitative RT-PCR (Fig. 3F) revealed a 75% decrease in FXYD5 expression in MDCK-ENaC-induced cells (n = 6, P < 0.0001). This suggests that overexpression of ENaC subunits, which results in increased Na+ extrusion via the Na+-K+-ATPase, may downregulate FXYD5 expression in epithelia.


Figure 3
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  		Fig. 3. Epithelial Na+ channel (ENaC) activation increases Na+-K+-ATPase expression and activity but downregulates FXYD5. MDCK-ENaC cells were incubated for 48 h in serum-free media (control; A and C) or induction media (induced; B and D) to stimulate the expression of the ENaC. Cells were incubated with antibodies against the {alpha}1-subunit of the Na+-K+- ATPase, stained using anti-mouse IgG antibodies labeled with Alexa Fluor 568 (red), and imaged for 150 ms. Nuclei were counterstained with Hoechst dye (blue). E: Na+-K+-ATPase activity was assessed by 86Rb+ uptake and demonstrated a 6-fold increase in MDCK-ENaC-induced cells compared with controls (n = 10). *P < 0.0001. F: quantitative RT-PCR analysis revealed a 75% decrease in FXYD5 expression in induced vs. control cells (F). Data were normalized to GAPDH expression (n = 6). *P < 0.0001.

 
FXYD5 is decreased in the lungs of Scnn1b transgenic mice. To confirm these findings in an in vivo model, mice overexpressing Scnn1b (ENaC β-subunit) were examined. These mice exhibit accelerated Na+ absorption in lower airway epithelia and demonstrate some of the features of airway pathophysiology observed in CF (20). Quantitative RT-PCR analysis of lung tissue from Scnn1b-positive mice revealed a 40% decrease in FXYD5 expression compared with that shown in control littermates (Fig. 4A) (n = 4, P < 0.0001). Immunoblot analysis of crude membrane preparations confirmed that FXYD5 expression was decreased in Scnn1b-positive mouse lung tissue (Fig. 4B). Because of the multiple cell types present in total lung preparations, these results may underestimate the actual downregulation of FXYD5 specifically in lung epithelia. From our kinetic analysis of FXYD5 effects on Na+-K+-ATPase pump activity and our biochemical data indicating that FXYD5 is negatively regulated in vitro and in vivo by Na+ hyperabsorption, we speculated that FXYD5 expression would be decreased in CF airway epithelia.


Figure 4
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  		Fig. 4. FXYD5 is decreased in lung tissue from mice that overexpress the Scnn1b transgene. Transgenic mice overexpressing the Scnn1b (ENaC β-subunit) transgene were assessed for FXYD5 expression. A: quantitative RT-PCR analysis of lung tissue from ENaC+/– mice exhibited a significant 40% decrease in FXYD5 expression (n = 4; *P < 0.0001) compared with wild-type control littermates. Data were normalized to GAPDH expression. B: representative immunoblot analysis of crude membrane preparations of ENaC+/– vs. ENaC–/– lung tissue. The immunoblot was cut in half and stained with either FXYD5 antibody 562 or actin as a loading control. Shown is a decrease in FXYD5 expression in ENaC+/– mouse lung.

 
FXYD5 is Upregulated in CF Airway Epithelia

FXYD5 is upregulated in the nasal epithelia of CF mice. CF murine nasal epithelium is the airway tissue that most closely approximates the ion transport abnormalities observed in human CF lung and nasal epithelia, including ENaC upregulation. In addition, the nasal epithelium of mice can be dissected from the underlying tissues and studied in isolation. Therefore, we utilized nasal epithelium from mice homozygous for the S489x mutation in Cftr for analysis of FXYD5 mRNA and protein expression. Quantitative RT-PCR analysis of nasal epithelia from S489x–/– mice revealed a significant (P < 0.001), nearly threefold increase in FXYD5 transcription compared with epithelia from wild-type littermates (Fig. 5A). To test whether this increase in FXYD5 mRNA transcription was accompanied by an increase in FXYD5 protein level, we developed a polyclonal antibody that recognized the mature (highly glycosylated) form of murine FXYD5 at ~27 kDa (supplemental data). (Supplemental data for the article is available at the Am J Physiol Lung Cell Mol Physiol website.) Immunoblot analysis of membrane fractions from nasal epithelia confirmed an increase in mature FXYD5 expression from S489x–/– CF mice compared with wild-type littermates (Fig. 5B).


Figure 5
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  		Fig. 5. FXYD5 is upregulated in the nasal epithelia of S489x–/– cystic fibrosis (CF) mice. A: quantitative RT-PCR analysis revealed that FXYD5 normalized to GAPDH expression was increased in nasal epithelia from S489x–/– CF mice compared with S489x+/+ littermate controls (+/+: n = 4; –/–: n = 5) *P < 0.001. B: immunoblot, representative of 4 independent experiments, of membrane preparations of nasal epithelium from S489x+/+ and S489x–/– mice using FXYD5 562 antisera and actin loading control.

 
FXYD5 is increased in lungs of CF mice. Upper and lower airway epithelia from the lungs of {Delta}F508–/– CF mice were retrieved for immunohistochemical analysis. Similar to our observations in the nasal epithelia of S489x–/– CF mice, FXYD5 expression was increased in the upper airway epithelia of {Delta}F508 CF mice compared with wild-type littermates (Fig. 6, A and B). Interestingly, FXYD5 immunoreactivity was observed within the cytoplasm and at the cell surface in upper airway epithelia, similar to the pattern of tumor and nontumor staining in squamous epithelia observed by us and others (14, 17). Although FXYD5 was increased in CF mice in the lower airway epithelium, here it appeared to localize to the cell membrane (Fig. 6D, arrow), indicating a possible cell-type-specific mechanism governing membrane insertion. No-primary-antibody control tissue showed no staining (Fig. 6, E and F). Similar results were observed in lung tissue from S489x–/– mice (data not shown). Immunoblot analysis performed on crude membrane fractions from {Delta}F508–/– CF lung tissue confirmed that FXYD5 is increased in the CF lung (Fig. 6G).


Figure 6
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  		Fig. 6. FXYD5 is increased in airway epithelia of CF mice. Upper airway and lung tissues were collected from adult {Delta}F508–/– mice (B, D, and F) and their wild-type (Wt) littermate controls (A, C, and E). A–D: representative images taken from upper airway (A and B) or lung (C and D) and stained with affinity-purified 562 FXYD5 antibody. Arrows denote positively stained airway and alveolar epithelial cells. E and F: representative of no-primary-antibody upper airway controls. Images taken under x40 objective lens. G: representative immunoblot demonstrating increased FXYD5 expression in crude membrane preparations from {Delta}F508 CF mice compared with wild-type control littermates.

 
CFTR inhibition upregulates FXYD5 in human epithelia. To test whether increased FXYD5 expression also occurs in human CF epithelium, we obtained nasal scrapings from CF and control patients and performed quantitative RT-PCR for FXYD5 mRNA. FXYD5 is increased in nasal epithelia from CF patients (P < 0.02; Fig. 7A) compared with controls. We also studied human airway epithelial cells cultured at the air-liquid interface and nasal scrapes from control and CF patients. Cells from the same donor grown in this manner with and without the inhibitor are well matched for other genetic and environmental influences and constitute a valid test of the effect of lack of CFTR function in isolation from other factors. Previous studies have shown that treatment of non-CF HTE cells grown on filters at the air-liquid interface in the presence of the CFTR inhibitor I-172 for 72 h resulted in markedly reduced CFTR activity, no increase in ENaC activity, and increased inflammatory response (27). HTE cells treated for 3 days with CFTR I-172 display a significant (P < 0.001) 40% increase in FXYD5 expression compared with control cultures, and this increase was completely abrogated by treatment with the NF-{kappa}B inhibitor PDTC (P < 0.01) (Fig. 7B). Similarly, stimulation of HTE cells with TNF-{alpha}-IL-1β for 1 h increased FXYD5 expression by ~60% (P < 0.001) after 4 h, which was blocked by pretreatment with the NF-{kappa}B inhibitor BAY 11 7085 (P < 0.01) (Fig. 7C). Therefore, FXYD5 is increased in human CF airway epithelia and in CF mice, possibly because of alterations in proinflammatory signaling observed in the CF airway.


Figure 7
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  		Fig. 7. CFTR inhibition upregulates FXYD5 in human airway epithelia. A: RNA was prepared from 4 CF and 7 non-CF human nasal scrapings as described in MATERIALS AND METHODS. Parallel samples were used to normalize copy number according to %epithelial cells retrieved in scrapings, which varied from 84 to 95% or from 77 to 98% in CF or non-CF samples, respectively. *P < 0.02. B: quantitative RT-PCR analysis of FXYD5 in human tracheal epithelial (HTE) cells treated with CFTR inhibitor (Inhib)-172 for 3 days increased FXYD5 expression, which was abrogated by including NF-{kappa}B inhibitor pyrrolidinecarbodithioate (PDTC) (control: n = 11; Inhib-172: n = 7; PDTC: n = 4). *P < 0.001 vs. control; #P < 0.01 vs. Inhib-172. C: quantitative RT-PCR analysis of FXYD5 in HTE cells 4 h after stimulation with TNF-{alpha}-IL-1β. Increased FXYD5 expression after TNF-{alpha}-IL-1β stimulation was inhibited by pretreatment with the NF-{kappa}B inhibitor BAY 11 7085 (control: n = 10; TNF-{alpha}-IL-1β: n = 9; TNF-{alpha}/IL-1β + BAY 11-7085: n = 6). *P < 0.001 vs. control; #P < 0.01 vs. TNF-{alpha}/IL-1β. Data were normalized to GAPDH and analyzed by one-way ANOVA using Newman-Keuls posttest analysis.

 

   DISCUSSION
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Together with previous studies, our results indicate that FXYD5 specifically associates with the Na+-K+-ATPase and increases the catalytic efficiency of the pump for Na+ and K+. Similar to other reports, we observed that FXYD5 increased Na+-K+-ATPase pump activity (18). However, our 86Rb+ uptake studies demonstrate that FXYD5 increased the apparent affinity for cytoplasmic Na+ twofold (K0.5 = 9.24 ± 2.68 mM for vector, 4.53 ± 1.54 mM for FXYD5-transfected cells), whereas Vmax values were not significantly changed. In contrast, FXYD5 induced a significant 60% decrease in the apparent affinity for K+ (K0.5 0.07 ± 0.01 mM for vector, 0.12 ± 0.01 mM for FXYD5) and increased Vmax twofold. Stable transfection of vector or FXYD5 did not affect surface expression of the Na+-K+-ATPase, indicating that FXYD5 specifically altered pump kinetics. These effects could be due to either a change in Na+ binding sites or the rate constants that stabilize the Na+ binding conformation of the Na+-K+- ATPase. Because we observed a decrease in the apparent affinity and increase in Vmax for extracellular K+, this suggests that FXYD5 increases the rate of K+ deocclusion: E2(K)ATP->E1ATP. Furthermore, Scatchard analysis indicates that FXYD5 decreases the negative cooperativity observed in Na+ binding, suggesting that FXYD5 affects the E1P-E2P conformational equilibrium by shifting toward E1P (9). The notion that the functional role of FXYD5 is to increase the catalytic efficiency of the Na+-K+-ATPase is reinforced by a 50–80% increase in the specificity constants of the Na+-K+-pump for both cations. These observations have implications for tissues such as the kidney and lung, where Na+ homeostasis is important for water absorption and where cells that express FXYD5 might be expected to have a higher catalytic turnover and increased Na+ absorption (Fig. 8).


Figure 8
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  		Fig. 8. Model of FXYD5 role in Na+ absorption in CF airway epithelia. Because of loss of CFTR-mediated Cl– efflux and resulting ENaC-mediated Na+ hyperabsorption, Na+-K+-ATPase expression and activity are increased in CF airway epithelia. Expression of FXYD5, a regulator of the Na+-K+-ATPase, is also upregulated in CF epithelia and increases the rate of Na+ absorption along the ENaC/Na+-K+-ATPase axis. As a result, FXYD5 contributes to the chronic dehydration of the protective airway surface liquid that lines the airway.

 
We used both in vitro and in vivo models of increased Na+ absorption to evaluate FXYD5 expression. MDCK-ENaC cells stably express the {alpha}, β, and {gamma} ENaC subunits on induction, and we demonstrated by immunofluorescence and 86Rb+ uptake that ENaC induction also increased Na+-K+-ATPase expression and activity. Interestingly, there was a significant, 75% decrease in FXYD5 expression, suggesting that Na+ hyperabsorption downregulates FXYD5. This result was confirmed by quantitative RT-PCR and immunoblot analyses in tissues from lungs of mice that specifically overexpress ENaC in airway epithelia. A similar decrease in FXYD4 (CHIF) expression has been observed in mouse kidney medullary collecting duct cells during hyperkalemic conditions experienced in acute tubular necrosis (35). In these two examples, conditions that led to a marked upregulation of Na+-K+-ATPase resulted in downregulation of the FXYD congener. This may represent an attempt to restore homeostasis, shifting the fine-tuning effects of FXYD5 and decreasing the catalytic efficiency in favor of higher enzyme activity.

In CF airway epithelia, there is marked increases in ENaC-mediated hyperabsorption of Na+, and early studies indicated that there was an upregulation of the Na+-K+-ATPase as well (36). From our observations that upregulation of ENaC, either over 48 h or chronically in a mouse model, downregulates FXYD5 expression, we expected FXYD5 expression to be also downregulated in CF. However, in multiple CF models, FXYD5 expression was increased. FXYD5 was significantly increased almost threefold in nasal epithelia of CF mice by quantitative RT-PCR, on immunoblots from murine nasal epithelia, and by immunohistochemical staining and immunoblots of lung epithelia of CF mice. Although in CF mouse lung ENaC activation is less than that seen in the human condition, ENaC upregulation is clearly demonstrated in CF mouse nose. The findings in mice were mirrored in human nasal scrapes, where FXYD5 is upregulated in CF compared with controls. Upregulation of ENaC activity is prominent in human CF nasal epithelium. Finally, in HTE cells grown at the air-liquid interface, application of the CFTR inhibitor I-172 for 72 h resulted in upregulation of FXYD5, confirming in another human model that lack of CFTR function increases FXYD5 expression. In this model, ENaC upregulation is not observed, but a CF inflammatory phenotype does occur (27). In these cells, inhibition of NF-{kappa}B with PDTC returned FXYD5 expression to control values, supporting the notion that the upregulation of FXYD5 in CF might be entrained not by the increased Na+ load but by the proinflammatory state of the airways. To further investigate this possibility, we stimulated HTE cells with the proinflammatory cytokines TNF-{alpha} and IL-1β and saw a significant increase in FXYD5 mRNA expression, which was blocked by another inhibitor of NF-{kappa}B. FXYD5 has been shown to increase cell motility and has been implicated in the regulation of proinflammatory cytokines such as monocyte chemotactic protein 1 (MCP-1) (25). Interestingly, the MCP-1 gene has a {kappa}B site upstream of its promoter, suggesting that increased NF-{kappa}B activity observed in CF epithelia may also contribute to the MCP-1 autoregulatory loop previously described to be affected after FXYD5 inhibition and that these effects are downstream, feedforward regulators of FXYD5 expression.

In patients with CF, a prominent hypothesis for the pathogenesis of the airway disease postulates that the airway surface liquid (ASL) that lines the epithelia of the lung is dehydrated because of increased ENaC activation (20, 24), which promotes bacterial adherence, infection, and inflammation. Given that our kinetic data indicate that FXYD5 increased the catalytic efficiency of the pump for Na+ and K+, increased FXYD5 expression may contribute to further ASL dehydration by modulating Na+-K+-ATPase activity to increase transepithelial Na+ absorption down the ENaC-Na+-K+-ATPase axis (Fig. 8). Furthermore, the inflammatory phenotype in CF may contribute to ASL dehydration by increasing FXYD5 expression, as well as being exacerbated by it. We conclude that FXYD5 modulates Na+-K+-ATPase pump activity, increasing transepithelial Na+ absorption, and suggest that increased FXYD5 expression observed in CF airway epithelia may therefore contribute to airway surface dehydration.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants P30 DK-27651 and T32 HL-07418 and by the Cystic Fibrosis Foundation. Pamela B. Davis gratefully acknowledges support from the Arline and Curtis Garvin Research Professorship.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Alma Wilson, Christian VanHeeckeren, Veronica Peck, and the Cystic Fibrosis Animal Core for technical assistance. We also thank Dr. Thomas Kelley for assistance in preparing mouse nasal epithelium and Yongyi Qian for help with immunohistochemistry.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. J. Miller, Dept. of Pediatrics, BRB Rm. 801, Case Western Reserve Univ., 2109 Adelbert Rd., Cleveland, OH 44106 (e-mail: txm20{at}case.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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