Interferon-
decreases barrier function in T84 cells by
reducing ZO-1 levels and disrupting apical actin
Adel
Youakim and
Minoo
Ahdieh
Department of Biochemistry, Immunex Corporation, Seattle, Washington
98101
 |
ABSTRACT |
The effects of
interferon-
(IFN-
) on tight junctions in T84 human intestinal
epithelial cells were investigated. Treatment of T84 cells with IFN-
caused a dose- and time-dependent increase in monolayer permeability as
assessed by transepithelial electrical resistance measurements.
Examination of specific proteins associated with tight junctions by
immunoblotting and confocal microscopy revealed changes in the
expression levels and localization of some of these proteins after
exposure of the cells to IFN-
. Specifically, IFN-
treatment
resulted in an almost total loss of zonula occludens (ZO)-1, whereas the levels of ZO-2 and occludin showed
relatively modest decreases compared with untreated cells. Loss of ZO-1
was associated with the altered localization of ZO-2 and occludin. In
IFN-
-treated cells, ZO-2 and occludin were diffusely distributed, whereas, in control cells, they, along with ZO-1, were predominantly localized to the tight junctions. Analysis of ZO-1 protein and RNA by
pulse chase and RT-PCR, respectively, showed an increase in protein
turnover, a decrease in protein synthesis, and a reduction in RNA
levels following IFN-
treatment. In contrast to ZO-1, ZO-2 and
occludin did not show any major changes in these parameters. In
addition, the organization of actin in the apical and tight junction
regions, but not in the mid- or basal regions, of the cells was also
perturbed by IFN-
treatment of cells. Time-course analysis of
IFN-
-induced alterations in ZO-1 expression and apical actin
perturbation indicated that these two effects were intimately linked
and could not be dissociated. These results suggest that IFN-
affects barrier function in T84 cells by decreasing the levels of ZO-1
and perturbing apical actin organization, which leads to a
disorganization of the tight junction and an increase in paracellular permeability.
tight junctions; epithelial barrier; zonula occludens-1; zonula
occludens-2; occludin; actin
 |
INTRODUCTION |
THE INTESTINAL EPITHELIUM forms a relatively
impermeable barrier between the lumen and the submucosa. This barrier
function is maintained by a complex of proteins composing the tight
junction that is located at the subapical aspect of the lateral
membranes. Tight junctions comprise numerous proteins, with the best
characterized being zonula occludens (ZO)-1, ZO-2, and occludin (for
reviews, see Refs. 2 and 3). ZO-1 and ZO-2 are cytoplasmic proteins (18, 32), whereas occludin is a putative transmembrane protein (9).
Recently, several new proteins, ZO-3 (12), claudin-1 and -2 (8), and
junctional adhesion molecule (JAM) (25), have also been shown to be
associated with tight junctions; the first is cytoplasmic, and the
latter two are transmembrane proteins. Although the precise interplay
of these proteins within the tight junction complex has not been fully
elucidated, recent findings suggest a complex series of interactions
among them. ZO-1 and ZO-2 appear to interact, as they
coimmunoprecipitate when using either anti-ZO-1 or anti-ZO-2 antibodies
(18). ZO-1, ZO-3, and occludin also appear to associate, based on in
vitro binding studies (10, 12). The COOH terminus of occludin is
necessary for the localization of this molecule to the tight junction
of polarized cells (9). Therefore, it is possible that this portion of
occludin contains a targeting sequence necessary for delivery to the
tight junction and that binding of the COOH terminus to ZO-1 is
required to retain occludin at the tight junction. The association of
ZO-1 with tight junctions and binding to occludin is mediated by two separate domains within the
NH2-terminal half of ZO-1 (7). In
addition to associating with other tight junction proteins, ZO-1 also
interacts with the cytoskeleton either by binding to spectrin (16) or
by binding directly to actin (15). The actin binding activity of ZO-1
has been mapped to the COOH-terminal half of the molecule (7). Thus
ZO-1 may act as a direct or indirect link between the transmembrane
component of the tight junction, occludin, and the cytoskeleton. The
interaction of tight junctions with the cytoskeleton is critical to the
regulation of barrier function, and modulation of the cytoskeleton may
influence paracellular flow through the tight junctions (20, 21, 30).
Disruption of the epithelial barrier in the gut is a hallmark of
inflammatory bowel disease and intestinal infections. Although it
remains unclear as to whether barrier breakdown is an initiating event
or a consequence of inflammation, it is readily apparent that loss of
the barrier contributes to propagation and exacerbation of
inflammation. Barrier breakdown can be elicited by a number of agents,
including bacteria, cells of the immune system, and proinflammatory
cytokines. In the complex milieu of the gut, it is likely that a
combination of factors contributes to barrier disruption. This is an
issue of significant importance for the treatment of gut inflammation
because an understanding of how the epithelial barrier is regulated in
disease may allow the development of therapeutic agents that prevent
breakdown or enhance recovery of normal barrier function. A number of
in vitro model systems, including the T84 human intestinal epithelial
cell line, have been developed that enable the study of epithelial
barrier regulation. T84 cells form a polarized, impermeable monolayer
that possesses many of the functional characteristics of intestinal
epithelial cells in vivo, including vectorial solute transport and
barrier function (6, 22). Previous studies have demonstrated that T84
cells treated with cytokines, such as interferon-
(IFN-
) (1, 15),
show decreased barrier function. IFN-
is greatly elevated in human
intestinal disease and undoubtedly contributes to the inflammatory
cascade, which includes barrier disruption. However, the effects of
IFN-
on tight junctions, the cellular complex responsible for
barrier function, have yet to be determined. The objective of this
study was to determine if IFN-
alters epithelial barrier of T84
cells by influencing the organization and composition of the tight
junctions. The results show that, indeed, IFN-
disrupts barrier
function by dramatically decreasing the levels of ZO-1, perturbing the
actin cytoskeleton in the region of the tight junction, and causing the
mislocalization of ZO-2 and occludin.
 |
MATERIALS AND METHODS |
Cell culture.
T84 cells were maintained and grown in DMEM-Ham's F-12 (DMEM-F12; 1:1)
containing 10% heat-inactivated fetal bovine serum and penicillin,
streptomycin, and glutamine (Life Technologies, Gaithersburg, MD).
Cells were routinely passaged when they reached 50-75%
confluence. For growth on porous filters, cells were grown in the same
medium and plated at 3 × 105
cells/100 µl or 5 × 106
cells/2 ml on 6.5-mm or 24-mm Transwell filters (clear polystyrene, 0.4 µm pore size; Costar, Cambridge, MA), respectively. For growth on the
6.5-mm filters, cells were plated "upside down" so that their
basolateral membranes were exposed to the upper chamber of the filter
cups. The transepithelial electrical resistance (TER) of cells grown on
filters was measured with an epithelial voltohmmeter (World Precision
Instruments, Sarasota, FL). Cells were used only if their TER was
>1,000
· cm2. Cells
with stable TER readings >1,000
· cm2 were
treated with IFN-
(107 U/mg;
Genzyme, Boston, MA) added to the basolateral side of the filters for
the indicated times. In some instances, cells were allowed to recover
from IFN-
treatment by washing three times in medium and by
maintaining in complete medium without IFN-
.
Preparation of detergent-soluble and -insoluble fractions.
T84 cell lysates were prepared by differential detergent solubility as
previously described (29).
Immunoblotting of protein fractions.
NP-40-soluble and -insoluble protein fractions were
separated on a 10% Tris-glycine polyacrylamide gel (Novex, San Diego, CA), transferred to nitrocellulose (0.45 µm; Bio-Rad, Richmond, CA),
and blocked for 4-6 h in PBS-0.1% Tween 20-5% dried milk (PTM). The blots were then incubated overnight at 4°C with primary antibodies (ZO-1, ZO-2, occludin, and E-cadherin; Zymed Laboratories, South San Francisco, CA) diluted 1:1,000-1:2,000 in PTM. The blots were then washed in PBS, incubated at room temperature for 90 min with
biotinylated secondary antibody (Molecular Probes, Eugene, OR) diluted
1:1,000 in PTM, followed by horseradish peroxidase-conjugated streptavidin (Sigma, St. Louis, MO) diluted 1:10,000 in PTM, and then
processed for enhanced chemiluminescence detection (Amersham, Arlington
Heights, IL).
Lambda phosphatase treatment of proteins.
Protein (25-100 µg) from the NP-40-soluble and -insoluble
fractions was immunoprecipitated with 5 µg of antibody (anti-ZO-1, anti-ZO-2, or anti-occludin) and 50 µl of protein G-agarose (Pierce Chemicals, Rockford, IL) for 4 h at 4°C. The antibody-protein G
complex was washed three times in NP-40-insoluble protein buffer without phosphatase inhibitors and then brought up in 100 µl of the
same buffer. Manganese chloride was added to a final concentration of 2 mM, and 800 units of lambda phosphatase (400,000 U/ml, New England
Biolabs, Beverly, MA) were added. The samples were incubated at
30°C for 90 min with frequent mixing and washed three times with
NP-40-insoluble protein, and the bound proteins were eluted from the
protein G beads by boiling in reducing SDS-PAGE sample buffer. The
samples were then processed as described above for immunoblotting of proteins.
Pulse-chase analysis and immunoprecipitation of ZO-1.
T84 cells were grown on 75-mm Transwell filter inserts and treated with
100 ng/ml IFN-
for various times. Cells were metabolically labeled
for 4 h with Redivue Pro-mix 35S
cell labeling mix (180 µCi/ml, sp act <1,000 Ci/mmol, Amersham) in
20 ml cysteine- and methionine-free DMEM (Life Technologies) containing
5% dialyzed fetal bovine serum. One-half of the filter inserts from
each sample (0 h chase group) was processed as described in
Immunoblotting of protein fractions to
isolate the detergent-soluble and -insoluble fractions. The other half
of the inserts was washed three times with complete DMEM-F12 containing
10% serum and five times the normal concentration of cysteine and
methionine and then maintained in this medium for an additional 12 h
(12-h chase group) before being processed for isolation of the
different protein fractions. IFN-
was present during the entire
pulse-chase period in the appropriate samples. Protein concentration
was determined by microbicinchoninic acid procedure (Pierce
Chemicals). Incorporation of radioactivity was measured by
cold TCA precipitation.
ZO-1 and occludin were immunoprecipitated from the NP-40-insoluble
protein fraction, whereas ZO-2 was immunoprecipitated from the
NP-40-soluble protein fraction. Briefly, 50-100 µg of protein were incubated with 10 µg of antibody overnight at 4°C. This
material was added to 50 µl of protein G-agarose (Pierce Chemicals)
and incubated for 2 h. The antibody-protein G complex was washed three times in NP-40-insoluble protein buffer and two times in RIPA buffer
(150 mM NaCl containing 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and
50 mM Tris, pH 8.0). The bound proteins were eluted with 30 µl of
SDS-PAGE reducing sample buffer and fractionated on a 10% Tris-glycine
polyacrylamide gel, which was subsequently fixed and dried and
quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Analysis of ZO-1 and ZO-2 expression by RT-PCR.
Total RNA was extracted from T84 cells with TRIzol reagent (Life
Technologies) using the manufacturer's protocol. RNA samples were
treated with DNase (10 units; Genhunter, Nashville, TN) before the
RT-PCR procedure. For RT-PCR, 0.5 µg of total RNA was reverse transcribed with 200 units Moloney murine leukemia virus RT (Life Technologies) that was primed with either 100 ng of random hexamer (Life Technologies) or 0.5 µg of oligo(dT) (Life Technologies) in 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl2, 2 mM DTT containing 10 units RNase inhibitor (Boehringer Mannheim, Indianapolis, IN), and 0.5 mM each dNTPs for 1 h at 42°C. The reaction mixture was heat
inactivated at 72°C for 15 min and then brought to 100 µl final
volume with water. PCR reactions were performed with 6.25 µl of cDNA
from the RT reaction, 10 units Taq
polymerase (Boehringer Mannheim), 1.5 mM
MgCl2, 0.4 µM each primer, 200 µM each dNTP, and 5 µCi
[
-32P]dCTP (sp act
3,000 Ci/mmol; Amersham) in 250 µl total volume divided into 25-µl
aliquots. The primer sets for ZO-1 (forward primer,
5'-CGGTCCTCTGAGCCTGTAAG-3'; reverse primer,
5'-GGATCTACATGCGACGACAA-3') and for ZO-2 (forward primer,
5'-GCCAAAACCCAGAACAAAGA-3'; reverse primer,
5'-ACTGCTCTCTCCCACCTCCT-3') were both designed to amplify the long and short isoforms of both proteins (16, 17). PCR was
performed in a Stratagene Robocycler Gradient 96 (Stratagene, La Jolla,
CA) by heating the samples at 94°C for 5 min for one cycle,
followed by 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min for the indicated number of cycles. Aliquots were taken after every
other cycle between cycles 14 and
30. Ten microliters of each PCR
reaction was electrophoresed on a 6% or 10%
polyacrylamide-Tris-borate-EDTA gel, dried, and quantified
on a PhosphorImager.
Immunofluorescence confocal analysis.
Cells were analyzed by confocal microscopy as previously described
(14).
 |
RESULTS |
IFN-
treatment of T84 cells decreased TER.
T84 cells grown on filter inserts formed polarized, impermeable
monolayers that possessed relatively high TER. Treatment of the cells
with different doses (10, 30, and 100 ng/ml) of IFN-
added to the
basolateral side of the monolayer resulted in a time- and
dose-dependent decrease in TER (Fig. 1).
Monolayers treated with the highest concentration of IFN-
(100 ng/ml) showed a 40% and 80% decrease in TER after 24 and 48 h of
treatment, respectively, relative to control cells. In contrast, cells
treated with the lower doses of IFN-
showed less of a decrease in
TER at 24 and 48 h, but, by 72 h, both showed an overall decline
(~80%) in TER that was comparable to the high dose of IFN-
. To
confirm that the changes in TER were a reflection of altered
paracellular permeability, [3H]mannitol flux
measurements were performed. In cells treated with 100 ng/ml IFN-
,
mannitol flux was increased ~10fold above untreated cells at 48 h
(5.2 vs. 0.55 nmol · h
1 · cm
2,
respectively; data not shown). Cells treated with the other two doses
of IFN-
showed a similar increase in mannitol flux relative to
untreated cells at 72 h (data not shown). Thus the decrease in TER
induced by IFN-
is at least partially due to increased paracellular
permeability.

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Fig. 1.
Interferon- (IFN- ) treatment of T84 cells decreases
transepithelial electrical resistance (TER) in a time- and
dose-dependent manner. T84 cells grown on polystyrene filters were
treated with different concentrations of IFN- added basolaterally.
At the indicated times, TER measurements were taken. Results represent
an average of 9 filters per time point. In all instances, baseline TER
at time 0 was >1,000
· cm2. ,
Untreated; , 10 ng/ml IFN- ; , 30 ng/ml IFN- ; , 100 ng/ml
IFN- .
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IFN-
treatment reduced the levels of ZO-1 in T84
cells.
To examine the effects of IFN-
on proteins associated with the tight
junctions, ZO-1, ZO-2, and occludin were analyzed by immunoblotting.
Cells were separated into NP-40-soluble and -insoluble fractions as
previously described (29). According to this method, cells are first
solubilized with NP-40 to remove proteins that are not associated with
the cytoskeleton. Subsequently, the NP-40-insoluble material is
solubilized with SDS. This latter fraction contains the cytoskeleton
and associated proteins, including those found in tight junctions.
Proteins in these two fractions were collected from untreated T84 cells
and cells treated for 72 h with different doses of IFN-
and
processed for immunoblotting. The blots were probed with antibodies to
ZO-1, ZO-2, occludin, and E-cadherin. Although E-cadherin is not
associated with tight junctions, its expression was analyzed because
disruption of E-cadherin-mediated adhesion events has been shown to
prevent tight junction formation (11). As shown in Fig.
2, a number of changes were apparent in the
various proteins following IFN-
treatment. In untreated cells, ZO-1
was associated almost exclusively in the insoluble fraction. Treatment
of the cells with IFN-
resulted in a substantial decrease (7- to
15-fold, 3 experiments) in ZO-1 levels in the insoluble fraction. The
fact that ZO-1 protein was not detected in the soluble fraction after
IFN-
treatment suggests that this protein was not being
"chased" from the insoluble fraction; rather, the expression of
this protein was reduced. In contrast to ZO-1, ZO-2 in untreated cells
was found in both the soluble and insoluble fractions. However, there
appeared to be two forms of ZO-2 in the soluble fraction and only one,
corresponding to the higher-molecular-weight form, in the insoluble
fraction. The possible structural significance of the different species
of ZO-2 will be addressed later. Treatment of the cells with IFN-
appeared to have only relatively modest effects (20-40% decrease,
3 experiments) on the amount of ZO-2 in the soluble fraction. In
contrast, ZO-2 in the insoluble fraction decreased dramatically,
similar to what was observed with ZO-1. Quantitation of ZO-2 in the
soluble fraction of the IFN-
-treated samples did not show an
increase in the proportion of the upper band as would be expected if
this protein were being displaced from the insoluble to the soluble
fraction. However, this could simply be a reflection of the modest
decrease in overall ZO-2 levels induced by IFN-
, which may have
masked any accumulation of the higher-molecular-weight species in the
soluble fraction.

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Fig. 2.
Differential detergent solubility of tight junction and adhesion
proteins and effects of IFN- treatment on protein expression. T84
cells were treated for 72 h with the indicated concentrations of
IFN- and then extracted as described in MATERIALS
AND METHODS. NP-40-soluble (S) and NP-40-insoluble (I)
fractions were immunoblotted with antibodies to zonula occludens
(ZO)-1, ZO-2, occludin, and E-cadherin.
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Occludin, the only known transmembrane component of tight junctions,
was also distributed in the soluble and insoluble fractions. However,
the nature of the proteins in the two fractions was quite different. A
major protein species was found in the soluble fraction, whereas, in
the insoluble fraction, a heterogeneous mixture of discrete,
higher-molecular-weight polypeptides was detected. IFN-
treatment of
the cells showed a relatively modest reduction (25-40%, 3 experiments) in occludin in the soluble and insoluble fractions. Furthermore, there did not appear to be a change in the pattern of the
protein bands in either fraction following IFN-
treatment.
The cell adhesion molecule E-cadherin was found primarily in the
soluble fraction, although a significant proportion was also found in
the insoluble fraction. These results are somewhat surprising, since
the adhesive properties of E-cadherin are dependent on its cytoskeletal
association; they suggest that, in T84 cells, only a portion of
E-cadherin is functionally involved in mediating cell adhesion or
simply that the association of E-cadherin with the cytoskeleton in
these cells may not be as strong as that of tight junction proteins
under the conditions used for extraction. Nonetheless, treatment with
IFN-
did not seem to alter the expression of E-cadherin in either
fraction. Thus it appears that IFN-
treatment of T84 cells induces a
dramatic decrease in ZO-1 levels, whereas the effects on other proteins
are relatively modest.
Previous studies have shown that a number of the tight junction
proteins are phosphorylated. To determine the contribution of
phosphorylation to the complexity of the protein patterns observed, particularly with regard to ZO-2 and occludin, the samples were treated
with lambda phosphatase before immunoblotting (Fig.
3). Lambda phosphatase cleaves
phosphates on serine, threonine, and tyrosine residues (34). ZO-1,
present in only the insoluble fraction, showed no change in mobility
after phosphatase treatment, indicating either an absence or a low
level of phosphorylation. This observation is consistent with an
earlier study showing that ZO-1 in Madin-Darby canine kidney (MDCK)
cells with high transepithelial resistance was not highly
phosphorylated (30). Similarly, neither ZO-2 in the soluble nor ZO-2 in
the insoluble fraction was altered by treatment with the phosphatase.
This result raises the possibility that the two species of ZO-2 protein
in the soluble fraction may represent the long
(
+) and short
(
) isoforms of the
protein (4) or that the lower-molecular-weight band is simply a
proteolytic product of the higher-molecular-weight band. These
possibilities will be addressed in later sections of this study.

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Fig. 3.
Phosphatase treatment of tight junction proteins. NP-40-soluble (S) and
NP-40-insoluble (I) fractions from untreated T84 cells were
immunoprecipitated with the appropriate antibodies, treated without
( ) or with (+) lambda phosphatase (PPase) and then immunoblotted
as described in MATERIALS AND METHODS.
Results shown are with proteins from untreated cells, but similar
results were obtained with samples from IFN- -treated cells.
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Occludin showed the most dramatic effect following phosphatase
treatment. Although occludin in the soluble fraction was unaffected by
the phosphatase, occludin in the insoluble fraction was reduced from a
broad, heterogeneous protein profile to a lower-molecular-weight protein that migrated similarly to the one in the soluble fraction. Thus these results show that occludin in the insoluble fraction of T84
cells is heavily phosphorylated, as has been shown previously for
occludin in the tight junction fraction of MDCK cells (29).
To further assess how IFN-
affects ZO-1 expression, the levels of
ZO-1 were measured at different times after treatment with 100 ng/ml
IFN-
. Within 24 h of IFN-
treatment, ZO-1 levels decreased by
~50% (Fig. 4), which
correlates with the decrease in TER (Fig. 1). By 48 and 72 h
of treatment, ZO-1 levels were undetectable by immunoblotting (Fig. 4).
Thus it appears that the loss of ZO-1 coincides with the decrease in
barrier function. However, barrier function was not totally abolished
even in the absence of detectable ZO-1 (cf. Figs. 1 and 4). These
results suggest that other proteins may compensate for the loss of ZO-1
and may thus contribute to the maintenance of barrier function, albeit
at a reduced level.

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Fig. 4.
ZO-1 levels decrease with increasing time of treatment with IFN- .
T84 cells were treated with 100 ng/ml IFN- for the indicated times.
Cells were extracted as in Fig. 1, and immunoblotting to detect ZO-1
was performed.
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IFN-
treatment decreases ZO-1 protein synthesis and
stability.
To determine how IFN-
treatment reduces ZO-1 expression,
the biosynthesis of ZO-1 protein was examined. T84 cells were analyzed by pulse-chase metabolic labeling with
35S-labeled amino acids and
immunoprecipitation of ZO-1, ZO-2, and occludin (Fig.
5). In untreated cells, a single band
corresponding to ZO-1 was detected at 0 and 12 h of chase (Fig. 5,
top). Quantitation of these bands
showed that the intensity of the 12-h chase sample was ~0.75 times
that of the 0-h chase sample. Thus the approximate half-life of ZO-1 in
untreated T84 cells is ~24 h. T84 cells treated with IFN-
for 24 h
before metabolic labeling also showed a single band corresponding to
ZO-1. However, the intensity ratio of the 12-h chase to the 0-h chase
was 0.60, indicating a half-life of ~15 h. Thus IFN-
treatment of
the T84 cells appears to increase the turnover rate of ZO-1 by almost
40%. No detectable ZO-1 was immunoprecipitated from cells pretreated
with IFN-
for 48 h (or 72 h, data not shown). In contrast to ZO-1,
ZO-2 (Fig. 5, middle) showed no
change in half-life between untreated cells and cells treated with
IFN-
for 24 h (half-life of ~24 h in both cases). Interestingly, in cells treated for 48 h with IFN-
, the half-life of
ZO-2 decreased to ~15 h. The fact that ZO-2 shows a more rapid turnover rate at about the same time that ZO-1 levels become
undetectable may indicate that ZO-2 is somewhat more unstable in the
absence of ZO-1. Occludin (Fig. 5,
bottom) showed no change in protein turnover rate between untreated and IFN-
-treated cells at any of the
time points. In all cases, a broad, heterogeneous collection of bands
corresponding to occludin was detected. The ratio of signal intensity
at the 12-h chase compared with the 0-h chase was about equal in all
cases and indicated a half-life of ~24 h, similar to ZO-1 in
untreated cells. These results indicate that the stability of ZO-1,
ZO-2, and occludin are affected differently by treatment with IFN-
.

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Fig. 5.
Pulse-chase analysis of tight junction proteins in untreated and
IFN- -treated T84 cells. T84 cells, treated for indicated times with
100 ng/ml IFN- , were analyzed following pulse-chase metabolic
labeling, and immunoprecipitation of tight junction proteins was
performed as described in MATERIALS AND
METHODS. Relative intensity refers to the ratio of
signal intensity at the 12-h chase to 0-h chase in each sample. Note
that in the ZO-1 48-h IFN- treatment no protein band was detected.
OCC, occludin.
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The rate of protein biosynthesis can also be measured in metabolically
labeled cells, by comparing the intensity of the radiolabeled protein
bands at 0 h of chase between untreated and IFN-
-treated cells. In
the case of ZO-1, the ratio of signal intensity at the 0-h chase
between cells treated with IFN-
for 24 h and untreated cells was
~0.5, indicating a 50% decrease in the rate of ZO-1 biosynthesis.
The dramatic reduction in the biosynthetic rate of ZO-1 is made more
apparent by the fact that cells treated with IFN-
for 48 h had no
detectable ZO-1 protein. In contrast, the ratios of ZO-2 signal
intensities of cells treated for 24 and 48 h with IFN-
were 1.0 and
0.95, respectively, relative to untreated cells. Similarly for
occludin, relative to untreated cells, the ratios were 0.93 and 0.84 for cells treated with IFN-
for 24 and 48 h, respectively. Thus the
biosynthesis of ZO-1 is severely reduced, whereas ZO-2 and occludin are
relatively unaffected by IFN-
treatment. These observations are
consistent with the immunoblotting data that showed that only ZO-1
protein levels are reduced by IFN-
treatment, whereas ZO-2 and
occludin are not. Furthermore, these results show that IFN-
treatment of T84 cells appears to specifically repress ZO-1 biosynthesis.
IFN-
treatment decreases the transcription of ZO-1.
The effect of IFN-
treatment on the mRNA levels of ZO-1 was also
examined. The levels of RNA were measured using a semiquantitative RT-PCR rather than Northern blotting because RT-PCR is useful for
measuring small changes in mRNA levels and also because it can resolve
splice variants that differ only slightly in size. This latter point is
an issue for ZO-1 and ZO-2 because each gene encodes at least two
splice variants that differ by ~240 and 108 nt, respectively. These
variants are very difficult to resolve by Northern analysis given the
relatively large sizes of the mRNAs for these genes (~7.5 and 5 kb
for ZO-1 and ZO-2, respectively). [32P]dCTP was
incorporated into the PCR reaction, and the labeled product that was
synthesized was analyzed at various cycle numbers. The cycle number at
which the labeled product appeared was a measure of the relative
abundance of that product. In untreated cells, the labeled DNA fragment
of ZO-1 was first detectable at cycle 26 but did not appear until cycle
28 in the IFN-
-treated cells (Fig.
6A).
With increasing cycle number, this difference remained apparent (Fig.
6). Thus the decrease in ZO-1 mRNA levels was consistent with the
decrease observed in the levels of ZO-1 protein. In contrast, and as a
control for possible sample variation, when levels of ZO-2 mRNA were
measured by RT-PCR (Fig. 6), a labeled product of equal intensity was
observed in both control and IFN-
-treated samples beginning at
cycle 26. In subsequent cycles, the
levels of ZO-2 remained about equal. This result indicates that
transcription of ZO-2 mRNA is unaffected by IFN-
and is consistent
with the relatively modest changes observed in ZO-2 protein levels. The primers chosen for the RT-PCR analysis flank the DNA sequences that
encode the splice variants of both ZO-1 and ZO-2 and are thus able to
detect the long and short isoforms of each protein. In both instances,
however, only one major DNA fragment was amplified that corresponds to
the long isoform of each protein (4, 33). Thus, in T84 cells, only the
+-form of ZO-1 and the
+-form of ZO-2 are expressed.
In the case of ZO-2, this observation, along with the fact that
phosphatase treatment had no effect on the protein, suggests that the
lower-molecular-weight band seen in the soluble fraction of the
immunoblot analysis may be a product of proteolysis.

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Fig. 6.
RT-PCR analysis of ZO-1 and ZO-2 mRNA in untreated and IFN- -treated
T84 cells. RNA from either untreated T84 cells or cells treated for 72 h with 100 ng/ml IFN- was processed for RT-PCR as described in
MATERIALS AND METHODS.
A: autoradiographs of the
32P-labeled PCR products. Expected
sizes of the amplified fragments are 371 and 212 bp for ZO-1 and ZO-2,
respectively. Numbers above the gels indicate the number of PCR cycles
used to obtain the product. B:
quantitation of the autoradiographs. , Untreated cells; ,
IFN- -treated cells.
|
|
Loss of ZO-1 alters the subcellular distribution of ZO-2 and
occludin.
To examine if the loss of ZO-1 affects the localization of ZO-2 and
occludin, a confocal microscopic analysis was undertaken (Fig.
7). In untreated cells, en face
(x-y)
sections showed that ZO-1 was localized to the cell boundaries (Fig.
7A), and, in vertical (x-z)
sections through the monolayer, it was localized to a discrete subapical region of the lateral membranes (Fig.
7a). This immunostaining pattern is
characteristic of proteins that are found at tight junctions. ZO-2 and
occludin showed very similar staining patterns (Fig.
7Bb and
Cc, respectively). E-cadherin also
stained cell boundaries in en face sections (Fig.
7D), but, in vertical sections, it
was located on the lateral membranes boundaries as expected for a
protein that mediates cell-to-cell interactions (Fig.
7d). Analysis of IFN-
-treated T84
cells showed that immunostaining for ZO-1 was very faint to
undetectable in en face and vertical sections (Fig.
7Ee). ZO-2 staining in treated cells
appeared more diffuse in en face staining (Fig.
7F) compared with untreated cells.
In vertical sections, the staining of ZO-2 was no longer localized to
the tight junctions; rather, it was diffusely scattered in the cells
(Fig. 7f). Occludin showed a similar
diffuse staining in en face sections (Fig.
7G), and it was also distributed
along the apical and lateral membranes in vertical sections (Fig.
7g). E-cadherin staining in
IFN-
-treated cells was similar to untreated cells, remaining at
regions of cell contact (Fig. 7Hh).
Thus, in untreated cells, ZO-1, ZO-2, and occludin were all localized to tight junctions. However, after IFN-
treatment, ZO-1 was lost and
the distribution of ZO-2 and occludin was no longer restricted to the
tight junctions. Thus these results raise the intriguing possibility
that the proper subcellular localization of ZO-2 and occludin does not
occur in the absence of ZO-1.

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|
Fig. 7.
Confocal analysis of untreated and IFN- -treated T84 cells stained
for ZO-1, ZO-2, occludin, and E-cadherin. Untreated T84 cells or cells
treated for 72 h with 100 ng/ml IFN- were processed for
immunofluorescence as described in MATERIALS
AND METHODS. Cells were then analyzed by confocal
microscopy. Shown are the en face
(x-y)
sections
(A-H)
and the corresponding vertical
(x-z)
sections
(a-h).
Untreated T84 cells
(A-D
and
a-d)
and IFN- -treated cells
(E-H
and
e-h)
were stained for ZO-1 (A,
E and
a,
e), ZO-2
(B, F
and b,
f), occludin
(C, G
and c,
g), and E-cadherin
(D, H
and d,
h). Arrows by the
x-z
sections indicate the apical (Ap) and basal (Ba) aspects of the cells.
Bar in a = 10 µm.
|
|
IFN-
treatment perturbs actin in the apical-tight
junction region of T84 cells.
It is well established that epithelial barrier function can also be
regulated by the actin-containing cytoskeleton (2). To determine if
IFN-
treatment of T84 cells also affects the cytoskeleton, the
distribution of actin was examined by confocal microscopy using
rhodamine-phalloidin (Fig. 8). In polarized
epithelial cells, actin is found in the microvillar projections, in the
terminal web, in a belt at the level of the adherens junctions, at
cortical regions below the plasma membranes, and in stress fibers on
the basal surface of the cells (26). There is also evidence that actin
is associated with the tight junctions (20). In control cells, actin
staining at the apical-tight junction level revealed a bright, dense,
punctate pattern representing staining in the microvilli and a dense
network of actin at the cell boundaries (Fig.
8A). At the midpoint of the cell, a
cortical actin ring was seen (Fig.
8B), whereas, at the basal end of
the cells, a dense network of stress fibers was detected (Fig.
8C). In IFN-
-treated cells, the
most striking differences observed in actin staining were seen at the
apical-tight junction level (Fig.
8D), where the punctate staining
seen in controls was absent. In addition, actin at the cell periphery
appeared discontinuous and not as sharply defined as in untreated
cells. In many instances, no actin staining was detected at the
apical-tight junction level (Fig. 8D). At the midcell and basal
levels, actin was found at the cell cortex and in a dense network of
stress fibers, (Fig. 8, E and F, respectively), reminiscent of what
was seen in untreated cells. These results indicate that the effects of
IFN-
on the actin cytoskeleton in T84 cells is limited to the
apical-tight junction-associated actin. The actin cytoskeleton in the
more basal regions of the cells is unaffected. This effect is not
simply due to a decrease in actin levels in IFN-
-treated cells,
since immunoblotting with anti-actin antibodies showed comparable
levels of actin in untreated and treated cells (data not shown). Thus
IFN-
may be perturbing the barrier function because of its effects
on actin as well as on ZO-1.

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Fig. 8.
Effects of IFN- treatment on the actin cytoskeleton. Untreated T84
cells or cells treated for 72 h with 100 ng/ml IFN- were stained
with rhodamine-phalloidin and analyzed by confocal microscopy. Vertical
sections
(x-z)
were taken off the monolayers to define the top and bottom of the
monolayer. En face sections
(x-y)
were then generated from selected planes in the vertical section.
Untreated cells
(A-C)
and IFN- -treated cells
(D-F)
are shown at the apical-tight junction level
(A,
D), midcell level
(B,
E), and basal level
(C,
F). Arrows in
D indicate regions where no actin
staining is detected. Bar in A = 10 µm.
|
|
The alterations in apical actin in IFN-
-treated T84 cells observed
in this study were more dramatic than previously described (5, 24).
This difference may be a reflection of clonal variability in the T84
cells used by the various laboratories. Alternatively, the stability of
the cytoskeleton may have been influenced by the matrices on which the
cells were grown (tissue culture-treated polystyrene filters in this
study vs. collagen-coated filters in the previous studies).
Loss of ZO-1 and apical actin disruption coincide temporally.
In an attempt to dissociate the effects of IFN-
on ZO-1 and actin
and also to determine if the effects of IFN-
were reversible, the
expression of ZO-1 and actin was examined by confocal microscopy at
various times during IFN-
treatment and after removal of IFN-
(Fig. 9). Disruption and recovery of the
epithelial barrier were assessed by TER measurements of cells treated
with 100 ng/ml IFN-
for 72 h and subsequent removal of IFN-
from
the medium for an additional 72 h (Fig.
9A). Treatment of the cells with
IFN-
showed a time-dependent decrease in TER, whereas removal of the
IFN-
showed a time-dependent recovery in TER. The fact that the
cells could reestablish epithelial barriers with high electrical
resistance when IFN-
was removed demonstrates that IFN-
is not
toxic to cells (1). Concurrent confocal analysis showed that changes in
ZO-1 and actin expression correlated well with barrier breakdown and
recovery (Fig. 9,
B-G).
Untreated T84 cells showed the characteristic ZO-1 and actin staining
pattern around the cell periphery (Fig. 9B). After 24 h of treatment with
IFN-
, the staining pattern was similar but of a decreased intensity
(Fig. 9C). At 48 and 72 h, however,
ZO-1 staining was almost undetectable as was actin staining at the
apical portion of the cells (Fig.
9D). Twenty-four hours after removal
of IFN-
, ZO-1 staining was very weak and discontinuous; however,
wherever there was ZO-1 staining, apical actin staining was also
detected (Fig. 9E). After 48 and 72 h of recovery, ZO-1 and apical actin staining increased concomitantly (Fig. 9, F and
G). Thus, by temporal analysis, the
expression of ZO-1 at tight junctions and the reorganization of actin
at the apical/tight junction region of the cells coincide.

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|
Fig. 9.
ZO-1 and actin expression during barrier breakdown and recovery. Cells
were treated with IFN- for 0-72 h. At 72 h, IFN- was removed
from the medium and the cells were allowed to recover for 24-72 h.
A: TER measurements during the course
of IFN- treatment (solid bar along
x-axis) and recovery after IFN-
removal from the culture medium (open bar along
x-axis).
B-G:
confocal analyses of ZO-1 and actin were performed during the
IFN- -induced barrier breakdown and subsequent recovery. Shown are
the en face sections
(x-y)
from the apical region of cells stained for ZO-1 (green) and actin
(red). B: untreated cells.
C: 24-h IFN- treatment.
D: 48 and 72 h IFN- treatment.
E: 24 h after removal of IFN- .
F: 48 h after removal of IFN- .
G: 72 h after removal of IFN- . Bar
in F = 5 µm.
|
|
 |
DISCUSSION |
Degradation of the intestinal epithelial barrier is a characteristic of
numerous gut disorders, including inflammatory bowel disease. The cause
of the breakdown is multifactorial and involves bacteria, cells of the
immune system, and cytokines. To better understand the contribution of
proinflammatory cytokines to regulation of the epithelial barrier, we
have examined the effects of IFN-
on T84 intestinal epithelial
cells. The findings in this study indicate that IFN-
acts on at
least two elements critical to barrier function. The first is to cause
a decrease in the expression of ZO-1, a key component of the tight
junction. The second is to alter the organization of the actin
cytoskeleton in the apical region of the cells.
The pivotal role of ZO-1 in the tight junction complex is reflected by
its binding to numerous other tight junction and cytoskeletal proteins.
ZO-1 has been shown to bind occludin (10), ZO-2 (18), ZO-3 (12), and
actin (7, 15). As such, ZO-1 may serve to organize the tight
junction complex and also to connect it to the actin cytoskeleton. A
recent study has demonstrated that ZO-1 does indeed bind to occludin,
via an NH2-terminal domain, and actin, via a COOH-terminal domain, thus acting as a bridge between the
plasma membrane and the cytoskeleton (7). Given its role in connecting
numerous proteins, one might predict that loss of ZO-1 would result in
a disorganization of the tight junction, such as aberrant localization
of ZO-2 and occludin. Similarly, if the organization of tight
junction-associated actin requires binding to ZO-1, then loss of ZO-1
should perturb actin at that site. These alterations are, in fact, what
is observed when T84 cells are treated with IFN-
. As the barrier
function of T84 cells is eliminated, there is an associated loss of
ZO-1 and a subsequent mislocalization of both ZO-2 and occludin as
observed by confocal microscopy. In the case of ZO-2, alterations in
subcellular distribution were also reflected in biochemical assays in
which the protein showed almost complete loss from the
detergent-insoluble, tight junction-enriched fraction after IFN-
treatment. This result supports the notion that ZO-2 association with
the tight junction can only occur in the presence of ZO-1. Occludin
shows a similar disorganized subcellular localization in the
IFN-
-treated cells. However, in contrast to ZO-2, occludin remains
in the detergent-insoluble fraction even in the absence of ZO-1. This
suggests that the insolubility of occludin may be due to its inherent
propensity to form oligmers (29). In addition to remaining in the
insoluble fraction, occludin from IFN-
-treated cells did not show
gross alterations in phosphorylation, suggesting that this modification
occurs independently of ZO-1 association and tight junction
localization. The mislocalization of occludin in cells with decreased
levels of ZO-1, however, is consistent with a previous report showing
that interaction of occludin with ZO-1 is required for the proper
subcellular localization of occludin to the tight junctions (10). Thus
it appears that ZO-1 plays an active role in maintaining the
localization of ZO-2 and occludin. Whether these proteins are targeted
to the tight junction but are unable to remain there because ZO-1 is
not present to anchor them at that site remains to be determined.
Treatment of T84 cells with IFN-
also altered the actin cytoskeleton
in the region of the tight junction. The importance of actin to the
maintenance and regulation of barrier function has been demonstrated in
numerous studies (20, 21, 30). It has been suggested that expansion or
contraction of the actin cytoskeleton may exert forces that tighten or
loosen the tight junctions, respectively (20). The tight junction
regulatory activity of actin appears to be localized to the apical
portion of the cytoskeleton. Thus, in MDCK cells treated with high
concentrations of cytochalasin D, only basal actin was perturbed and
barrier function was only modestly affected. In contrast, MDCK cells
treated with low concentrations of cytochalasin D in which both the
apical and basal pools of actin were perturbed show decreased barrier function (31). Bacterial infection of epithelial cells by
Clostridium difficile and
Salmonella typhymurium also alters
apical and tight junction-associated actin and subsequently decreases
epithelial barrier function (13, 17). With C. difficle, the effect is most likely due to inactivation
of Rho, a small GTP binding protein required for actin assembly (19).
Similar results are obtained by directly targeting Rho by transfection
of T84 cells with C. botulinum toxin
C3 exoenzyme (27). Interestingly, in this study, ZO-1 was also lost
from the tight junctions of toxin-transfected cells and was
redistributed in the cell cytoplasm; ZO-1 levels, however, were not
reduced compared with control cells. Thus, whereas the effects on
apical actin may be similar with bacterial infection and IFN-
treatment of T84 cells, the effects on ZO-1 are quite different,
suggesting that the mechanism of barrier disruption induced by these
various agents is not the same. Whether the effects of IFN-
on
apical actin are the result of Rho inactivation remains to be determined.
On the basis of the experiments in this study, it has not been possible
to dissociate the effects of IFN-
on ZO-1 from those on actin.
Because ZO-1 levels in IFN-
-treated cells are reduced and protein is
not simply mislocalized, it is tempting to speculate that loss of ZO-1
is critical to the disorganization of apical actin. Further support for
this hypothesis comes from the fact that agents whose mode of action is
primarily on actin organization disrupt barrier function rapidly (i.e.,
minutes to hours). In contrast, IFN-
-induced barrier breakdown
requires significantly longer periods (i.e., hours to days), suggesting
that the perturbation of actin, in this case, is secondary to the loss
of ZO-1. Nonetheless, because the two events, loss of ZO-1 and apical
actin disruption, are temporally linked, it is not possible to
unequivocally state that one event is responsible for the occurrence of
the other. In future studies, it may be possible to test whether loss
of ZO-1 influences apical actin organization by expressing ZO-1 from a
heterologous promoter and then measuring the effects of IFN-
treatment on barrier function. One would predict that if loss of ZO-1
was the initiating event in barrier disruption then overexpression of
ZO-1 should prevent barrier breakdown and actin reorganization.
It is intriguing that, despite the apparent lack of ZO-1,
IFN-
-treated T84 cells can still maintain some barrier function. This observation suggests that other proteins may contribute to barrier
function despite the loss of a critical component of the tight
junction. Redundancy of function in tight junctions was shown in cells
lacking occludin; these cells could still form tight junctions and form
impermeable barriers (28). In this case, proteins, such as claudin-1
and -2 (8) and JAM (24), may functionally substitute for occludin. What
is unclear is whether these proteins function at tight junctions within
the same complex as does occludin (i.e., they bind to ZO-1) or as part
of a "parallel" complex (i.e., ZO-1 independent). Similarly, in
this study, is the residual barrier activity seen in the absence of
ZO-1 the result of redundancy in ZO-1 function or is it due to a
ZO-1-independent complex of proteins? Further analysis of the
composition and dynamics of tight junctions will allow us to resolve
these questions.
 |
ACKNOWLEDGEMENTS |
We thank Lori Whittaker, Helen Hathaway, Douglas Williams, and Linda
Park for critical reading of the manuscript, Ann Bannister for
editorial assistance, and Mari Hall for assistance with graphics.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Youakim,
Dept. of Biochemistry, Immunex Corporation, 51 University St., Seattle,
WA 98101 (E-mail: ayouakim{at}immunex.com).
Received 7 December 1998; accepted in final form 3 February 1999.
 |
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