Am J Physiol Lung Cell Mol Physiol 294: L654–L664, 2008.
First published February 3, 2008; doi:10.1152/ajplung.00430.2007.
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
FXYD protein family, typified by the ␥-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 ␣- and ␤-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 sub-
sequently 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.
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: [email protected]).
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.
Na⫹-K⫹-ATPase; sodium transport
THE
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Miller TJ, Davis PB. FXYD5 modulates Na⫹ absorption and is
increased in cystic fibrosis airway epithelia. Am J Physiol Lung Cell
Mol Physiol 294: L654–L664, 2008. First published February 3, 2008;
doi:10.1152/ajplung.00430.2007.—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 MadinDarby 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-␬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.
FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
MATERIALS AND METHODS
Cell Lines
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 ⌬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 ␤,
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.
UCSF), prepared in DMSO, and diluted from a 1:1,000 stock. Similarly, the NF-␬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-␣ and 5
ng/ml IL-1␤ (Sigma) in 200 ␮l of HBSS (Invitrogen) added to the
apical surface. NF-␬B activation was inhibited by pretreating HTE
with 20 ␮M 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.
Human Tracheal Epithelial Cells and Treatment With CFTR
Inhibitor I-172
Creation of MDCK-hF5Flag Stable Cell Line
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 ⫻ 106 cells/1-cm2
filter, Transwell-clear polyester membrane; Costar, Corning, NY) as
previously described (8, 27) and allowed to differentiate in serumcontaining 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 ␮M CFTR inhibitor I-172 (a kind gift from Alan Verkman,
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 ␮g/ml zeocin and analyzed after 3-wk
incubation by fluorescence-activated cell sorting using the BDFACSAria (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 ␣1-Na⫹-K⫹-ATPase (clone 464.6; Millipore, Billerica, MA).
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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 ␣-,
␤-, and ␥-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 ␮M 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.
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FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
Table 1. Comparison of intracellular Na⫹ and extracellular K⫹ activation of Na⫹-K⫹-ATPase pump activity
in FXYD5-transfected MDCK cells
Kinetic Constants
K⫹
Na⫹
Model
Cell Line
K0.5, mM
Vmax
Specificity Constant
K0.5, mM
Vmax
Specificity Constant
Noncooperative (Eq. 1)
Sham
hF5Flag
Sham
hF5Flag
0.153
0.205
0.07
0.12
6512
12735
5356
10240
1
1.46
1
1.3
2.866
1.648
9.24
4.53
10905
9780
8858
7956
1
1.56
1
1.83
Cooperative (Eq. 2)
Values are averages ⫾ SE of 6 independent experiments. Values for apparent kinetic constants of human FXYD5-transfected Madin-Darby canine kidney
(MDCK) or sham control cells were calculated with the noncooperative and cooperative models of ligand binding (Eqs. 1 and 2, respectively, in MATERIALS AND
METHODS). Specificity constants were calculated according to Eq. 3.
Total RNA (0.5 ␮g) was utilized to synthesize cDNA with the
first-strand cDNA synthesis kit (Roche Molecular Biochemicals,
Mannheim, Germany). cDNA synthesis was performed with oligop(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⬘-TCACCTGTGGCGATTCAG-
GCAAAATTGAGACAA (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
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-␣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, ␣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 ␣1-Na⫹-K⫹-ATPase membrane preparations shown in G normalized to
actin, demonstrating no significant (NS)
change in ␣1-Na⫹-K⫹-ATPase.
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Quantitative RT-PCR
FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
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
tained with a ⫻63 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.
86
Rb⫹ 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 ␮M monensin and 50 ␮M
bumetanide. For assays that varied intracellular Na⫹-dependent
86
Rb⫹ uptake, the wash and incubation solutions were similar except
that 2 mM RbCl, 10 ␮M monensin, and 2–70 mM NaCl were used
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 ␮g/ml;
Bethyl Labs) or mouse anti-␣1-subunit Na⫹-K⫹-ATPase antibody (1
␮g/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 obAJP-Lung Cell Mol Physiol • VOL
Fig. 2. Ouabain-sensitive 86Rb⫹ uptake in MDCK-hF5Flag cells. MDCK-hF5Flag
(Œ) or MDCK-sham control (■) cells were preincubated 15 min with or without
100 ␮M 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 ␮M; 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 ouabainsensitive 86Rb⫹ (K⫹) influx rates were fit to Eq. 1 (noncooperative model) and
represent 6 independent experiments ⫾ SE. Kinetic constants are summarized
in Table 1.
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Adult ⌬F508 mice and their wild-type littermates were killed, and
the lungs were fixed by perfusion using 2% paraformaldehyde in 1⫻
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 ␮m) 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 ␮g/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 1⫻ 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).
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FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
with choline chloride added to maintain isotonicity. Cells were then
preincubated in assay buffer in the presence or absence of 100 ␮M
ouabain for 10 min at 37°C and washed twice. The assay was initiated
by the addition of 25 ␮l (1–2 ␮Ci 86RbCl) to 225 ␮l 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 ␮l 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
86
Rb⫹ uptake observed between clones. Ouabain-sensitive 86Rb⫹
uptake was calculated as the difference between total and ouabaininsensitive 86Rb⫹ accumulation, and samples were normalized to
protein content using the DC protein assay (Bio-Rad).
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):
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:
␯ ⫽ V max关S兴n/共K⬘ ⫹ 关S兴n兲
(2)
where K0.5 ⫽ K’ 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
1/n
Specificity constant ⫽ Vmax共S兲/K0.5共S兲
⫹
(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.
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 StudentNeuman-Kuels regression analysis for pairwise comparisons. The
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 ␣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
86
Rb⫹ 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.
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Kinetic Analysis
␯ ⫽ V max/共1 ⫹ Ks/关S兴兲n
FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
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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
FXYD5 Modulates Na⫹-K⫹-ATPase Ion Transport
Fig. 4. FXYD5 is decreased in lung tissue from mice that overexpress the
Scnn1b transgene. Transgenic mice overexpressing the Scnn1b (ENaC ␤-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.
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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.
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.
FXYD5 alters Na⫹-K⫹-ATPase pump kinetics in MDCKhF5Flag 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 ␮M bumetanide and in
the presence or absence of 100 ␮M 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 vectortransfected 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 FXYD5transfected 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
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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 fluorescenceactivated 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 ␣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 ␣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 ␣-subunit of the Na⫹-K⫹-ATPase, and this
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FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
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
Fig. 6. FXYD5 is increased in airway epithelia of CF mice.
Upper airway and lung tissues were collected from adult
⌬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 ⫻40 objective lens.
G: representative immunoblot demonstrating increased
FXYD5 expression in crude membrane preparations from
⌬F508 CF mice compared with wild-type control littermates.
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FXYD5 is downregulated after ENaC activation in MDCKENaC cells. To investigate whether increased Na⫹-K⫹ATPase activity affects FXYD5 expression, we utilized a
stable cell line that expresses high levels of the ␣-, ␤-, and
␥-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 shortcircuit 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 MDCKENaC 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 MDCKENaC-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.
FXYD5 is decreased in the lungs of Scnn1b transgenic mice.
To confirm these findings in an in vivo model, mice overexpressing Scnn1b (ENaC ␤-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
FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
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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.
FXYD5 is Upregulated in CF Airway Epithelia
AJP-Lung Cell Mol Physiol • VOL
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-␬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-␣-IL-1␤. Increased FXYD5 expression after TNF-␣-IL-1␤ stimulation was inhibited by pretreatment with the NF-␬B inhibitor BAY 11 7085
(control: n ⫽ 10; TNF-␣-IL-1␤: n ⫽ 9; TNF-␣/IL-1␤ ⫹ BAY 11-7085: n ⫽
6). *P ⬍ 0.001 vs. control; #P ⬍ 0.01 vs. TNF-␣/IL-1␤. Data were normalized
to GAPDH and analyzed by one-way ANOVA using Newman-Keuls posttest
analysis.
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-␬B inhibitor PDTC (P ⬍ 0.01) (Fig. 7B). Similarly,
stimulation of HTE cells with TNF-␣-IL-1␤ for 1 h increased
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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).
FXYD5 is increased in lungs of CF mice. Upper and lower
airway epithelia from the lungs of ⌬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 ⌬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-typespecific mechanism governing membrane insertion. No-primaryantibody 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 ⌬F508⫺/⫺ CF lung tissue
confirmed that FXYD5 is increased in the CF lung (Fig. 6G).
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.
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FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
FXYD5 expression by ⬃60% (P ⬍ 0.001) after 4 h, which was
blocked by pretreatment with the NF-␬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.
DISCUSSION
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.
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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)ATP3 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).
We used both in vitro and in vivo models of increased Na⫹
absorption to evaluate FXYD5 expression. MDCK-ENaC cells
stably express the ␣, ␤, and ␥ 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 ENaCmediated 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-␬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
FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
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.
GRANTS
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.
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further investigate this possibility, we stimulated HTE cells
with the proinflammatory cytokines TNF-␣ and IL-1␤ and saw
a significant increase in FXYD5 mRNA expression, which was
blocked by another inhibitor of NF-␬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 ␬B site upstream of its promoter, suggesting that
increased NF-␬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.
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FXYD5 IS UPREGULATED IN CYSTIC FIBROSIS
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