1 CURE: Digestive Diseases Research Center, West Los Angeles Veterans Affairs Medical Center, School of Medicine, University of California, Los Angeles 70073; and 2 Department of Surgery and Physiology, School of Medicine, University of California, San Francisco, California 94143
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ABSTRACT |
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Previous studies found that
monolayers formed from canine oxyntic epithelial cells in primary
culture displayed remarkable resistance to apical acidification and
both mitogenic and migratory responses to epidermal growth factor (EGF)
treatment. In our present studies, we found that EGF increased
transepithelial resistance (TER) but not short-circuit current in these
monolayers. Parallel effects of EGF on decreasing mannitol flux and
increasing TER implicate direct regulation of paracellular
permeability. EGF acting at either apical and basolateral receptors
rapidly increased TER, but the apical response was sustained whereas
the basolateral response was transient. 125I-labeled EGF
binding revealed specific apical binding, but receptor numbers were
25-fold lower than on the basolateral surface. Both apical and
basolateral EGF activated tyrosine phosphorylation of EGF receptors
(EGFR), -catenin, and cellular substrate as evident on confocal
microscopy. Although apical EGF activated a lesser degree of receptor
autophosphorylation than basolateral EGF, phosphorylation of
-catenin was equally prominent with apical and basolateral receptor
activation. Together, these findings indicate that functional apical
and basolateral EGFR exist on primary canine gastric epithelial cells
and that these receptors regulate paracellular permeability. The
sustained effect of apical EGFR activation and prominent
phosphorylation of
-catenin suggest that apical EGFR may play a key
role in this regulation.
epidermal growth factor receptors; transforming growth factor receptors; gastric mucosal defense; paracellular pathway
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INTRODUCTION |
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EPIDERMAL GROWTH
FACTOR (EGF) is a 53-amino acid polypeptide that regulates the
proliferation and differentiation of a variety of cell types, including
polarized epithelial cells. EGF is present in salivary glands, duodenal
Brunner's glands, and mucosal epithelial cells after injury (1,
28, 36). Several other EGF family members are present in gastric
mucosa, including transforming growth factor- (TGF-
), which is
found in parietal cells (2, 5). Other members of the EGF
family present in gastrointestinal mucosa include amphiregulin and
heparin-binding EGF (1, 20).
EGF peptide family members are endogenous to the apical and basolateral gastric environment. These endogenous peptides appear to enhance mucosal repair, healing, and resistance to injury (6, 13, 17, 26). EGF itself is secreted at the luminal surfaces of salivary glands and duodenal Brunner's glands and is present in milk (3, 13). In addition, EGF is relatively stable in gastric juice, although some degradation occurs to smaller forms that retain moderate physiological activity (24). The physiological importance of luminal EGF is supported by observations (18, 21, 25, 31) that removing submandibular glands enhances susceptibility of the gastric mucosa to injury, whereas adding exogenous luminal EGF has protective effects. These data provide indirect evidence that functional EGF receptors (EGFR) are present on the luminal surface of gastric cells, where they appear to regulate aspects of mucosal defense and repair. However, it is also possible that EGF undergoes translocation to basolateral receptors (29).
Although it is clear that EGF acts predominantly at the basolateral surfaces of polarized epithelia, several lines of evidence indicate that EGF can also act at apical surface receptors. Low amounts (2-15%) of EGF total binding activity have been detected on the apical surfaces of several cultured cell lines (14, 15). Studies have focused on the classical EGFR, which is a 170-kDa transmembrane glycoprotein composed of an extracellular ligand binding domain and a cytoplasmic tyrosine kinase domain. EGFR have been immunohistochemically detected on the apical membranes of compacted eight-cell stage mouse embryos, trophoblastic cells from human placenta, and injured or diseased kidney cells (12, 34). However, the stability, receptor activation, and functional consequences of apical EGFR remain unclear. Model systems are limited for investigating apical receptors.
In our present studies, we have used a primary culture model of polarized canine gastric mucosal cells to examine the effects of EGF on paracellular permeability and to investigate the presence and functions of apical EGFR. In previous studies (4), we found these monolayers to display marked resistance to apical but not basolateral acidification, thus mimicking an important property of mucosal defense in vivo (27). We (4) further showed that apical acidification increased transepithelial resistance (TER) and decreased mannitol flux, indicating that the paracellular pathway was a critical point of resistance to apical acid. We now show that functional EGFR are located on the apical, as well as basolateral, surfaces of primary gastric mucosal cells and that both apical and basolateral EGFR mediate ligand-induced decreases in paracellular permeability and tyrosine phosphorylation of apical junction proteins.
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METHODS |
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Materials.
Materials for cell culture were obtained from sources outlined
previously (6). Transwell inserts were from Costar
(Cambridge, MA). 125I-labeled EGF and
125I-labeled TGF- were purchased from Biomedical
Technology (Stoughton, MA), and human recombinant EGF was from R&D
Systems (Minneapolis, MN). Anti-EGFR antibody MAb528 (Ab-1) was
purchased from Oncogen Science (Cambridge, MA). An epithelial
voltohmmeter (EVOM) Millicell-ERS was from Millipore (Bedford, MA).
Affinity-purified rabbit anti-ZO-1 was from Zymed Laboratories (San
Francisco, CA). Anti-phosphotyrosine (monoclonal IgG2bk)
and agarose-conjugated anti-phosphotyrosine were from Upstate
Biotechnology (Lake Placid, NY). A monoclonal antibody
phosphotyrosine-PY 20 used in the Western blot and anti-
-catenin (mouse IgG1) were from Transduction Laboratories (Lexington, KY). Other
chemicals were from Sigma Chemical (St. Louis, MO).
Tissue dispersion, cell separation, and culture. Enzyme-dispersed canine oxyntic mucosal cells were separated by elutriation and cultured on collagen-coated Transwell filter inserts (6 or 12 well; Costar), as described previously (4-6). When cells became confluent cultures, they contained predominantly chief cells (~55%) and parietal cells (~25%), as indicated by immunologic detection of pepsinogen and H+-K+-ATPase, respectively (18). The remaining ~10% of the cells are likely to be surface and mucous neck cells, proliferative cells, and occasional endocrine cells. Cells were cultured in DMEM-Ham's F-12 (1:1) plus 20 mM HEPES, 100 µg/ml amikacin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2% calf serum. Cultures were fed with the same medium every 48 h until confluent (~72 h). Monolayers were switched to serum-free medium (R0) for 6-16 h before growth factor studies were performed.
Electrophysiological measurement of TER. Monolayer TER was monitored using an EVOM with chopstick electrodes. This technique allows repeated measurements of monolayer TER in a sterile condition for a prolonged period of time. Comparative studies using Ussing chambers as described previously (4) were selectively performed to ensure that similar results were obtained with both methods.
Mannitol flux. Apical-to-basolateral mannitol flux was determined by adding [3H]mannitol to the apical medium as described previously (4). Aliquots from the basolateral medium at the indicated time intervals were sampled to determine the radioactivity.
125I-EGF binding.
Binding studies were performed on monolayers with TER >2,500
· cm2 as described previously (5).
125I-EGF was added either apically or basolaterally, as
indicated, in the absence or presence of various dilutions of either
cold EGF or monoclonal EGFR antibody MAb528. Unless otherwise
mentioned, MAb528 at the concentration of 20 nM (1:25 dilution of the
stock) was used in the studies. Binding was performed at 37°C for 45 min. At the end of binding, aliquots from the counterlateral side of
the 125I-EGF label were taken to determine the amount of
label crossing the monolayers. After extensive washing of the
monolayers, membranes were cut off from the inserts, and the
radioactivity was counted in a gamma counter. The total binding was
determined after subtracting the membrane blank (tracer incubated with
filter without cells) and expressed as the percentage of maximal
binding. 125I-EGF binding studies were also studied at
4°C for 2, 4, and 8 h of incubation.
125I-TGF- binding.
Selective binding studies were repeated with 125I-TGF-
label. The binding conditions were the same as those for
125I-EGF binding studies.
Immunostaining of junctional tyrosine phosphorylation. Monolayers were either treated with MAb528 or with apical EGF before paraformaldehyde (3.7%) fixation. Cells were coincubated with primary antibodies specific for ZO-1 (rabbit polyclonal, 1:1,000; Zymed) and phosphotyrosine (monoclonal, 1:50; Upstate Biotechnology) and then immunofluorescently stained using corresponding FITC- (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA) and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (1:50, Vector Laboratories, Burlingame, CA). Confocal microscopy (MRC1000, Bio-Rad Laboratories, Hercules, CA) was used to better visualize the apical tyrosine phosphorylation. Photomicrographs were generated by superimposing three adjacent horizontal (Z) optical sections of 0.5- to 1.0-µm thickness centered on the section displaying the most intense signal for the apical junctional marker ZO-1.
Immunoprecipitation and Western blot.
EGF-mediated tyrosine phosphorylation of the EGFR and -catenin was
assessed using immunoprecipitation and Western blot. Monolayers were
placed in serum-free medium overnight and then apically or basolaterally treated with EGF for 0-20 min. Cells were washed with ice-cold PBS containing phenylmethylsulfonyl fluoride (PMSF) (1 mM) and sodium vanadate (100 µM). Cells were then lysed in buffer A [50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton
X-100, 1.5 mM MgCl2, 10 mM EDTA, 10% glycerol, and
protease and phosphatase inhibitor cocktails (10 µg/ml aprotinin and
leupeptin, 1 mM PMSF, 1 mM Na3VO4, 100 µM
p-nitrophenylphosphate, and 10 mM NaF)] for 30 min in a
cold room. Soluble proteins were immunoprecipitated with
antiphosphotyrosine antibodies bound to protein A/G-agarose beads
(4G10, Upstate Biotechnology). Precipitated proteins were resuspended
in sample buffer, resolved by SDS-PAGE (Novex Electrophoresis, San
Diego, CA), transferred to nitrocellulose membranes (Bio-Rad Laboratories), and probed with primary antibodies to
antiphosphotyrosine (PY20, Transduction Laboratories) to detect EGFR or
with antibody to
-catenin (Transduction Laboratories).
Immunoreactive bands were visualized by enhanced chemiluminescence
(Amersham International) with horseradish peroxidase-conjugated
secondary antibodies (Bio-Rad Laboratories).
Data analysis. Kinetic binding data were analyzed using GraphPad Prism nonlinear regression software programs (San Diego, CA). Results are expressed as means ± SE. Statistical significance of differences between mean values was assessed using the Student's paired t-test with P < 0.05 (Statistix, NH Analytical, Roseville, MN).
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RESULTS |
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EGF-mediated regulation of
TER and paracellular permeability.
Canine oxyntic cells were cultured on Transwell filters until
confluent. When the TER was >1,000 · cm2,
monolayers were switched to serum-free medium for at least 6 h
before growth factor studies were performed. Monolayers were exposed to
5 nM EGF at either the apical or basolateral surface. Both treatments
resulted in detectable increases in TER within 2 min after adding EGF
(Fig. 1A) and a maximal
150-200% increase in TER within 30 min. Basolateral addition
consistently evoked greater increases in TER than apical addition.
However, the response to basolateral EGF was transient; TER returned to
baseline within 2-3 h with basolateral treatment, whereas the
effects of apical EGF were relatively stable over an experimental
period of several hours.
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Inhibition of EGF effects by immunoblocking EGFR with monoclonal
antibody MAb528.
We also investigated the effects of blocking EGF-induced increases in
TER by anti-receptor antibodies. In previous studies with canine
epithelial monolayers plated in plastic culture dishes, we
(18) found that the immunoblocking monoclonal antibody
against the human EGFR (MAb528) totally inhibited TGF--stimulated
thymidine incorporation and migration but did not reduce the effects of cytokines unrelated to EGF. Figure 2
illustrates that ipsilateral (same side) addition of MAb528 (1:25
dilution) suppressed increases in TER induced by low doses (1 nM) of
either apical or basolateral EGF. These immunoblocking effects of
MAb528 were surmounted by higher doses (50 nM) of EGF (Fig. 2). MAb528
at a 1:25 dilution appeared somewhat more effective in blocking
basolateral than apical receptor-mediated increases in TER. Monoclonal
antibody against somatostatin CURE S6 (7, 35) at a similar
dose did not alter TER effects of either apical or basolateral EGF
treatments. Thus apical and basolateral receptors immunologically
related to the human EGFR mediate ligand-dependent increases in TER.
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Binding of 125I-EGF to apical and
basolateral sites.
We (5) previously reported that both 125I-EGF
and 125I-TGF- bound to gastric epithelial monolayers in
plastic culture plates. We now find that 125I-EGF
specifically bound to high-affinity binding sites on both apical and
basolateral surfaces of monolayers grown on Transwell inserts. The
binding to both surfaces was blocked by ipsilateral, but not
contralateral, addition of MAb528 (Fig.
3, A and B). At the
end of the 45-min binding experiment, only 0.26 ± 0.04%
(mean ± SE; n = 4) of 125I
radioactivity was detected in the contralateral bathing medium, indicating that apical and basolateral membrane domains were completely separate and that EGF was not translocated to the contralateral surface
under these conditions. We found that basolateral EGFR specifically
bound ~25-fold more 125I-EGF than apical receptors
(10,000 ± 4,300 vs. 390 ± 120 counts · min
1 · filter
1;
mean ± SE; n = 3). This low proportion of apical
to total EGFR is similar to reports (14, 15) for other
cell types.
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Apical EGF induces apical and junctional tyrosine
phosphorylation.
We next examined protein tyrosine phosphorylation in response to apical
EGF treatment (Fig. 4). Cells exposed to
apical EGF for 20 min were colabeled with antibodies against
phosphotyrosine and antibodies against the tight junction protein ZO-1
to mark the apical/basolateral membrane interface. Immunofluorescent
confocal microscopy revealed marked increases in apical and
perijunctional tyrosine phosphorylation in response to apical EGF (Fig.
4B) compared with control cells (Fig. 4A).
Enhanced tyrosine phosphorylation was detected in most but not all
cultured cells, suggesting that within this mixed cell model, not all
cell types respond equally to apical EGF treatment. Nevertheless, these
results provide further evidence that apical EGFR are functional and
stimulate tyrosine kinase activity on ligand binding. In these
experiments, control cells (Fig. 4A) were treated with
MAb528 to reduce background signals due to endogenous secretion of
TGF- by parietal cells (5). However, we also detected
increased tyrosine phosphorylation in EGF-treated cells compared with
cells not exposed to MAb528 (data not shown, n = 3).
Although we focused our studies on the apical region, as marked by
ZO-1, phosphorylation at deeper sites within cells was also evident. We
did not detect changes in ZO-1 intensity among control, EGFR-blocked,
or apically EGF-treated monolayers.
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DISCUSSION |
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Our data demonstrate that exogenous EGF at the physiological concentrations added to the apical or basolateral surface of canine gastric monolayers in primary culture causes a rapid increase in TER and a corresponding fall in paracellular permeability, as evidenced by reduced mannitol flux. The tight linear relationship between 1/TER and mannitol flux supports the conclusion that changes in paracellular permeability underlie changes in TER under these conditions and that EGF-induced change in TER reflects direct effects on paracellular permeability.
The response to apical EGF was stable for hours. In contrast, the response to basolateral EGF was transient, returning to baseline after ~2 h. It is unclear why the effects of EGF on TER and mannitol flux are more stable with apical compared with basolateral treatment. It is possible that EGF is more rapidly internalized and degraded at basolateral EGFR or that downregulation of EGFR is more prominent at basolateral receptors. Regardless, the temporal correlation, evident by sustained apical and transient basolateral EGFR effects on both TER and mannitol flux, further supports the conclusion that increases in TER reflect decreased paracellular permeability.
Our data provide several lines of evidence supporting the existence of EGFR on the apical surface of these monolayers that are closely related to classical EGFR. 125I-EGF specifically bound to sites on the apical surface. However, these apical sites were sparse compared with basolateral receptors. Immunoblockade of EGFR by the anti-human EGFR antibody MAb528 displaced binding from both apical and basolateral sites. In addition, MAb528 blocked EGF-induced increases in TER at both apical and basolateral receptors. Anti-EGFR treatment inhibited the effects of low concentrations of EGF on TER, and this inhibition could be surmounted by higher concentrations of EGF. Apparent differences in MAb528 blockade of apical binding reflected the high proportion of nonspecific binding to the lower concentration of apical receptors. Taken together, these findings indicate that apical EGFR are present on these monolayers and that they are closely related to basolateral EGFR. Although mechanisms were not elucidated, studies (19) using polarized kidney epithelial cells with overexpressed apical EGFR demonstrated differences in EGFR downregulation and endocytosis between basolateral and apical EGFR. Studies of internalization would be difficult on our monolayers because of the low concentrations of apical EGFR.
Controls indicated that the apical and basolateral compartments of these culture inserts were functionally separated. Ipsilateral but not contralateral addition of MAb528 inhibited EGF binding to basolateral and apical receptors, respectively. Furthermore, the 125I-EGF tracer did not significantly cross the monolayer in these culture inserts.
It is well established that protein tyrosine phosphorylation mediates activation by EGFR, a process initiated by autotyrosine phosphorylation of EGFR itself (23). Tyrosine phosphorylation of EGFR substrates involved in the mitogenic pathway (SHC, ERK1, and ERK2) was detected in response to both apical and basolateral EGFR activation in fibroblast and polarized epithelial cells (9, 19, 22). These data suggest that in some experimental models for the mitogenic response to EGF apical EGFR may activate similar pathways to basolateral EGFR.
Although tyrosine phosphorylation has been proposed to play a role in tight junction regulation, supporting evidence remains limited. Selective protein kinase and tyrosine phosphatase inhibitors have been observed to alter protein phosphorylation, junction morphology, and paracellular permeability (8, 11, 33), but the specificity of these effects is controversial.
We used immunohistochemistry to study the effects of EGF on tyrosine-phosphorylated proteins (32). Confocal microscopy of monolayers grown on filter inserts revealed a typical apical junctional pattern with antibodies to ZO-1. The addition of EGF did not obviously alter the intensity or distribution of ZO-1 staining. In control monolayers or monolayers treated with MAb528, anti-phosphotyrosine antibodies detected proteins scattered throughout the cell. However, after treatment with apical or basolateral EGF, confocal microscopy indicated early, marked enhancement of apical and perijunctional tyrosine phosphoprotein immunoreactivity. Induction of tyrosine phosphorylation by apical EGFR activation provided additional evidence for the biological activity of these apical receptors.
We also used immunoprecipitation and Western blotting to study tyrosine
phosphorylation of EGFR and -catenin (16) in response to apical and basolateral EGFR activation. Autophosphorylation of the
180-kDa EGFR was found with both apical and basolateral EGF treatment.
However, basolateral EGFR activation produced much more pronounced
receptor autophosphorylation, reflecting the greater number of
basolateral EGFR.
Apical and basolateral EGF treatment also induced tyrosine
phosphorylation of the 92-kDa -catenin protein. Despite the
considerable difference in receptor density and autophosphorylation
between apical and basolateral EGF treatment, apical and basolateral
EGFR activation produced a roughly comparable degree of phosphorylation of
-catenin. These findings showing that apical EGFR activation induces tyrosine phosphorylation of EGFR and
-catenin provide further support for the potential physiological relevance of apical EGFR in gastric cells. These findings also suggest that apical EGFR may
phosphorylate certain junctional proteins, such as
-catenin, more
efficiently than basolateral receptors.
The mechanisms coupling EGFR activation to regulation of paracellular
permeability remain to be unraveled. EGF-dependent tyrosine phosphorylation of -catenin has previously been observed (10, 30) in a variety of transformed or immortalized cell lines. However, in these models (10, 30), phosphorylation
correlated with decreased cell adhesion or increased permeability. Our
findings suggest that EGF induces tyrosine phosphorylation of
-catenin simultaneously with a decrease in paracellular
permeability. We can only speculate that apical EGFR exert
physiological regulation of paracellular permeability and that tyrosine
phosphorylation of
-catenin plays a role in these actions of EGF.
Our primary cell culture monolayers provide a model for dissecting the
relevance and mechanisms of growth factor regulation of paracellular
permeability via effects at the apical junctional complex. This model
appears to be physiologically relevant by virtue of the presence of an apical barrier to acid that mimics this critical in vivo function of
the gastric mucosa and responsiveness to regulation by growth factors.
The physiological importance of the regulation of paracellular permeability and apical EGFR remains to be determined. Our data indicate that apical EGFR on canine gastric monolayers specifically bind ligands, exhibit tyrosine kinase activity, and mediate a decrease in paracellular permeability. Other experiments in our laboratory have shown that EGF-dependent decreases in paracellular permeability correlate with increased resistance to apical acidification (unpublished observations). We postulate that apical EGFR exert physiologically relevant actions in gastric mucosa, regulating the paracellular pathway, and thereby decreasing the permeability to acid and contributing to the remarkable ability of gastric mucosal glandular cells to withstand the acidic environment of the gastric lumen.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-30444 and DK-19984 and by the Medical and Research Services of the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. C. Chen, Veterans Affairs Wadsworth Hospital Center, Bldg. 115, Rm. 215, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: mcychen{at}ucla.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 March 1999; accepted in final form 22 August 2000.
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