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J Biol Chem, Vol. 274, Issue 47, 33449-33454, November 19, 1999
From the Department of Medicine, University of California,
San Diego, California 92103
We have previously demonstrated that epidermal
growth factor (EGF) inhibits calcium-dependent chloride
secretion via a mechanism involving stimulation of phosphatidylinositol
3-kinase (PI3-K). The muscarinic agonist of chloride secretion,
carbachol (CCh), also stimulates an antisecretory pathway that involves
transactivation of the EGF receptor (EGFR) but does not involve PI3-K.
Here, we have examined if ErbB receptors, other than the EGFR, have a
role in regulation of colonic secretion and if differential effects on
ErbB receptor activation may explain the ability of the EGFR to
propagate diverse signaling pathways in response to EGF
versus CCh. Basolateral, but not apical, addition of the
ErbB3/ErbB4 ligand The movement of water across intestinal epithelial cells is a
passive process driven by the active transport of ions. Under normal
circumstances sodium and chloride are actively absorbed from the
intestine, creating an osmotic gradient favorable for fluid absorption.
However, several pathological conditions are characterized by
alterations in intestinal ion transport processes, consequently
resulting in abnormalities of fluid transport (1). In such conditions,
it is thought that the absorptive capacity of the intestine is
overwhelmed by excessive fluid secretion, leading to the clinical
manifestation of secretory diarrhea. Conversion of the epithelium from
a net absorptive to a secretory state can occur through the actions of
neuronal, hormonal, and immunologically derived substances that are
released, for example, during conditions of bowel inflammation.
Typically, such agents promote chloride secretion by binding to
specific receptors on the surface of epithelial cells, thereby
increasing levels of intracellular second messengers, such as cyclic
nucleotides and calcium. This, in turn, activates prosecretory
signaling pathways, which ultimately interact with, and activate, the
transport proteins that comprise the chloride secretory mechanism (1,
2).
In addition to prosecretory signaling pathways, it is becoming evident
that mechanisms also exist within epithelial cells that result in
down-regulation of chloride secretion (3). For example, treatment of
epithelial cells with epidermal growth factor (EGF)1 results in inhibition
of subsequent chloride secretory responses to
calcium-dependent agonists, such as carbachol (CCh) (4). This effect of EGF is mediated by activation of phosphatidylinositol 3-kinase (PI3-K) (5). Furthermore, we have recently demonstrated that,
in addition to stimulating secretion, agonists such as CCh also
stimulate tyrosine kinase-dependent signaling pathways that limit the extent of ongoing secretory responses (6). These pathways
involve transactivation of the EGFR and subsequent activation of the
extracellular signal-regulated kinase isoforms of mitogen-activated protein kinase. However, CCh-induced activation of the EGF receptor (EGFR), unlike that induced by EGF itself, does not result in an
increase in the lipid kinase activity of PI3-K (5). One possible
explanation for this apparent differential propagation of signaling
pathways by the EGFR in response to activation by EGF, or
transactivation by CCh, respectively, may lie in the complex nature of
molecular interactions between the EGFR and its related growth factor
receptors, which together constitute the ErbB family of receptor
tyrosine kinases.
The EGF receptor (EGFR; ErbB1) is the prototypic member of the ErbB
family. To date, three other members of this family have been
identified: ErbB2 (p185erbB2/neu), ErbB3
(p180erbB3), and ErbB4
(p180erbB4) (7-9). Despite the large degree of
structural homology between them, ErbB family members differ from each
other in their patterns of expression, ligand specificity, and
intracellular substrates. Ligands that bind to and activate ErbB
receptors can broadly be divided into two classes, those that bind the
EGFR, such as EGF and transforming growth factor- Although overexpression of ErbB receptors, particularly ErbB2, has been
correlated with the development of epithelial tumors (15, 17, 18),
there is little information regarding potential roles for ErbB
receptors in acute regulation of epithelial function. Therefore, in the
present study, we have employed the T84 colonic epithelial
cell line to examine a possible role for ErbB family members, other
than the EGFR, in regulation of intestinal chloride secretion and to
examine the possibility that differential formation of receptor dimer
complexes may underlie the ability of the EGFR to propagate diverse
inhibitory signals in response to activation of the receptor by EGF
itself, or in response to transactivation by CCh.
Materials--
Recombinant human Cell Culture--
Methods for maintenance of T84
cells in culture were as described previously (19). Briefly,
T84 cells were grown in Dulbecco's modified Eagle's
medium/F-12 medium (JRH, Lenexa, KS) supplemented with 5% newborn calf
serum. Cells were passaged by trypsinization. For Ussing
chamber/voltage clamp experiments, approximately 5 × 105 cells were seeded onto 12-mm Millicell transwell
polycarbonate filters. For Western blotting/immunoprecipitation
experiments, approximately 106 cells were seeded onto 30-mm
Millicell transwell polycarbonate filters. Cells seeded onto
filters were cultured for 10-15 days prior to use.
Electrophysiological Studies--
Monolayers of T84
cells were mounted in Ussing chambers (window area = 0.6 cm2) and bathed in oxygenated (95% O2,
5% CO2) Ringer's solution at 37 °C. The composition of
the Ringer's solution was (in mM): 140 Na+,
5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 120 Cl Immunoprecipitations and Western Blotting--
T84
cell monolayers grown on filters were washed (twice) with Ringers'
solution, allowed to equilibrate for 30 min at 37 °C, and then
stimulated with agonists for the times indicated. The reaction was
stopped by washing in ice-cold phosphate-buffered saline, and the cells
were lysed in ice-cold lysis buffer (1% Triton X-100, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain, 100 µg/ml
phenylmethylsulfonyl fluoride, 1 mM
Na+-vanadate, 1 mM NaF, and 1 mM
EDTA in phosphate-buffered saline) for 45 min. Cells were then scraped
into microcentrifuge tubes and spun at 12,000 rpm for 10 min, and the
pellet was discarded. Samples were assayed for protein content (Bio-Rad
protein assay kit) and adjusted so that each sample contained an equal
amount of protein. For immunoprecipitation studies, lysates were
incubated with the appropriate dilution of immunoprecipitating antibody for 1 h at 4 °C, followed by another 1-h incubation at 4 °C
with protein A-Sepharose. Lysates were then centrifuged for 3 min at 15,000 rpm, and the supernatant was discarded. The pellets were then
washed twice in lysis buffer and twice in phosphate-buffered saline and
were then resuspended in 2× gel loading buffer (50 mM
Tris, pH 6.8, 2% SDS, 200 mM dithiothreitol, 20%
glycerol, 0.2% bromphenol blue). Samples were boiled for 3 min and
then loaded onto a polyacrylamide gel, and proteins were separated by
electrophoresis. Resolved proteins were transferred overnight at
4 °C onto a polyvinylidene membrane (NEN Life Science Products). After transfer the membrane was preblocked with a 1% solution of
blocking buffer (Upstate Biotechnology Inc.) for 30 min, followed by a
1-h incubation with the appropriate concentration of primary antibody
in 1% blocking buffer. After washing (three times for 10 min each) in
Tris-buffered saline with 1% Tween (TBST), membranes were then
incubated for 30 min in horseradish peroxidase-conjugated secondary
antibody (anti-mouse or anti-rabbit IgG/horseradish peroxidase;
Transduction Laboratories, Lexington, KY) in 1% blocking buffer.
This was followed by three 10-min washes in TBST. Immunoreactive proteins were detected using a chemiluminescence detection kit (Roche
Molecular Biochemicals). Densitometric analysis was carried out using
NIH Image software.
Statistical Analyses--
All data are expressed as means±S.E.
for a series of n experiments. Student's t tests
or analysis of variance (ANOVA) with the Student-Newman-Keuls
post hoc test were used to compare mean values as
appropriate. p values <0.05 were considered to represent significant differences.
Heregulin Inhibits CCh-stimulated Chloride Secretion in
T84 Cells--
First, we set out to determine if ErbB
receptors, other than the EGFR, are functionally expressed in colonic
epithelial cells. To do this, we examined the effects of the
ErbB3/ErbB4 ligand, Heregulin Stimulates Tyrosine Phosphorylation and Dimerization of
ErbB2 and ErbB3 in T84 Cells--
We next examined the
effects of the growth factor on protein tyrosine phosphorylation in
T84 cells. Basolateral HRG (100 ng/ml) induced a
time-dependent increase in the tyrosine phosphorylation of
high molecular mass (~180 kDa) proteins (Fig.
2), with a maximal effect occurring at
approximately 15 min. In order to determine if these proteins
correspond to ErbB receptors activated in response to HRG, experiments
were next carried out in which cells were stimulated with HRG (100 ng/ml); lysates were immunoprecipitated with antibodies to EGFR, ErbB2,
or ErbB3; and Western blots were analyzed with anti-phosphotyrosine.
HRG was found to stimulate tyrosine phosphorylation of both ErbB2 and
ErbB3, but had no effect on tyrosine phosphorylation of EGFR (Fig.
3). Expression of ErbB4 in
T84 cells was not detected (data not shown). Further
experiments were carried out in which cells were stimulated with HRG
(100 ng/ml), lysates were immunoprecipitated with anti-ErbB3, and
immunoprecipitated proteins were analyzed by Western blotting with
anti-ErbB2. These experiments revealed that, in addition to stimulating
tyrosine phosphorylation of ErbB3 and ErbB2, HRG also stimulates the
formation of ErbB3/ErbB2 receptor dimers (Fig.
4).
PI3-K Mediates the Inhibitory Effect of HRG on CCh-stimulated
Isc--
We next went on to determine the signaling
pathway mediating the effects of HRG on CCh-stimulated secretion.
First, in voltage clamp experiments we found that the inhibitory
effects of maximally effective concentrations of EGF (100 ng/ml) and
HRG (100 ng/ml) on CCh-stimulated chloride secretion were not additive,
implying EGF and HRG exert their antisecretory effects via a common
signaling pathway (Fig. 5A).
Since previous studies from our laboratory have demonstrated that PI3-K
mediates the inhibitory effects of EGF on CCh-stimulated secretion, we
therefore examined a possible role for PI3-K in also mediating the
inhibitory effects of HRG. Cells were stimulated with HRG (100 ng/ml),
cell lysates were immunoprecipitated with anti-ErbB3 or with
anti-ErbB2, and immunoprecipitated proteins were analyzed by Western
blotting with antibodies to the p85 subunit of PI3-K. The data
demonstrate that HRG increased co-immunoprecipitation of p85 with both
ErbB2 and ErbB3 (Fig. 5, C and D), indicating HRG
likely stimulates activation of PI3-K. Thus, we examined the effects of
the PI3-K inhibitor, wortmannin, on HRG-mediated inhibition of
CCh-stimulated chloride secretion. Pretreatment of voltage-clamped
T84 cells with wortmannin (50 nM) completely
reversed the inhibitory effect of HRG on CCh-stimulated Isc
(Fig. 5D). Of note, and as previously reported (5),
wortmannin (50 nM) did not significantly alter responses to
CCh alone.
EGF and CCh Differentially Stimulate ErbB Receptor Tyrosine
Phosphorylation, Dimerization, and Recruitment of p85 to ErbB Receptor
Complexes in T84 Cells--
While both EGF and CCh
stimulate activation of the EGFR in T84 cells, only EGF
stimulates increases in PI3-K activity (5). We therefore set out to
determine if differential stimulation of ErbB receptors might underlie
the ability of the EGFR to propagate diverse signals in response to
stimulation by EGF versus CCh. Cells were stimulated with
either CCh (100 µM) or EGF (100 ng/ml), and cell lysates
were immunoprecipitated with antibodies to EGFR, ErbB2, or ErbB3.
Immunoprecipitates were analyzed by Western blotting with
anti-phosphotyrosine. As previously reported (6), both CCh and EGF
stimulated EGFR phosphorylation (Fig.
6A). However, only EGF
stimulated an increase in tyrosine phosphorylation of ErbB2 (Fig.
6B) and neither agonist appeared to have an effect on ErbB3
phosphorylation, although ErbB3 phosphorylation could readily be
detected in HRG-stimulated cells (Fig. 6C).
In similar experiments, EGF- and CCh-stimulated T84 cell
lysates were immunoprecipitated with anti-EGFR, followed by Western blotting with anti-ErbB2. Data from these studies revealed that only
EGF, and not CCh, stimulated the formation of EGFR/ErbB2 receptor
dimers (Fig. 7A). Finally,
experiments were carried out to determine if the apparent difference in
the ability of EGF and CCh to stimulate activation of ErbB2 might
underlie the difference in their ability to stimulate PI3-K.
T84 cell monolayers were stimulated with EGF (100 ng/ml) or
CCh (100 µM), and lysates were immunoprecipitated with
antibodies to EGFR or ErbB2. Western blots were then probed with
antibodies to the p85 subunit of PI3-K. We found that, although both
EGF and CCh stimulated recruitment of p85 to the EGFR (Fig.
7B), only EGF stimulated the recruitment of p85 to ErbB2
(Fig. 7C).
In the present study we provide further evidence that the ErbB
family of growth factor receptors plays an important role in regulation
of intestinal epithelial ion transport. In addition to the EGFR, we
have shown that colonic epithelial cells also express ErbB3 and ErbB2
and that activation of these receptors with HRG, a growth factor that
is expressed in the intestinal mucosa (21), results in inhibition of
calcium-dependent chloride secretory responses. In
vivo, such an effect on chloride secretion would be accompanied by
a reduction in net fluid secretion into the intestinal lumen. The
effects of HRG appear to be mediated by the formation of ErbB2/ErbB3
receptor dimers, since HRG increased tyrosine phosphorylation of both
these receptor types and increased co-immunoprecipitation of ErbB2 with
ErbB3. Since no known ligands bind directly to ErbB2, the formation of
ErbB2/ErbB3 dimers is likely brought about by HRG first binding to
ErbB3 followed by recruitment of ErbB2 to form the receptor dimer
complex. This particular dimer combination has been shown to mediate
the effects of HRG in many other cell types and is believed to be the
most catalytically active of ErbB dimer complexes (7, 18, 22-24). It
is also noteworthy that, in T84 cells, HRG did not
stimulate phosphorylation of the EGFR, indicating that there is a
similar selectivity and specificity of growth factor/ErbB receptor
interactions in the intestinal epithelium to that seen in other tissues
(9, 14).
Similar to EGF (5), the effects of HRG in inhibiting CCh-stimulated
chloride secretion appear to be mediated via stimulation of PI3-K
activity, an enzyme that mediates the effects of HRG in several other
cell types (18, 23-25). This conclusion is based on the observations
that (i) the inhibitory effects of HRG and EGF on CCh-stimulated
secretion were not additive, implying a common mechanism of action,
(ii) HRG stimulated the recruitment of PI3-K to the ErbB2/ErbB3
receptor complex, and (iii) an inhibitor of PI3-K activity, wortmannin
(26), completely reversed the inhibitory effects of HRG on
CCh-stimulated secretion. Although wortmannin has been demonstrated to
alter the activities of other enzymes, such as phospholipase D and
phospholipase A2 (33, 34), it is unlikely that either of
these enzymes are involved in mediating the effects of HRG on chloride
secretion since both phospholipase A2 and phospholipase D
potentiate, rather than inhibit, calcium-dependent secretory responses (35, 40). How PI3-K might exert an inhibitory effect on chloride secretion is currently unknown, but studies from our
laboratory indicate that EGF-stimulated PI3-K activity may target
basolateral K+ channels to inhibit the extrusion of
K+ across the basolateral membrane, a step that is
essential for epithelial chloride secretion to occur (3). It is
possible that this effect of PI3-K may, in turn, be mediated by protein kinase C since this enzyme also appears to have a role in negative regulation of epithelial secretion and has been shown to function downstream of PI3-K in other cell types (27-29). Studies are currently under way in our laboratory to more fully elucidate possible
interactions between PI3-K, protein kinase C, and epithelial transport
processes in T84 cells.
The present studies demonstrate that, depending on the activating
agonist, stimulation of the EGFR results in the formation of different
receptor dimer complexes. Activation of the EGFR by its cognate ligand,
EGF, not only leads to increased tyrosine phosphorylation of the EGFR,
but also results in increased phosphorylation of ErbB2, accompanied by
the formation of EGFR/ErbB2 receptor dimer complexes. The formation of
this complex is rapid, occurring within 1 min, is stable for prolonged
periods of time, and thus mirrors the time course of the inhibitory
effects of EGF on calcium-stimulated chloride secretion (4). In
contrast, transactivation of the EGFR by CCh is not accompanied by
appreciable stimulation of ErbB2 phosphorylation nor does it bring
about the formation of EGFR/ErbB2 receptor dimers. This is in contrast
to previous studies in rat fibroblasts where G-protein-coupled receptor
agonists, such as lysophosphatidic acid and thrombin, were found to
stimulate both EGFR and ErbB2 phosphorylation (30). This apparent
difference in the responsiveness of ErbB2 receptors to
G-protein-coupled receptor-mediated tyrosine phosphorylation in
fibroblasts and colonic epithelial cells underlines the inherently
heterogenous nature of signaling within the ErbB family of receptor
tyrosine kinases.
It is not yet clear how activation of the EGFR by EGF itself, or
transactivation by CCh, leads to differential phosphorylation of ErbB2
and recruitment to the EGFR. However, it is likely that this may be due
to differential phosphorylation of tyrosine residues on the EGFR (13).
Whereas the intrinsic tyrosine kinase activity of the EGFR mediates
autophosphorylation of the receptor in response to EGF, a different
mechanism exists for EGFR phosphorylation in response to
G-protein-coupled receptor agonists, which may involve Src family
tyrosine kinases as signaling intermediates (31, 32). These two
different mechanisms for EGFR activation presumably result in different
patterns of tyrosine phosphorylation, which, in turn, may dictate the
nature of receptor dimers formed, and/or signaling proteins recruited,
in response to stimulation by different agonists (13). Furthermore,
since only EGF, and not CCh, stimulated recruitment of the p85 subunit
of PI3-K to ErbB2, our current data suggest that it is this difference
in ErbB2 activation and recruitment to the EGFR that likely underlies the ability of the EGFR to differentially signal to PI3-K in response to EGF and CCh in T84 cells (5). However, it is interesting to note that, even though it does not stimulate the lipid kinase activity of the enzyme, CCh does induce association of PI3-K with the
EGFR. This is in agreement with our previous data, which showed that
CCh increases the amount of both the p85 and p110 subunits of PI3-K in
anti-phosphotyrosine immunoprecipitates from T84 cells (5).
However, even though CCh stimulates PI3-K recruitment to the EGFR, it
is unclear what the physiological significance of this effect may be
since, as mentioned above, it does not appear to stimulate the lipid
kinase activity of the enzyme (5).
In summary, we have shown that, in addition to the EGFR,
T84 colonic epithelial cells also express the ErbB2 and
ErbB3 members of the ErbB family of receptor tyrosine kinases. Similar
to EGF, treatment of T84 cells with the ErbB3 ligand, HRG,
results in inhibition of subsequent calcium-dependent
chloride secretory responses. The effects of HRG appear to be mediated
by the recruitment of PI3-K to ErbB3/ErbB2 receptor dimers. As depicted
in Fig. 8, we propose that ErbB receptor
expression in the intestinal epithelium may provide a means by which
diversification, and integration, of antisecretory signaling pathways
can be achieved in response to different growth factors and hormones.
These studies, along with emerging evidence to suggest a potential role
for growth factors, such as EGF, in healing mucosal ulcers associated
with intestinal inflammation (37-39), may provide the basis for novel approaches in the treatment of intestinal inflammatory disorders.
We thank Glenda Wheeler-Loessel for assistance
with manuscript submission.
*
This work was supported by a career development award from
the Crohn's and Colitis Foundation of America (to S. J. K.)
and by Grant DK28305 from the National Institutes of Health (to K. E. B.). These studies were presented in part at the 1998 Annual Meeting of the American Gastroenterological Association, and have been
published in abstract form (Keely, S. J., and Barrett, K. E. (1998) Gastroenterology 114, A385).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.
§
Faculty member, Biomedical Sciences Ph.D. Program, University of
California, San Diego, School of Medicine.
The abbreviations used are:
EGF, epidermal
growth factor;
EGFR, epidermal growth factor receptor;
CCh, carbachol;
HRG, heregulin;
Isc, short circuit current;
PI3-K, phosphatidylinositol 3-kinase;
ANOVA, analysis of variance.
ErbB2 and ErbB3 Receptors Mediate Inhibition of
Calcium-dependent Chloride Secretion in Colonic Epithelial
Cells*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-heregulin (HRG; 1-100 ng/ml) inhibited
secretory responses to CCh (100 µM) across
voltage-clamped T84 epithelial cells.
Immunoprecipitation/Western blot studies revealed that HRG (100 ng/ml)
stimulated tyrosine phosphorylation and dimerization of ErbB3 and
ErbB2, but had no effect on phosphorylation of the EGFR. HRG also
stimulated recruitment of the p85 subunit of PI3-K to ErbB3/ErbB2
receptor dimers, while the PI3-K inhibitor, wortmannin (50 nM), completely reversed the inhibitory effect of HRG on
CCh-stimulated secretion. Further studies revealed that, while both EGF
(100 ng/ml) and CCh (100 µM) stimulated phosphorylation
of the EGFR, only EGF stimulated phosphorylation of ErbB2, and neither
stimulated ErbB3 phosphorylation. EGF, but not CCh, stimulated the
formation of EGFR/ErbB2 receptor dimers and the recruitment of p85 to
ErbB2. We conclude that ErbB2 and ErbB3 are expressed in
T84 cells and are functionally coupled to inhibition of
calcium-dependent chloride secretion. Differential dimerization with other ErbB family members may underlie the ability of
the EGFR to propagate diverse inhibitory signals in response to
activation by EGF or transactivation by CCh.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and those which
bind to ErbB3 and ErbB4, the heregulins (HRG). ErbB2 has been
classified as an orphan receptor due to the lack of a known ligand that
binds this receptor. ErbB receptors also differ in their kinase
activity; whereas ErbB3 lacks intrinsic kinase activity, ErbB2 is the
most catalytically active member of the ErbB family. The transduction of extracellular signals via ErbB receptors is a complex process, involving what can be considered as both lateral and vertical signaling
pathways. Generally, upon ligand binding, ErbB receptors undergo
autophosphorylation and dimerization to form catalytically active homo-
or heterodimers, with ErbB2 being the preferred partner for the other
three activated receptors (10-12). Depending on the type of dimer
formed, specific sets of SH2-containing proteins interact with the
dimer complex, ultimately resulting in the activation of different
intracellular effector proteins, such as mitogen-activated protein
kinase, PI3-K, and phospholipase C-
. The multiplicity of ErbB
receptor ligands, possible combinations of ligand-stimulated receptor
dimers, and the existence of multiple downstream effector proteins,
confers this family of receptors with an enormous potential for
regulation and diversification of intracellular signaling pathways (9,
11, 13-16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HRG (R&D Systems,
Minneapolis, MN), carbachol (Sigma), epidermal growth factor (Genzyme,
Cambridge, MA), mouse monoclonal anti-human EGF receptor, mouse
anti-recombinant rat ErbB3 rabbit polyclonal anti-human p85, and mouse
monoclonal anti-phosphotyrosine antibodies (Upstate Biotechnology Inc.,
Lake Placid, NY), rabbit polyclonal antibodies against a peptide
corresponding to the carboxyl terminus (1169-1186) of human ErbB2
(Santa Cruz Biotechnology, Santa Cruz, CA), and Tris-glycine
electrophoresis gels (Bio-Rad) were obtained from the sources
indicated. All other reagents were of analytical grade and were
obtained commercially.
, 25 HCO3, 2.4 H2PO4, 0.4 HPO42, and 10 glucose. Monolayers were
voltage-clamped to zero potential difference by the application of
short-circuit current (Isc). Under these conditions,
changes in Isc (
Isc) in response to agonists are wholly reflective of electrogenic chloride secretion (20).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HRG, on Isc responses to CCh across
voltage-clamped monolayers of T84 cells. Pretreatment of
T84 cells with basolateral HRG (100 ng/ml; 20 min) resulted
in significant attenuation of subsequent Isc responses to
basolateral CCh (100 µM) (Fig.
1A). Maximal responses to CCh
were 45.7 ± 4.7 µA/cm2 and 24.8 ± 2.8 µA/cm2 in the absence and presence of HRG, respectively
(p < 0.001; n = 9). The effects of
basolateral HRG were concentration-dependent, with a
maximal effect occurring at approximately 100 ng/ml (Fig. 1B). Of note, a residual response to CCh persisted that was
insensitive to even the highest concentration of HRG tested. These data
are reminiscent of those previously reported for EGF (4). HRG was without effect on CCh-stimulated Isc when added to the
apical side of the monolayer. Responses to CCh (100 µM)
were 48.3 ± 4.8 µA/cm2 and 44.7 ± 6.2 µA/cm2 in the absence and presence of apical HRG (100 ng/ml), respectively (n = 6). HRG alone had no effect
on basal Isc.
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Fig. 1.
Heregulin inhibits CCh-stimulated chloride
secretion in T84 cells. A, pretreatment of
voltage-clamped T84 cell monolayers with basolateral HRG
(100 ng/ml; 15 min) significantly attenuated subsequent responses to
basolateral CCh (100 µM) added at time zero
(p < 0.001; n = 9). HRG alone had no
effect on basal Isc. 
, CCh alone; 
, CCh + HRG.
B, the effects of HRG were
concentration-dependent with a maximal inhibitory effect
occurring at approximately 100 ng/ml (n = 7-9 for each
concentration tested).
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Fig. 2.
Heregulin stimulates protein tyrosine
phosphorylation in T84 cells. T84 cell
monolayers, grown on permeable supports, were treated with basolateral
HRG (100 ng/ml) for the times indicated, and cell lysates were analyzed
by Western blotting with anti-phosphotyrosine. In comparison to
control, unstimulated monolayers (C), HRG stimulated a
time-dependent tyrosine phosphorylation of proteins with
calculated molecular sizes of approximately 180 kDa as indicated by the
arrow. This blot is representative of four similar experiments.
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Fig. 3.
Heregulin stimulates tyrosine phosphorylation
of ErbB2 and ErbB3, but not EGFR, in T84 cells. Cells
were stimulated with HRG (100 ng/ml) for the times indicated in the
figure and were then immunoprecipitated with antibodies to EGFR
(A), ErbB2 (B), or ErbB3 (C).
Immunoprecipitated proteins were then analyzed by Western blotting with
anti-phosphotyrosine. Densitometric analysis of the data is represented
in panel D (n = 3-4 for each
experiment). We found that, while HRG had no effect on phosphorylation
of EGFR, it did stimulate tyrosine phosphorylation of both ErbB2 and
ErbB3 (p < 0.001 by ANOVA in each case). Control
experiments (panel A) revealed that
phosphorylation of EGFR was detected readily in lysates of cells
treated with EGF (100 ng/ml) for 1 min.
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Fig. 4.
Heregulin stimulates dimerization of ErbB2
and ErbB3 in T84 cells. A, cells were
stimulated with HRG (100 ng/ml) for the times indicated and cell
lysates were then immunoprecipitated with antibodies to ErbB3.
Immunoprecipitated proteins were analyzed by Western blotting with
anti-ErbB2. Densitometric analysis of the data is presented in
panel B. HRG significantly increased the
co-immunoprecipitation of ErbB2 with ErbB3 (p < 0.05 by ANOVA, n = 3) with a time course similar to that
seen for HRG-stimulated ErbB3 and ErbB2 tyrosine phosphorylation
(cf. Fig. 3).
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Fig. 5.
Phosphatidylinositol 3-kinase mediates the
inhibitory effect of HRG in T84 cells. A,
voltage-clamped T84 cell monolayers were pretreated with
either HRG (100 ng/ml), EGF (100 ng/ml), or a combination of both, for
20 min prior to stimulation with CCh (100 µM). Both HRG
and EGF alone significantly inhibited subsequent Isc
responses to CCh. However, their effects were not found to be additive,
implying both growth factors activate a common antisecretory signaling
pathway (n = 5; *, p < 0.05, **,
p < 0.01 by ANOVA followed by Student-Newman-Keuls
post hoc test). In further experiments cells were
stimulated with HRG for the times indicated and cell lysates were
immunoprecipitated with anti-ErbB2 (B) or anti-ErbB3
(C). Immunoprecipitated proteins were then analyzed by
Western blotting with antibodies to the regulatory p85 subunit of
PI3-K. Our data indicate that PI3-K is recruited to both ErbB2 and
ErbB3 in response to stimulation with HRG. These blots are
representative of three similar experiments. D, further
experiments revealed that the PI3-K inhibitor, wortmannin (50 nM), reversed the inhibitory effect of HRG (100 ng/ml) on
CCh (100 µM)-stimulated chloride secretion across
voltage-clamped monolayers of T84 cells. **, significant
difference from cells stimulated with CCh alone (n = 6;
p < 0.01 by ANOVA followed by Student-Newman-Keuls
post hoc test).
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[in a new window]
Fig. 6.
EGF and CCh differentially stimulate ErbB
receptor phosphorylation in T84 cells. Cells were
stimulated with CCh (100 µM) or EGF (100 ng/ml) for the
times indicated and lysates were immunoprecipitated with antibodies to
EGFR (A), ErbB2 (B), or ErbB3 (C).
Immunoprecipitated proteins were then Western blotted with
anti-phosphotyrosine. Although both CCh and EGF stimulated
phosphorylation of EGFR (p < 0.05 and
p < 0.001, respectively, by ANOVA), only EGF was found
to stimulate phosphorylation of ErbB2 (p < 0.005).
Neither CCh nor EGF had any effect on ErbB3 phosphorylation, although,
as can be seen in panel C, tyrosine
phosphorylation of the receptor was readily detected in response to HRG
(100 ng/ml; 15 min). Panels D and E
show the densitometric analysis of these data (n = 3-6
for each experiment).
View larger version (32K):
[in a new window]
Fig. 7.
EGF, but not CCh, stimulates dimerization of
ErbB2 with the EGFR and recruitment of p85 to ErbB2 in T84
cells. Cells were stimulated with either CCh (100 µM) or EGF (100 ng/ml). A, cell lysates were
immunoprecipitated with anti-EGFR and immunoprecipitated proteins were
analyzed with anti-ErbB2. Only EGF, and not CCh, stimulated increases
in co-immunoprecipitation of ErbB2 with the EGFR. This blot is
representative of three separate experiments. Finally, CCh- or
EGF-stimulated cell lysates were analyzed by immunoprecipitation with
anti-EGFR (B) or anti-ErbB2 (C), followed by
Western blotting with antibodies to the p85 subunit of PI3-K. While
both EGF and CCh stimulated recruitment of p85 to the EGFR, only EGF
stimulated recruitment of p85 to ErbB2. Each blot is also
representative of three to five similar experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 8.
Negative regulation of
Ca2+-dependent chloride secretion by the ErbB
family of receptor tyrosine kinases. Agonists such as CCh
stimulate epithelial chloride secretion via a mechanism involving
increases in intracellular Ca2+ (heavy solid
lines). However, the magnitude and duration of CCh-stimulated
secretory responses are intrinsically limited by a signaling pathway
involving CCh-stimulated transactivation of the EGFR and subsequent
activation of the extracellular signal-regulated kinase isoforms of
mitogen-activated protein kinase (light dotted lines).
Chloride secretion in response to
Ca2+-dependent agonists may also be limited by
growth factors, such as EGF and HRG, which via the formation of
EGFR/ErbB2 and ErbB3/ErbB2 heterodimers, respectively, lead to
activation of PI3-K and subsequent down-regulation of the secretory
response (heavy dashed lines). Please note that the
convergence of the signaling pathways shown on an apical chloride
channel is not necessarily intended to imply a direct effect of these
mechanisms on a chloride channel protein, but rather (for simplicity)
an effect (either positive or negative) on the overall chloride
secretory mechanism. This could also involve effects on basolateral
membrane transport pathways, such as potassium channels.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence and reprint requests should be addressed:
University of California San Diego Medical Center, 8414, 200 W. Arbor
Dr., San Diego, CA 92103. Tel.: 619-543-3726; Fax: 619-543-6969;
E-mail: skeely@ucsd.edu.
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