Page
1 of 43 in PresS. Am J Physiol Gastrointest Liver Physiol (September 7, 2006). doi:10.1152/ajpgi.00297.2006
Articles
Na,K-ATPase regulates tight junction permeability through occludin
phosphorylation in pancreatic epithelial cells
Sigrid A. Rajasekaran1, Sonali P. Barwe1, Jegan Gopal1, Sergey Ryazantsev2, Eveline E.
Schneeberger5 and Ayyappan K. Rajasekaran1, 3, 4*
1
Department of Pathology and Laboratory Medicine, 2Department of Biological
Chemistry, 3Molecular Biology Institute, 4Jonsson Comprehensive Cancer Center, David
Geffen School of Medicine, University of California, Los Angeles, California 90095,
USA; 5Molecular Pathology Unit, Massachusetts General Hospital East, Charlestown,
MA 02129
*
Correspondence should be addressed to:
Ayyappan K. Rajasekaran
Department of Pathology and Laboratory Medicine
Room 13-344 CHS
University of California, Los Angeles
Los Angeles, California 90095
Phone (310) 825-1199
Fax (310) 267-2410
Email: arajasekaran@mednet.ucla.edu
Running Head: Na,K-ATPase and tight junctions
Copyright © 2006 by the American Physiological Society.
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2
Abstract
Tight junctions are crucial for maintaining the polarity and vectorial transport
functions of epithelial cells. We and others have shown that Na,K-ATPase plays a key
role in the organization and permeability of tight junctions in mammalian cells, and
analogous septate junctions in Drosophila. However, the mechanism by which Na,KATPase modulates tight junctions is not known. In this study, using a well-differentiated
human pancreatic epithelial cell line HPAF-II, we demonstrate that Na,K-ATPase is
present at the apical junctions and forms a complex with protein phosphatase (PP)-2A, a
protein known to be present at tight junctions. Inhibition of Na,K-ATPase ion transport
function reduced PP2A activity, hyper-phosphorylated occludin, induced rearrangement
of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na,K-ATPase is required for controlling the tight
junction gate function.
Key words: Na,K-ATPase
1-subunit,
Na,K-ATPase
phosphorylation, protein phosphatase-2A, pancreas
1-subunit,
occludin
Page 3 of 43
3
Introduction
Polarized epithelial cells form a permeability barrier between two biological
compartments, the integrity of which is maintained by intercellular junctional complexes
composed of tight junctions (TJ), adherens junctions, and desmosomes. TJs (or zonula
occludens) are the most apical component of the intercellular junctional complexes and
serve as a gatekeeper of the paracellular pathway. The TJ multicomponent protein
complexes are composed of transmembrane, scaffolding, and signaling proteins, and
integrate diverse processes such as cell polarity, cell proliferation, and tumor suppression
(27). A functional TJ is crucial to maintain the barrier function of epithelia and
dysregulation of this barrier function has been reported in a variety of diseases such as
ischemic retinopathies, pulmonary edema, inflammatory bowel disease, rheumatoid
arthritis, and nephropathies leading ultimately to dysfunction of the affected organ (12).
In pancreas, the disruption of the tight paracellular seals has been associated with early
events in acute pancreatitis (26), and altered expression and localization of various TJ
proteins have been described in primary and metastatic pancreatic cancer (13, 15, 32).
Although it has been recognized that understanding the mechanisms that regulate the
multicomponent, multifunctional complex of the TJ is a fundamental cell biology
question, less is known about the mechanisms that regulate TJs in pancreas due to the
lack of a suitable cell culture model.
Na,K-ATPase is an ubiquitously expressed oligomeric protein composed of two
essential polypeptide subunits, the catalytic -subunit (~112 kDa) (29) and the -subunit
Page 4 of 43
4
(~55 kDa) (28). An optional -subunit (~7 kDa), a member of the FXYD protein family,
functions as a tissue-specific modulator of Na,K-ATPase function (4). Four
-subunit
and three -subunit isoforms have been described, of which the most abundant are
1
(hereafter referred as NaK-
1
and
and NaK- ). Localized to the basolateral plasma
membrane, Na,K-ATPase catalyzes an ATP-dependent transport of three sodium ions out
and two potassium ions into the cell per pump cycle to maintain Na+ and K+ gradients
across the plasma membrane. This Na+ and K+ homeostasis is necessary to regulate the
functions of the various ion and solute transporters in epithelial cells. We have shown
that in addition to its epithelial transport function, Na,K-ATPase plays a fundamental role
in the formation and maintenance of epithelial TJs in mammalian cells (21, 22). Recently,
Violette et al. (35) showed that Na,K-ATPase is a potent regulator of TJ formation and
function during mouse preimplantation development. This role of Na,K-ATPase in the
structural organization of polarized epithelial cells seems to be conserved since studies in
Drosophila have shown that both Na,K-ATPase
- and
-subunit are crucial for the
structure and function of septate junctions, which are functionally similar to TJs in
epithelial cells (10, 19). However, the mechanism by which Na,K-ATPase regulates TJs
in polarized epithelial cells is not known.
A wide array of growth factors, cytokines, drugs and hormones have been shown
to affect the TJ barrier function through a variety of mechanisms including the regulation
of TJ protein expression at the transcriptional level or by endocytosis as well as
regulating TJ protein function by post-translational mechanisms such as phosphorylation
(12). The TJ protein occludin is a 504 amino acid polypeptide with a molecular mass of ~
Page 5 of 43
5
60 kDa. A hydropathy plot analysis predicts a tetraspan membrane protein that forms two
extracellular loops with N- and C-termini located intracellularly. The C-terminal domain
of occludin is rich in serine, threonine, and tyrosine residues that are targets for a number
of protein kinases such as atypical protein kinase C (aPKC) (17) and the nonreceptor
tyrosine kinase c-Yes (5), and protein phosphatases that include the serine/threonine
protein phosphatase 2A (PP2A) (17). The PP2A holoenzyme consists of a catalytic
subunit (C), a structural subunit (A) and a regulatory subunit (B). Several B subunit
families that modulate PP2A catalytic activity, substrate specificity, and subcellular
localization have been identified (30). PP2A containing the B
regulatory subunit is a
major PP2A isoform involved in cell growth and cytoskeletal regulation in numerous cell
types, and is localized to and regulate TJ functions (17).
We have established the human pancreatic adenocarcinoma cell line HPAF-II as a
polarized cell culture model to study TJs in pancreas (20). HPAF-II cells are ductal
pancreatic cancer cells that express Muc 1 and Muc 4 mucin genes and secrete high
levels of Muc1 mucin. Using this cell line, we now provide evidence for the first time
that in mammalian cells Na,K-ATPase is localized to the apical junctions (in addition to
its well-described basolateral localization), and associates with PP2A. Inhibition of the
Na,K-ATPase ion transport function reduced PP2A activity, hyper-phosphorylated
occludin, and induced rearrangement of TJ strands. The resulting increase in TJ
permeability to ionic and non-ionic molecules suggests that Na,K-ATPase activity is
required in controlling the TJ gate function in pancreatic epithelial cells.
Page 6 of 43
6
Materials and Methods
Cell culture, transepithelial electrical resistance (TER), and antibodies. Human
HPAF-II cells were kindly provided by Dr. Reber (University of California, Los Angeles,
CA) and were cultured in RPMI supplemented with 10% fetal bovine serum, 2 mM Lglutamine, 25 U/ml penicillin, 25 µg/ml streptomycin, and 100 µM non-essential amino
acids as described (20). For experiments, the cells were seeded on Costar Transwells with
0.4-µm pore size (Corning, Corning, NY) and allowed to grow until a TER of more than
1000
.
cm2 developed, usually 3-6 days. Co-immunoprecipitation and GST-pull down
assays were done using confluent monolayers grown on 100 mm culture dishes.
For K+-free conditions, HPAF-II cells were washed twice with K+-free buffer
(140 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 20 mM Hepes, 10 mM Glucose, pH 7.4,
10% FBS dialyzed against the K+-free buffer), and then incubated with this buffer
generally for 3 hours unless noted otherwise (21, 22), a timepoint at which the cells are
fully viable and the inhibition of Na,K-ATPase is completely reversible. For K+repletion, K+-free buffer was replaced by culture medium and the cells were incubated at
37oC, 5% CO2 for the times indicated. Control cells received a media change at the same
time. For ouabain treatment, cells were treated with varying concentrations of ouabain
(Sigma Chemical Co., St. Loius, MO) dissolved in DMSO added to the culture medium
usually for 7 hours. HPAF-II cells treated with DMSO alone were used as control cells
for these experiments.
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TER was measured as described previously (20-22) with an EVOM Epithelial
Voltohmeter (World Precision Instruments, Sarasota, FL). To obtain the TER values in
.
cm2, the background resistance value of a blank filter without cells was subtracted from
the measured values and then the values were normalized to the area of the filter.
Antibodies against ZO-1, occludin, claudin-4, and E-cadherin were obtained from
Invitrogen Corporation (Carlsbad, CA) and -catenin, anti-PP2A catalytic
antibodies
were from BD Biosciences (San Jose, CA). Antibodies against NaK-
(M8-P1-A3 for
immunoprecipitations and M7-PB-E9 for immunoblotting) and NaK-
(M17-P5-F11)
were kindly provided by Dr. William Ball Jr., University of Cincinnati Medical Center,
Cincinnati, OH. Horseradish peroxidase (HRP)-conjugated secondary anti-mouse and
anti-rabbit antibodies were purchased from Cell Signaling Technology Inc. (Beverly,
MA),
FITC-conjugated
secondary
antibodies
from
Jackson
ImmunoResearch
Laboratories Inc. (West Grove, PA), and gold-conjugated secondary antibodies from Ted
Pella Inc. (Redding, CA).
Immunoblot
analysis,
immunoprecipitations,
and
-phosphatase
( -PPase)
treatments. Cell lysates were prepared as described earlier (23). Briefly, confluent
monolayers of HPAF-II cells grown on Transwells were lysed in a buffer containing 95
mM NaCl, 25 mM Tris, pH 7.4, 0.5 mM EDTA, 2% SDS, 1 mM phenylmethylsulfonyl
fluoride, and 5 µg/ml each of antipain, leupeptin, and pepstatin. The lysates were clarified
by centrifugation at 13,000 rpm for 10 min. The supernatants were collected and total
protein was estimated using the Bio-Rad DC reagent (Bio-Rad Laboratories, Hercules,
Page 8 of 43
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CA) as per manufacturer’s instructions. Equal amounts of protein (100 µg) were
separated by SDS-PAGE. Primary antibodies were diluted 1:1000 in PBS containing 10%
non-fat dry milk. Horseradish peroxidase (HRP)-conjugated secondary anit-mouse or
anti-rabbit antibodies were used at a dilution of 1:2000 in phosphate buffered saline
(PBS)/10% non-fat dry milk. Protein bands were detected by ECL (Perkin Elmer Life
Sciences, Boston, MA).
-PPase treatment of occludin and claudin-4 was done as described previously
(21). Occludin or claudin-4 was immunoprecipitated from control or Na,K-ATPaseinhibited cells lysed in a buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1%
Triton-X-100, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycholate, 0.2 mM sodium
vanadate, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and 5 µg/ml each of antipain,
leupeptin, and pepstatin. The immunoprecipitates were washed and then incubated with
-PPase (New England Biolabs, Inc., Beverly, MA) at 30oC for 30 min according to the
manufacturer’s instructions. Proteins were resolved by SDS-PAGE and immunoblotted as
described above.
GST pull-down assays and co-immunoprecipitations. In vitro binding assays were
done as described (1, 3). Briefly, the cytoplasmic domain of NaK- containing amino
acids 1-35 (NaK- N-GST) and the N-terminus of NaK- containing amino acids 1-93
(NaK-
N-GST) were cloned in pGEX-5X vector (Invitrogen, Carlsbad, CA) and
transformed into E. coli BL-21 cells. Expression of recombinant protein was induced by
the addition of 0.25 mM isopropylthiogalactoside (IPTG) for 2 hours. Bacterial cell
Page 9 of 43
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pellets were lysed in a buffer containing 50 mM Tris.HCl, pH 8.0, 100 mM NaCl, 2 mM
MgCl2, 250 µg/ml lysozyme, 1 mM PMSF, and 10 µg/ml each of antipain, leupeptin and
pepstatin. The GST-fusion protein was bound to glutathione-coupled agarose beads
(Pharmacia Biotech, Piscataway, NJ) for 1 hour at 4oC and the amount of coupled fusion
protein was estimated using Coomassie-stained SDS-PAGE gels. HPAF-II cell lysates
were prepared in a buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Triton
X–100, 1 mM EDTA, 1 mM EGTA, 1 mM sodium glycerolphosphate, 1 mM sodium
orthovanadate, 1 mM PMSF, 5 µg/ml each of antipain, leupeptin, pepstatin. The lysates
were clarified by centrifugation at 10,000 rpm for 10 min at 4oC. The protein
concentrations of the cleared supernatants were determined and equal amounts of protein
were incubated with purified GST-fusion proteins at 4oC for 16 hours. Bound proteins
were detected by immunoblotting as described above.
For co-immunoprecipitations, equal protein from cell lysates prepared as
described above were incubated on a rotator overnight with antibody bound to protein A
agarose beads at 4oC. The proteins bound to the beads were separated by SDS-PAGE
and co-immunoprecipitating proteins were analyzed by immunoblotting.
Transmission electron microscopy (TEM), freeze fracture and immunoelectron
microscopy. For TEM, HPAF-II monolayers were grown to confluence on Transwells.
Control or Na,K-ATPase-inhibited cells were fixed in 2.5% glutaraldehyde in 0.1 M
sodium cacodylate buffer, pH 7.4, for 2 hours at room temperature. The samples were
Page 10 of 43
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then processed by conventional procedures for electron microscopy. Ultrathin sections
were contrasted with uranyl acetate and lead citrate.
Freeze fracture analysis was done as described previously (20). Briefly, confluent
monolayers of HPAF-II cells grown on Transwells were fixed in 2% glutaraldehyde in
PBS for 30 minutes at 4oC. After rinsing with Dulbecco’s PBS, the cells were scraped
from the filters and infiltrated with 25% glycerol in 0.1 M cacodylate buffer. The cells
were pelleted, frozen in liquid nitrogen slush and freeze fractured at –115oC in a Balzers
400T freeze-fracture unit (Balzers, Liechtenstein). The replicas were cleaned with sodium
hypochlorite and examined with a Philips 301 electron microscope (Philips, Einthoven,
Holland).
For immunoelectron microscopy, HPAF-II cells grown on 35mm tissue culture
dishes were fixed in ice-cold methanol. After washing with PBS/1 mM MgCl2/0.1 mM
CaCl2/0.2 % bovine serum albumin (PBS-CM-BSA), the cells were incubated with
primary antibodies against NaK-
and occludin for 16 hours at 4oC. The cells were
washed three times with PBS-CM-BSA containing 0.075% saponin (Sigma Chemical
Co., St. Louis, MO) and then incubated for 1 hour at room temperature with goldconjugated anti-mouse (10 nm; NaK- ) and anti-rabbit (5 nm; occludin) secondary
antibodies diluted 1:6 in PBS-CM/saponin. After washing with PBS-CM/saponin, the
cells were fixed in 2% glutaraldehyde in cacodylate buffer, scraped off the plate and cell
pellets were processed by conventional electron microscope procedures. To compare the
staining intensity of NaK-
at the apical junctional region and at the lateral plasma
Page 11 of 43
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membrane, the numbers of 10 nm gold particles were counted in 32 and 29 fields of 0.25
µm x 0.25 µm localized to the respective regions. The data represent the means ±
standard error of number of gold particles per field.
Paracellular diffusion studies. Confluent monolayers of HPAF-II cells with TER values
of at least 1000
.
cm2 were chosen for these studies. Cells in culture medium, treated
with ouabain or under K+-depleted conditions were assessed for paracellular diffusion of
3
H-Inulin or 3H-Mannitol as described previously (20, 21). 2 µCi 3H-Inulin or 2µCi 3H-
Mannitol (Amersham Corp., Arlington Heights, IL) in 500 µl culture medium with or
without ouabain or K+-free medium were added to the apical side of the filter; the basal
compartment contained 1 ml of respective medium without tracer. The cells were
incubated for 60 min at 37oC before equal-volume aliquots of apical and basal
compartment media were collected. The samples were counted in a liquid scintillation
counter and permeability was calculated with P = (X)B/(X/µl)i/A/T where XB are counts
per minute in the basal chamber; (X/µl)i is the initial concentration in the apical chamber,
A is the area of the filter in cm2 and T is the time in minutes as described previously (21,
22).
PP2A activity. Control and Na,K-ATPase inhibited HPAF-II cells on Transwells were
lysed in a buffer containing 20 mM imidazole-HCl, pH 7.0, 2 mM EDTA, 2 mM EGTA,
1 mM benzamidine, 1 mM PMSF, 10 µg/ml trypsin inhibitor and 10 µg/ml each of
antipain, leupeptin, and pepstatin. Equal amounts of protein were used for
immunoprecipitation of the catalytic subunit of PP2A. PP2A activity was determined by
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dephosphorylation of the specific phosphopeptide R-K-pT-I-R-R using the Ser/Thr
Phosphatase Assay Kit 1 (Upstate Biotechnology, Lake Placid, NY) according to
manufacturer’s instructions, and free phosphate released was determined using the
Malachite Green Assay included in the kit.
Immunofluorescence and confocal microscopy. Immunofluorescence and confocal
microscopy were done as described previously (20, 22, 23). HPAF-II cells grown on
Transwells were fixed in methanol at –20oC and then incubated with primary antibodies
diluted 1:1000 in PBS-CM-BSA for one hour at room temperature. FITC-conjugated
secondary antibodies were used at a dilution of 1:100. Epifluorescence pictures were
captured with a SPOT CCD camera and SPOT imaging software, version 4.0.4
(Diagnostic Instruments Inc., MI) attached to an Olympus AX70 microscope. Confocal
microscopy was done with an Olympus laser scanning confocal microscope and images
were generated by the Fluoview Image Analysis software (version 2.1.39) as described
(22).
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Results
To test whether Na,K-ATPase enzyme activity is necessary for TJ function in
HPAF-II cells, we inhibited the enzymatic activity of Na,K-ATPase by two independent
methods, using the specific pharmacological inhibitor ouabain, and by K+-depletion (21,
22). While ouabain binds to and inhibits the enzyme irreversibly, Na,K-ATPase can be
re-activated after K+-depletion by addition of K+ to test for reversibility of observed
effects. Transepithelial electrical resistance (TER), a measure of the ion permeability of
TJs, was drastically decreased in K+-depleted cells (Fig. 1A). While the TER of untreated
control cells was 1694 ± 43
.
.
cm2, the TER of K+-depleted cells dropped to 896 ± 60
cm2 within one hour and reduced to 289 ± 9
.
cm2 after two hours. This TER decrease
was reversible upon K+-repletion. In ouabain treated cells, TER decreased in a dose
dependent manner (Fig. 1B) indicating that inhibition of Na,K-ATPase leads to increased
ion permeability of TJs in HPAF-II cells. TJ permeability to non-ionic molecules in
Na,K-ATPase inhibited cells was determined by tracer studies using
3
H-inulin
(hydrodynamic radius ~10-14 angstroms) and 3H-mannitol (hydrodynamic radius ~4
angstroms) (Fig. 1C). Ouabain caused a dose dependent increase of TJ permeability for
both inulin and mannitol, with 50 µM ouabain having an effect similar to K+-depletion.
The TJ permeability to inulin and mannitol was reversible upon K+-repletion. Taken
together, these results demonstrated that inhibition of Na,K-ATPase increases the
permeability of both ionic and non-ionic molecules through the paracellular space in
HPAF-II cells.
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We then tested whether the observed permeability changes are associated with
altered TJ organization. Immunofluorescence of the TJ proteins ZO-1, occludin, and
claudin-4 in ouabain-treated and K+-depleted cells did not reveal significant differences
compared to control cells at the light microscopic level (Fig. 2A). Further, confocal
microscope vertical sections showed lateral localization of -catenin in control and Na,KATPase inhibited cells indicating that the polarity is not affected (Fig. 2B). However,
transmission electron microscopy revealed that upon Na,K-ATPase inhibition by both
K+-depletion and ouabain, the TJ contact points were reduced as compared to the
extensive TJs observed in control cells (Fig. 3A). K+-repleted cells showed a morphology
similar to untreated control cells. In freeze fracture electron micrographs, the TJ network
in control HPAF-II cells was somewhat variable with most segments forming a network
of TJ strands (Fig. 3B, control), while approximately 10% of the remaining junctional
length was comprised of segments composed of condensed TJ strands (data not shown).
In K+-depleted cells the frequency of segments with condensed strands increased to 20%,
which upon K+-repletion, returned to approximately 8% of the total TJ length.
To test whether the rearrangement of TJ strands upon inhibition of Na,K-ATPase
is associated with any changes in TJ protein levels we performed immunoblot analysis of
ZO-1, occludin and claudin-4 (Fig. 4A). We did not find any substantial differences in
the levels of either of these TJ proteins or in the levels of the adherens junction proteins
E-cadherin and -catenin, both of which have been shown to regulate TJs (Fig. 4A).
Furthermore, there was no difference in -catenin immunofluorescence staining pattern
and intensity (Fig. 2B), -catenin tyrosine phosphorylation (data not shown), or in the
Page 15 of 43
15
amounts of
-catenin co-immunoprecipitating with E-cadherin between control and
Na,K-ATPase inhibited cells (data not shown), suggesting that increased TJ permeability
in Na,K-ATPase inhibited cells might not be due to compromised adherens junction
function.
Since we did not observe any change in TJ protein expression, we checked for
post-translational modifications in TJ proteins. In MDCK cells, occludin migrates as
multiple band clusters of slow migrating high molecular mass occludin forms that are
phosphorylated on Ser residues, and of fast migrating, low molecular mass,
dephosphorylated occludin species (37). In control HPAF-II cells occludin migrated as a
doublet (Fig.4A). Following inhibition of Na,K-ATPase the intensity of the high
molecular mass occludin form increased with a concominant decrease in the low
molecular mass occludin form (Fig.4A, lanes 2 and 6). To test whether occludin is being
hyper-phosphorylated
upon
inhibition
of
Na,K-ATPase,
we
treated
occludin
immunoprecipitates with -protein phosphatase ( -PPase) prior to immunoblot analysis.
In the absence of -PPase, control cells showed two distinct bands (Fig. 4B). Upon PPase treatment, the molecular mass of the upper band (arrow) shifted to a mass similar
to the lower band, confirming that the upper band is the hyper-phosphorylated form of
occludin. In K+-depleted cells, the lower mass band almost completely disappeared,
indicating that most of the occludin in Na,K-ATPase inhibited cells was hyperphosphorylated, as assessed by its sensitivity to -PPase treatment. In K+-repleted cells,
the intensity and phosphorylation of occludin was similar to that of untreated control
cells. Ouabain showed a dose dependent effect in the levels of hyper-phosphorylated
Page 16 of 43
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occludin. We observed a similar occludin hyper-phosphorylation in several other human
epithelial cell lines with inhibited Na,K-ATPase function (data not shown). Although
altered phosphorylation of other TJ proteins has been shown to be associated with
compromised TJ function (2), we did not observe hyper-phosphorylation of claudin-4
(Fig. 4C) or ZO-1 (data not shown) under electrophoretic conditions in which a molecular
mass shift upon phosophorylation of both of these proteins has been observed previously
(17). These data indicated that increased TJ permeability in Na,K-ATPase-inhibited
HPAF-II cells might be associated with hyper-phosphorylation of occludin.
Occludin has been shown to be phosphorylated at serine/threonine as well as
tyrosine residues and is a target for a number of protein kinases and protein phosphatases
(9). Immunoprecipitation of occludin and immunoblotting using anti-phosphotyrosine
antibody revealed no band, suggesting that increased phosphorylation of occludin in
Na,K-ATPase inhibited cells is not due to tyrosine phosphorylation (data not shown). A
previous report by Nunbhakdi-Craig et al (17) had identified occludin as a target of
aPKC
mediated serine/threonine phosphorylation in MDCK cells. However, Na,K-
ATPase inhibition in HPAF-II cells did not change aPKC activity and we were not able
to confirm that occludin hyper-phosphorylation upon Na,K-ATPase inhibition in these
cells was due to increased aPKC activity (data not shown). To identify other kinases
possibly involved in the phosphorylation of occludin, we treated HPAF-II cells with
inhibitors of kinases such as PKC (Bisindolylmaleimide), Erk1/2 (PD98059) as well as
the PI3-kinase pathway (LY294002), prior to the inhibition of Na,K-ATPase. None of the
above mentioned inhibitors could prevent the hyper-phosphorylation of occludin in
Page 17 of 43
17
Na,K-ATPase inhibited HPAF-II cells (data not shown). Next, we decided to test the role
of PP2A, since recent studies revealed that PP2A is localized to TJs and is involved in the
regulation of the TJ function in mammalian cells (17). In HPAF-II cells, the specific
PP2A inhibitor calyculin A caused a dose dependent increase in occludin
phosphorylation (Fig. 5A) and at 25 nM concentration most of the occludin was
phosphorylated. The effect of calyculin on occludin phosphorylation was reversible
following washout of the inhibitor (Fig. 5B). As observed in Na,K-ATPase inhibited
cells, occludin hyper-phosphorylation in calyculin A treated cells was accompanied by
increased TJ permeability (data not shown). These results suggested that inhibition of
PP2A might be involved in the hyper-phosphorylation of occludin and we tested PP2A
activity in Na,K-ATPase function compromised cells using a spectrophotometric enzyme
assay. In control cells, total PP2A activity in PP2A catalytic subunit immunoprecipitates
was determined as 6.85 ± 0.33 pmoles of phosphate release, which was reduced to 4.11 ±
0.40 pmoles of phosphate in immunoprecipitates of K+-depleted cells, a value similar to
the activity in calyculin A treated cells (Fig. 5C). The inhibition of PP2A activity by K+depletion was reversible upon addition of K+. These results indicated that inhibition of
Na,K-ATPase results in reduced PP2A activity, which in turn increases the occludin
phosphorylation leading to increased TJ permeability. Since PP2A is localized to TJs we
hypothesized that occludin might directly bind to PP2A to regulate the phosphorylation
of this protein. However, our co-immunoprecipitation experiments did not detect PP2A
associated with occludin (data not shown). Strikingly, we found that PP2A readily
coimmunoprecipitated with NaK- and to a lesser extent with NaK- , whereas control
IgG did not show any binding (Fig. 5D). It is possible that reduced NaK- binding to
Page 18 of 43
18
PP2A is due to reduced immunoprecipitation efficiency of the NaK- subunit antibody.
To rule out this possibility, we tested the association of PP2A with NaK- using a PP2A
catalytic subunit antibody for co-immunoprecipitation analysis. Na,K-
was clearly
detected in the PP2A immunoprecipitates. GST-pull down assays further confirmed that
PP2A associates with the N-terminus of NaK- in cell lysates of HPAF-II cells (Fig. 5E).
The association of NaK-
with PP2A was found in both control and Na,K-ATPase
inhibited cells (data not shown). Although the N-terminus of Na,K- did not pull down
PP2A, a recent study revealed that the Na,K- loop 4-5 binds to PP2A in a yeast twohybrid screen (18). These results indicate that PP2A is in a complex with Na,K-ATPase
and that normal Na,K-ATPase function is necessary to maintain PP2A activity.
Since PP2A is localized to TJs and since it forms a complex with Na,K-ATPase,
we tested whether Na,K-ATPAse is also localized to the TJs using immunogold labeling
and electron microscopy. NaK- (10 nm gold particles) and occludin (5nm gold particles)
distinctly codistributed at the apical junctions that include both tight and adherens
junction regions (Fig. 6C). In addition and as expected, NaK- was also localized to the
entire lateral plasma membrane but clearly excluded from the desmosomes or the apical
plasma membrane indicating a specific immuno-labeling (Fig. 6A and C). Quantitative
analysis of the electron micrographs revealed a three fold difference in NaK- labeling
intensity with 1.7 ± 0.3 gold particles per unit area detected at the apical junctions (ApJ)
versus 5.6 ± 0.6 per unit area localized to the basolateral plasma membrane (LM) below
the adherens junction (Fig. 6B and C). The intensity labeling for occludin was 6.4 ± 0.5
gold particles per unit area in the ApJ region and 1.1 ± 0.3 gold on the lateral membrane
Page 19 of 43
19
(LM). The non-specific areas (NS) contained 0.1 ± 0.1 of each 10 nm and 5 nm gold
particles per area unit suggesting specific immunolabeling of NaK- and occludin on the
plasma membrane domains. We were not able to confirm the localization of NaK- to the
TJ region due to the failure of our antibody to detect NaK- by immuno-EM. However,
these results strongly indicate that at least Na,K- is localized to the apical junctions in
polarized pancreatic epithelial cells (in addition to the basolateral plasma membrane).
Page 20 of 43
20
Discussion
In this study, we provide the first evidence that in mammalian cells Na,K-ATPase
is localized to the apical junctional complex and associates with PP2A, a protein known
to regulate TJ function and localized to TJs. We demonstrate that inhibition of Na,KATPase results in the hyper-phosphorylation of occludin in a PP2A dependent manner.
Hyper-phosphorylation of occludin was accompanied by altered TJ structure at the
electron microscopy level and increased permeability to both ionic and nonionic solutes.
Thus, these results demonstrate that normal Na,K-ATPase enzyme activity is necessary
for the proper regulation of the phosphorylation status of occludin by PP2A compatible
for its function to regulate the permeability of TJs. Based on these results we suggest that
Na,K-ATPase is a key regulator of the TJ gate function in pancreatic epithelial cells.
Na,K-ATPase appears to regulate TJ permeability through different mechanisms
in different cell types. Upon inhibition of Na,K-ATPase activity in MDCK cells, TJ
permeability increases but is accompanied by de-phosphorylation of occludin (S. A.
Rajasekaran, unpublished observations). Interestingly, occludin de-phosphorylation in
MDCK cells is associated with increased TJ permeability (17). In primary cultures of
polarized retinal pigment epithelial cells (RPE), inhibition of Na,K-ATPase decreased
TER and increased permeability to non ionic molecules (21) similar to HPAF II cells.
Although there was a striking similarity in the TJ morphology by light and electron
microscopy in RPE and HPAF II cells, changes in the phosphorylation of occludin were
not detected in RPE cells (21). Whether this is due to the apical localization of Na,K-
Page 21 of 43
21
ATPase in RPE cells remains to be determined. In mouse blastocysts, inhibition of Na,KATPase by K+-depletion as well as ouabain treatment increased permeability and
compromised localization of ZO-1 and occludin at the plasma membrane (35). Thus it
appears while Na,K-ATPase has a conserved role in the regulation of TJ function in
mammalian cells, the mechanism by which the effect is manifested is distinctly different
in different cell types.
During Ca2+ induced TJ biogenesis, translocation of occludin and ZO-1 from the
cytosol to the plasma membrane is accompanied by increased phosphorylation of
occludin (8, 25, 31, 37). Using MDCK cells and a Ca2+ switch assay, Nunbhakdi-Craig et
al. (17) demonstrated that enhanced PP2A activity prevents TJ assembly whereas
inhibition of PP2A increased phosphorylation of occludin, ZO-1, and claudin-1 and
promoted localization of these proteins to the TJ and TJ assembly. These studies
suggested a critical role for PP2A in the regulation of TJs during their biogenesis. In
contrast, our studies were performed in HPAF-II cells with established TJs. Our results
reveal that inhibition of PP2A activity increases occludin phosphorylation as observed in
the study by Nunbhakdi-Craig et al. (17), but in sharp contrast disrupted TJ structure and
function. These differences could be due to diverse signaling in TJ biogenesis and the
maintenance of established TJs of fully polarized cells or due to cell type-specific or
species-specific differences. Indeed, we observed that in other human epithelial cells such
as Caco-2 and RT-4 occludin was hyper-phosphorylated upon inhibition of Na,K-ATPase
while occludin was de-phosphorylated in Na,K-ATPase inhibited MDCK cells of canine
origin (data not shown). Since Na,K-ATPase is a key enzyme necessary for the survival
Page 22 of 43
22
of a cell it is possible that different cell types have evolved different strategies to adapt to
the inhibition of this enzyme. However, these results clearly manifest a critical role for
PP2A in the regulation of TJ function. Although recent studies indicate that claudins play
a critical role in the structure and functions of TJs (24, 34), the finding that increased
occludin phosphorylation significantly alters their permeability suggests that occludin
might be involved in the fine regulation of the TJ permeability by integrating signals
obtained from Na,K-ATPase via PP2A, either through protein-protein interactions or by
responding to an increase in intracellular Na+ upon inhibition of the pump activity.
One of the striking findings reported in this study is the localization of NaK- to
the apical junctional complex. The quantification of the immunogold labeling indicates
that the density of Na,K-ATPase localized to the TJ is less than its density at the
basolateral plasma membrane. However, the significance of NaK- localization at the
apical region is not known. The fact that Na,K-ATPase binds to PP2A, a protein localized
to TJs and that regulates occludin phosphorylation, suggests that Na,K-ATPase, occludin
and PP2A might be in a complex at the apical junctional region. Consistent with this idea,
a recent study indicated that Na,K-ATPase co-sediments with fractions enriched in
occludin but not ZO-1 and claudin during epithelial polarization (36). These results
further support our idea that Na,K-ATPase, PP2A, and occludin might form an
independent complex at the TJ region. The Na,K-ATPase-PP2A-occludin complex might
form a membrane microdomain involved in cell signaling activity that regulates finetuning of TJ permeability. At present, it is not known whether Na,K- is localized to the
Page 23 of 43
23
TJ as well or whether NaK- is independently present at TJs. Future experiments are
necessary to further validate this point.
This study has relevance to pancreatic diseases such as pancreatitis and pancreatic
cancer. We showed that in HPAF-II cells Na,K-ATPase is a potent inhibitor of PP2A and
its level of inhibition is similar to calyculin A, a specific inhibitor of PP2A, suggesting
that in pancreatic epithelial cells Na,K-ATPase plays an important role in the regulation
of PP2A activity. Perturbation of Na,K-ATPase function might lead to reduced PP2A
activity and might be associated with loss of TJ functions in pancreatic diseases such as
pancreatitis. Disruption of the TJ paracellular permeability has been implicated in the
caerulein-induced acute pancreatitis (7, 26). However, whether this increase in
paracellular permeability is associated with changes in PP2A and Na,K-ATPase activity
needs to be determined.
In a recent study we presented evidence that NaK- , independently of Na,KATPase activity, triggers the formation of a scaffolding complex containing PI3-Kinase,
annexin II, and Rac1 which eventually signals downstream to suppress cell motility (3) In
addition to these proteins, it is also known that Na,K-ATPase binds to signaling proteins
like IP3R (38), PLC- 1 (38), and Src (11, 33) and to the spectrin–ankyrin cytoskeleton
(14, 16). These results are consistent with the idea that Na,K-ATPase might function as a
scaffolding signaling platform. We have shown earlier that the level of NaK- is reduced
in a highly transformed, poorly differentiated pancreatic carcinoma cell line lacking TJs
(6). Reduced expression of NaK-
in carcinoma might therefore, result in the
Page 24 of 43
24
deregulation of this scaffolding complex leading to loss of TJs and gain of invasive and
metastatic behavior of pancreatic cancer cells.
Page 25 of 43
25
Acknowledgements
We thank Dr. Enrique Rozengurt (UCLA) for useful suggestions during the initial phase
of this study. The expert technical assistance of Joanne McCormack is gratefully
acknowledged.
Grants
Supported by NIH grants to AKR (DK56216) and EES (HL25822).
Page 26 of 43
26
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Figure Legends
Figure 1. Increased tight junction permeability in Na,K-ATPase inhibited HPAF-II
cells. (A) Transepithelial electrical resistance (TER) in K+-depleted HPAF-II cells. +K+
indicates the addition of K+ in K+-repleted samples. (B) HPAF-II cells were treated with
100 nM, 2.5 µM , or 50 µM ouabain. TER measurements revealed a dose-dependent
reduction in TER as compared to DMSO-treated control cells. (C) Paracellular
permeability of 3H-Inulin and 3H-Mannitol in HPAF-II monolayers treated with indicated
concentrations of ouabain, in K+-depleted cells (-K+) and in K+-repleted cells (-K+/+K+).
All data represent the means ± SE of three independent experiments done in triplicate.
Figure 2. Localization of tight junction proteins and epithelial polarity upon
inhibition of Na,K-ATPase. (A) Immunofluorescence of TJ marker proteins ZO-1,
occludin, and claudin-4 reveal similar staining patterns in control, K+-depleted (-K+) (3
hours), K+-repleted (-K+/+K+) (3 hours depletion, 12 hours repletion), and ouabaintreated (50 µM) (7 hours) HPAF-II cells. (B) Polarized distribution of the basolateral
marker protein -catenin in confocal XY and XZ (vertical) sections in control and Na,KATPase inhibited cells. Bars, 10 µm.
Figure 3. Altered tight junction ultrastructure upon Na,K-ATPase inhibition in
HPAF-II cells (A) Transmission electron microscopy of control, K+-depleted (-K+) (3
hours), K+-repleted (-K+/+K+) (3 hours depletion, 12 hours repletion), and ouabaintreated (50 µM) (7 hours) HPAF-II cells. Inserts are higher magnification of the TJ
regions of each panel. Bar, low magnification, 0.5 µm; Bar, insert, 0.2 µm. (B) Freeze
Page 33 of 43
33
fracture replica of control, K+-depleted (-K+) and K+-repleted (-K+/+K+) HPAF-II cells.
Compressed TJ strands in K+-depleted cells are indicated (arrowheads). Bars, 80 nm.
Figure 4. Na,K-ATPase inhibition results in hyper-phosphorylation of the tight
junction protein occludin. (A) Immunoblot analysis of 100 µg whole cell lysates from
Na,K-ATPase inhibited HPAF-II cells for TJ proteins ZO-1, occludin, and claudin-4, and
adherens junction proteins E-cadherin and -catenin. Asterisk (*) indicates the shift in
molecular weight of occludin observed in Na,K-ATPAse inhibited cells. (B) Occludin is
hyper-phosphorylated in Na,K-ATPase inhibited cells. Occludin was immunoprecipitated
from control, K+-depleted (-K+) (3 hours), K+-repleted (-K+/+K+) (3 hours depletion, 12
hours repletion), and ouabain-treated (50 µM) (7 hours) HPAF-II cells. The
immunoprecipitates were either untreated or treated with
-protein phosphatase ( -
PPase), separated by SDS-PAGE and immunoblotted for occludin. Arrow indicates
phosphorylated occludin. (C) -PPase treatment of claudin-4 immunoprecipitates does
not result in a shift in electrophoretic mobility.
Figure 5. PP2A-dependent phosphorylation of occludin in HPAF-II cells. (A)
Calyculin A, a specific PP2A inhibitor, induces occludin hyper-phosphorylation. HPAFII cells were incubated with increasing amounts of calyculin A (Cal A) for 2 hours prior
to cell lysis. Occludin was immunoprecipitated and treated with or without -PPase prior
to SDS-PAGE and immunoblotting for occludin. Occludin was hyper-phosphorylated
(arrow) in a calyculin A dose-dependent manner. (B) Occludin phosphorylation upon
calyculin A treatment is reversible. After treatment with 25 nM Calyculin A for 1 hour,
Page 34 of 43
34
the drug was washed out and the cells were allowed to recover. An occludin immunoblot
of whole cell lysates is shown. (C) Inhibition of Na,K-ATPase activity suppresses PP2A
activity. The catalytic subunit of PP2A was immunoprecipitated from control, Na,KATPase inhibited cells (-K+) (3 hours), and Calyculin A (25 nM) treated cells. K+repletion (-K+/+K+) (3 hours depletion, 12 hours repletion) was included to show
reversibility. PP2A activity was determined by dephosphorylation of a PP2A-specific
peptide (see Materials & Methods). Data shown represent the means ± SE of three
independent experiments done in duplicate. A PP2A immunoblot shows that equal
amounts of PP2A were immunoprecipitated under all treatment conditions. (D) Coimmunoprecipitation experiments using anti-NaK- and NaK- antibodies followed by
immunoblotting for PP2A reveal that PP2A is in a protein complex with NaK- and
NaK- . PP2A immunoprecipitates were included to indicate the PP2A band. The
association of NaK-
with PP2A in a protein complex was confirmed by
immunoprecipitation with anti-PP2A antibody and immunoblotting with NaKantibody. Immunoprecipitates with an irrelevant IgG do not pull down PP2A or NaK- .
(E) GST-pull down assays reveal that PP2A binds to the N-terminus of NaK- but not the
NaK- N-terminus, indicating that a different NaK- cytoplasmic domain is involved in
PP2A binding. PP2A immunoprecipitated (IP) is included to indicate the PP2A band.
Figure 6. Na,K-ATPase localizes to the apical junctional complex. Immunogold
labeling of confluent HPAF-II monolayers for NaK- (10 nm gold) and occludin (5 nm
gold). (A) Low magnification of a cell-cell contact region. (B) A diagrammatic
representation of the regions used in the quantitation of the immunogold labeling. The
Page 35 of 43
35
membrane boundaries of the cells and the distribution of labeling are indicated by tracing
the image in A. NaK- is represented by circles and occludin by stars. The rectangle
indicates the area of higher magnification shown in (C). Since Na,K-
and occludin
labeling were observed at both the TJ and AJ, labeling in this region is considered
positive for labeling of apical junctions (ApJ), also indicated in (C). The labeling below
this region is considered positive for lateral membrane (LM). Areas in the cytoplasm of
neighboring cells (NS) were considered for non-specific labeling. Bars represent the
mean ዊ SE of gold particles in each defined area from 25 cell-cell contact regions
examined. (C) High magnification of the cell-cell contact region of two neighboring cells
as indicated in (B). The apical junctional region (ApJ) with tight junctions (TJ) and
adherens junctions (AJ) is indicated. The lateral plasma membrane region including a
desmosome is shown (LM). Note that NaK- is localized to the entire lateral membrane
including the apical junctional region, but is excluded from desmosomes. The histogram
in (B) shows the quantitation of the immunogold labeling. Bar in (A), 0.2 µm; (B) 0.1
µm.
Page 36 of 43
Increased tight junction permeability in Na,K-ATPase inhibited HPAF-II cells. (A)
Transepithelial electrical resistance (TER) in K+-depleted HPAF-II cells. +K+ indicates the
addition of K+ in K+-repleted samples. (B) HPAF-II cells were treated with 100 nM, 2.5
M , or 50
M ouabain. TER measurements revealed a dose-dependent reduction in TER
as compared to DMSO-treated control cells. (C) Paracellular permeability of 3H-Inulin and
3
H-Mannitol in HPAF-II monolayers treated with indicated concentrations of ouabain, in
K+-depleted cells (-K+) and in K+-repleted cells (-K+/+K+). All data represent the means ±
SE of three independent experiments done in triplicate.
Page 37 of 43
Localization of tight junction proteins and epithelial polarity upon inhibition of Na,KATPase. (A) Immunofluorescence of TJ marker proteins ZO-1, occludin, and claudin-4
reveal similar staining patterns in control, K+-depleted (-K+) (3 hours), K+-repleted (+
K /+K+) (3 hours depletion, 12 hours repletion), and ouabain-treated (50
M) (7 hours)
HPAF-II cells. (B) Polarized distribution of the basolateral marker protein ß-catenin in
confocal XY and XZ (vertical) sections in control and Na,K-ATPase inhibited cells. Bars, 10
m.
Page 38 of 43
Altered tight junction ultrastructure upon Na,K-ATPase inhibition in HPAF-II cells (A)
Transmission electron microscopy of control, K+-depleted (-K+), K+-repleted (-K+/+K+),
and ouabain-treated HPAF-II cells. Inserts are higher magnification of the TJ regions of
each panel. Bar, low magnification, 0.5
m; Bar, insert, 0.2
m. (B) Freeze fracture
replica of control, K+-depleted (-K+) and K+-repleted (-K+/+K+) HPAF-II cells.
Compressed TJ strands in K+-depleted cells are indicated (arrowheads). Bars, 80 nm.
Page 39 of 43
Na,K-ATPase inhibition results in hyper-phosphorylation of the tight junction protein
occludin. (A) Immunoblot analysis of 100
g whole cell lysates from Na,K-ATPase
inhibited HPAF-II cells for TJ proteins ZO-1, occludin, and claudin-4, and adherens
junction proteins E-cadherin and
-catenin. Asterisk (*) indicates the shift in molecular
weight of occludin observed in Na,K-ATPAse inhibited cells. (B) Occludin is hyperphosphorylated in Na,K-ATPase inhibited cells. Occludin was immunoprecipitated from
control, K+-depleted (-K+) (3 hours), K+-repleted (-K+/+K+) (3 hours depletion, 12 hours
repletion), and ouabain-treated (50
M) (7 hours) HPAF-II cells. The
immunoprecipitates were either untreated or treated with
-protein phosphatase ( PPase), separated by SDS-PAGE and immunoblotted for occludin. Arrow indicates
phosphorylated occludin. (C)
-PPase treatment of claudin-4 immunoprecipitates does
not result in a shift in electrophoretic mobility.
Page 40 of 43
PP2A-dependent phosphorylation of occludin in HPAF-II cells. (A) Calyculin A, a specific
PP2A inhibitor, induces occludin hyper-phosphorylation. HPAF-II cells were incubated
with increasing amounts of calyculin A (Cal A) for 2 hours prior to cell lysis. Occludin was
immunoprecipitated and treated with or without
-PPase prior to SDS-PAGE and
immunoblotting for occludin. Occludin was hyper-phosphorylated (arrow) in a calyculin A
dose-dependent manner. (B) Occludin phosphorylation upon calyculin A treatment is
reversible. After treatment with 25 nM Calyculin A for 1 hour, the drug was washed out
and the cells were allowed to recover. An occludin immunoblot of whole cell lysates is
shown. (C) Inhibition of Na,K-ATPase activity suppresses PP2A activity. The catalytic
subunit of PP2A was immunoprecipitated from control, Na,K-ATPase inhibited cells (-K+)
(3 hours), and Calyculin A (25 nM) treated cells. K+-repletion (-K+/+K+) (3 hours
depletion, 12 hours repletion) was included to show reversibility. PP2A activity was
determined by dephosphorylation of a PP2A-specific peptide (see Materials & Methods).
Data shown represent the means ± SE of three independent experiments done in
duplicate. A PP2A immunoblot shows that equal amounts of PP2A were
immunoprecipitated under all treatment conditions. (D) Co-immunoprecipitation
experiments using anti-NaKand NaKantibodies followed by immunoblotting for
PP2A reveal that PP2A is in a protein complex with NaKand NaK- . PP2A
immunoprecipitates were included to indicate the PP2A band. The association of NaKwith PP2A in a protein complex was confirmed by immunoprecipitation with anti-PP2A
Page 41 of 43
antibody and immunoblotting with NaKantibody. Immunoprecipitates with an
irrelevant IgG do not pull down PP2A or NaK- . (E) GST-pull down assays reveal that
PP2A binds to the N-terminus of NaKbut not the NaKN-terminus, indicating that a
different NaKcytoplasmic domain is involved in PP2A binding. PP2A
immunoprecipitated (IP) is included to indicate the PP2A band.
Page 42 of 43
Na,K-ATPase localizes to the apical junctional complex. Immunogold labeling of confluent
HPAF-II monolayers for NaK(10 nm gold) and occludin (5 nm gold). (A) Low
magnification of a cell-cell contact region. (B) A diagrammatic representation of the
regions used in the quantitation of the immunogold labeling. The membrane boundaries
of the cells and the distribution of labeling are indicated by tracing the image in A. NaKis represented by circles and occludin by stars. The rectangle indicates the area of
higher magnification shown in (C). Since Na,Kand occludin labeling were observed at
both the TJ and AJ, labeling in this region is considered positive for labeling of apical
junctions (ApJ), also indicated in (C). The labeling below this region is considered
positive for lateral membrane (LM). Areas in the cytoplasm of neighboring cells (NS)
were considered for non-specific labeling. Bars represent the mean ± SE of gold particles
Page 43 of 43
in each defined area from 25 cell-cell contact regions examined. (C) High magnification of
the cell-cell contact region of two neighboring cells as indicated in (B). The apical
junctional region (ApJ) with tight junctions (TJ) and adherens junctions (AJ) is indicated.
The lateral plasma membrane region including a desmosome is shown (LM). Note that
NaKis localized to the entire lateral membrane including the apical junctional region,
but is excluded from desmosomes. The histogram in (B) shows the quantitation of the
immunogold labeling. Bar in (A), 0.2
m; (B) 0.1
m.