Division of Gastroenterology, University Hospital, University of Nottingham, Nottingham NG7 2UH, United Kingdom
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ABSTRACT |
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The effect of chronic exposure to transforming growth factor-
(TGF-
) on bradykinin-stimulated acute prostanoid production and ion
secretion in monolayers of HCA-7 colony 29 colonic epithelial cells has
been studied. Monolayers synthesized prostaglandin
E2 (PGE2) at a basal rate of 2.10 ± 0.31 pg · monolayer
1 · min
1
over 24 h. Bradykinin
(10
8-10
5
M) dose dependently increased acute
PGE2 release by three orders of
magnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).
When monolayers were primed by a 24-h exposure to TGF-
, basal
PGE2 release rose to 6.31 ± 0.38 pg · monolayer
1 · min
1
(TGF-
concn 10 ng/ml; P = 0.001).
However, the stimulation of acute prostaglandin release, intracellular
cAMP, and increased SCC by bradykinin was significantly reduced by
preincubation with TGF-
. Priming with
PGE2
(10
8-10
6
M) over 24 h mimicked the effect of TGF-
on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF-
may
downregulate acute responses to bradykinin. In vivo, TGF-
could have
an important modulatory function in regulating secretion under
inflammatory conditions.
transforming growth factor-; cyclooxygenase; electrogenic ion
transport
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INTRODUCTION |
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ELECTROGENIC CHLORIDE secretion in intestinal
epithelial cells occurs when
Cl are actively transported
from the basolateral to the apical side of epithelial cells, and
excessive Cl
secretion in
vivo gives rise to secretory diarrhea (3). A number of inflammatory
mediators, including bradykinin (BK), eicosanoids such as prostaglandin
E2
(PGE2), histamine,
5-hydroxytryptamine, and a range of cytokines are known to mediate this
secretory response (26).
BK is a biologically active nine-amino acid peptide formed during
inflammation from protein kininogen and is known to stimulate electrogenic Cl secretion
in a variety of epithelial cells, including those of the
gastrointestinal tract (31), airways, and uterus (11). BK also acts
directly on colonic epithelial cells to stimulate Cl
secretion in vitro (1).
The effect of BK in directly stimulating electrogenic
Cl
secretion has best been
characterized for monolayers of the HCA-7 colony 29 colonic epithelial
cell line (5, 16). In this cell line, the effect of BK in stimulating
Cl
secretion is mediated
through cAMP-dependent pathways, pathways thought to be due to
eicosanoid production (13), although other mediators such as
intracellular Ca2+ also play a part.
Although much attention has focused on agents that stimulate
electrogenic Cl secretion,
relatively little is known of endogenous regulatory mechanisms that
will attenuate the secretory response. Epidermal growth factor (EGF)
has recently been recognized to have an important physiological role in
maintaining intestinal homeostasis (28) as well as stimulating
proliferation and growth. Growth factor receptors are found throughout
the human gastrointestinal tract and are primarily located in the
basolateral domain (4, 27, 34).
Transforming growth factor- (TGF-
) is a homolog of EGF and may be
the most important physiological ligand to the EGF receptor in the
intestinal mucosa (2, 12, 17). TGF-
has recently been shown to
enhance vectorial prostaglandin release from HCA-7 colony 29 cells when
applied to the basolateral compartment in which the EGF receptor
resides (4). Cyclooxygenase (COX) is the rate-limiting enzyme for the
conversion of arachidonic acid (AA) to prostanoids. Two isoforms of COX
are recognized: COX-1, which is constitutively expressed, and COX-2,
which is induced in pathological conditions by a range of chemicals
including cytokines and inflammatory mediators (22). TGF-
induces
COX-2 expression in the HCA-7 colony 29 cell line, resulting in
increased prostaglandin production (4). However, the effect of TGF-
on BK-mediated acute colonic epithelial ion transport and prostaglandin
synthesis is not known. Our original hypothesis was that the induction
of COX-2 expression in HCA-7 cells by TGF-
(4) could lead to both
enhanced acute PGE2 release and
acute secretory response to BK. In this study, we
therefore investigated the effect of TGF-
on BK-induced ion
transport and prostaglandin release in monolayers of HCA-7 colony 29 cells.
Although TGF- increased PGE2
release over 24 h, it did not enhance acute prostaglandin release or
the acute secretory response to BK. In fact, both prostaglandin release
and the acute Cl
secretory
response were significantly attenuated and BK-stimulated cAMP
production was reduced; these changes could be mimicked by exogenously
applied PGE2.
These findings suggest that TGF- may have an important role in
limiting the acute secretory response to BK during inflammation via a
prostanoid-dependent mechanism(s).
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METHODS |
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Cell culture. HCA-7 colony 29 (16) cells were grown in DMEM supplemented with 10% FCS, glutamine (0.29 mg/ml), ampicillin (8 µg/ml), penicillin (40 µg/ml), streptomycin (368 µg/ml), and nonessential amino acids in an atmosphere of 5% CO2 at 37°C. Cells were seeded on Snapwell and Transwell filters (polycarbonate membrane; pore size 0.45 µm; surface area 1 cm2; Costar) and formed confluent monolayers within 8-10 days, as assessed with an epithelial volt-ohmmeter (World Precision Instruments).
COX-1 and COX-2 Western blotting. HCA-7 cells were grown to confluence, lysed (10 mM PBS, 0.1% Triton X-100, 0.1% EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.03% leupeptin; Sigma), and centrifuged for 10 min. Cell lysates were separated by SDS-PAGE with COX-1 and COX-2 standards (Cayman Chemical, Ann Arbor, MI), transferred to nitrocellulose, and blocked with 5% nonfat dried milk in Tris-buffered saline (TBS). Membranes were incubated overnight in 0.1% Tween 20 in TBS with a 1:500 dilution of either COX-1 or COX-2 rabbit polyclonal antibodies (both from Cayman Chemical). The blots were washed, and biotinylated goat anti-rabbit IgG was added. Labeled bands were detected by an avidin-biotin complex (ABC) technique (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA).
Assay of basal and BK-induced prostaglandin synthesis.
To study prostaglandin production, cells were grown to confluence on
Transwell filters. After a change of the medium, cells were exposed to
basolateral TGF- or control medium for 24 h. The
supernatant was collected from the basolateral compartment to measure
PGE2 levels. Monolayers were
washed twice with fresh medium, then stimulated with basolateral BK
(10
6 M), and after 1 min
supernatant was collected for the
PGE2 assay. Dose-response curves
showing the effect of different concentrations of BK and the
attenuation of the response after preincubation with the COX inhibitor
piroxicam
(10
9-10
5
M) were constructed. The
PGE2 enzyme immunoassay (Amersham)
is based on the competition between unlabeled PGE and a fixed quantity of peroxidase-labeled PGE for a limited amount of the PGE-specific antibody.
Assay of BK-induced cellular cAMP production.
In subsets of experiments BK-induced cAMP production was measured by a
direct enzyme immunoassay (Amersham). Briefly, confluent monolayers of
HCA-7 cells grown on permeable supports and pretreated for 24 h
according to experimental conditions were washed twice with fresh
medium. After acute stimulation with BK
(106 M), membrane inserts
were transferred to new wells and the medium was replaced with lysis
reagent at a concentration of 0.25% dodecyltrimethylammonium bromide.
Cell lysate was collected, and cAMP levels were measured according to
the manufacturer's instructions. The detection range at room
temperature was 12.5-3,200 fmol/well.
Electrophysiology.
For electrophysiological studies, cells were treated with either
control medium or TGF- (10 and 100 ng/ml, respectively) for up to 24 h. Filters (area 1 cm2) were
placed into an Ussing chamber (World Precision Instruments) bathed in
oxygenated (95% O2-5%
CO2) Krebs-Henseleit solution (in mM: 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.0 MgSO4, 24.8 NaHCO3, 1.2 KH2PO4,
and 11.1 glucose) and maintained at 37°C. In some experiments, the
Cl
-containing salts of the
Krebs-Henseleit solution were replaced with (in mM) 117 sodium
gluconate, 4.7 potassium gluconate, and 2.5 calcium sulfate. The
epithelium was voltage-clamped to 0 mV by continuous application of a
short-circuit current (SCC) with a DVC-1000 dual-voltage clamp (World
Precision Instruments). Periodic constant-amplitude voltage pulses were
used to assess transepithelial resistance. Basal SCC
(µA/cm2) and resistance
(
· cm2)
were measured after the monolayers were allowed to equilibrate for 15 min. The changes in SCC (
SCCs) in response to BK, carbachol, and
forskolin administered to the basolateral side of the monolayer were
recorded. SCC was digitally recorded and analyzed with the Acqknowledge
III (BIOPAC Systems) data acquisition system.
SCCs were expressed in
units of microamperes per square centimeter. In some experiments
SCCs over a given time period were integrated and converted into
nanoequivalents by using the Faraday relationship (26.8 µA/cm2 = 1 µeq · cm
2 · h
1).
Materials.
BK, carbachol, forskolin, piroxicam, DMEM, and FCS were purchased from
Sigma; TGF- was purchased from R&D Systems.
Statistical analysis. Data are expressed as means ± SE. The unpaired, two-tailed Student t-test (adjusted for multiple testing by Bonferroni's correction) or a one-way ANOVA was used to determine the significance of differences between means. P < 0.05 was accepted as indicating statistical significance. The dose-response curves for BK and EC50 values were obtained by applying data to the curve-fitting program PRISM (Graphpad, San Diego, CA).
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RESULTS |
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Characteristics of HCA-7 cells.
HCA-7 cells expressed COX-1 and COX-2 proteins as shown by Western
blotting (Fig.
1A,
inset). They synthesized
PGE2 at a basal rate of 3.03 ± 0.45 ng · monolayer1 · 24 h
1, equivalent to an
average rate of 2.10 ± 0.31 pg · monolayer
1 · min
1.
HCA-7 epithelial cells exhibited a basal
SCC of 1.29 ± 0.21 µA/cm2
(n = 20) and a resistance of 138.1 ± 9.9
· cm2
(n = 20).
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Effect of BK.
BK applied to the basolateral compartments of monolayers of HCA-7 cells
caused a substantial increase in acute
PGE2 release (measured 1 min after
addition of the agonist) in a dose-dependent fashion of between 2.70 ± 0.45 (BK 108 M) and
6.30 ± 0.84 ng/monolayer (BK
10
5)
(n = 4-5; Fig.
1A). This was accompanied by a
rise in intracellular cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (n = 4;
P < 0.02; Fig. 3). BK stimulated
SCC in a dose-dependent fashion with an
EC50 = 2.43 × 10
7 M (Fig.
1B).
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Effect of TGF- preincubation on basal
PGE2 release and acute BK-stimulated
PGE2 release.
In previous experiments, the basolateral application of TGF-
increased COX expression and stimulated
PGE2 production over 24 h to
levels from 0.01 to 100 ng/ml, with a submaximal effect at 10 ng/ml
(4). In our experiments, TGF-
(10 ng/ml) administered to the
basolateral compartments of monolayers of HCA-7 cells similarly increased the basolateral release of
PGE2 over 24 h from 3.03 ± 0.45 (control) to 9.09 ± 0.54 ng/monolayer (rate 6.31 ± 0.38 pg · monolayer
1 · min
1;
n = 4;
P = 0.001).
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Effect of TGF- preincubation on subsequent
BK-stimulated ion transport.
Preincubation with TGF-
at 10 ng/ml for 24 h did not affect basal
SCC (1.67 ± 0.58 µA/cm2,
n = 20), although it was associated
with a significant increase in resistance from 138.1 ± 9.9 (control)
to 169 ± 16.2
· cm2
(n = 20;
P < 0.03). As with acute
PGE2 release after stimulation with BK, there was also an unexpected reduction in the
SCC response to subsequent BK challenge after a preconditioning with TGF-
for 24 h (Fig.
4B).
Compared with that for control cells, the maximal
SCC response to
BK was reduced by 57 ± 6% for cells conditioned by
TGF-
at 10 ng/ml (P < 0.01) and
by 60 ± 5.5% for cells conditioned by TGF-
at 100 ng/ml
(P < 0.01) (Fig.
5).
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Effect of exogenous PGE2 on responses to
BK.
To examine if exogenously administered prostaglandin could mimic the
effect of TGF-, monolayers of HCA-7 cells were preincubated with
PGE2 at concentrations
(10
8-10
6
M) in the range achieved after exposure to TGF-
in the experiments described above. Under these conditions, the BK-induced rise in cAMP
was significantly reduced (P < 0.05;
Fig. 3). This was accompanied by a significant dose-dependent reduction
in
SCC after exposure to PGE2
for 24 h compared with that for controls. This reduction was similar to
the TGF-
-induced downregulation of BK-stimulated ion transport (Fig.
5).
Time course of the effect of TGF- preincubation on
BK-stimulated ion transport.
To determine the duration of exposure of HCA-7 cells to TGF-
required to downregulate the secretory response to BK, TGF-
(10 ng/ml) was applied for different time periods (1, 6, 12, and 24 h) in
paired experiments before stimulation with BK. The area under the
response curve for the period after the addition of BK was measured
from 0 to 1 min (time of initial peak response) and is shown as the
percent modulation of the response from control monolayers. Exposure to TGF-
at 10 ng/ml caused a
reduction of the
SCC response to BK compared with that for the
control after 1 h of incubation, and this effect was maximal after 12 h
of incubation (Fig 6).
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Effect of AA on TGF--induced downregulation of ion
transport.
To investigate if TGF-
acted by depleting substrate for the acute
response to BK, TGF-
-treated monolayers were exposed to AA in Ussing
chambers for 30 min before stimulation with BK. AA (10
5-10
3
M) induced a slow increase in SCC (
SCC 2 µA/cm2), which returned to
baseline after 5 min. The responses to BK with and without AA in
control monolayers showed no difference (19.2 ± 2.36 vs. 19.8 ± 3.25 µA/cm2), and there was no
reversal of the downregulatory effect of TGF-
(Fig.
7), indicating that the effect of TGF-
was not due to the depletion of endogenous AA.
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Contribution of Cl secretion to
SCC.
To confirm previous reports that
Cl
secretion was the main
contributor to BK- and carbachol-stimulated
SCC in this cell line, ion substitution studies were performed. In
Cl
-free media,
SCCs in
response to BK and carbachol stimulation were reduced to 51.3 ± 7 (n = 6;
P < 0.02) and 55.4 ± 1.6%
(n = 6;
P < 0.02) of the response seen in
normal Krebs-Henseleit solution, confirming that the secretory response
to BK and carbachol was at least in part due to electrogenic
Cl
secretion
(23).
Specificity of the secretory response.
To investigate whether TGF- additionally affected the acute
secretory response of HCA-7 cells to other secretagogues and to test
the secretory capacity of HCA-7 cells, carbachol
(10
4 M; a cholinomimetic
agent that increases intracellular
Ca2+) and forskolin
(10
4 M; a direct adenylyl
cyclase activator) were added to controls and TGF-
-pretreated
monolayers. There was no significant reduction in the carbachol- or
forskolin-stimulated response in monolayers treated with TGF-
at 10 ng/ml for 24 h (Fig. 8). Likewise,
PGE2 in a dose range from
10
8 to
10
6 M had no significant
effect on the carbachol-induced
SCC (Fig. 8).
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DISCUSSION |
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In this study we have investigated the effect of TGF- on acute
secretory responses to BK in colonic epithelial cells. In the
intestine, lamina propria cells, including myofibroblasts and
mononuclear cells (11, 18, 33, 37), and epithelial cells
(5-7, 13, 20) have been previously implicated in the inflammatory
response to BK. BK activates cytosolic phospholipase A2 and increases
prostaglandin production thought to be primarily mediated by COX-1,
although COX-2 may participate in more chronic responses (22). We chose
the HCA-7 colony 29 cell line because previous studies had shown that
these cells express both COX-1 and COX-2 (4) and demonstrate a good
secretory response to BK (5-7, 13, 20). In addition, TGF-
induces COX-2 expression and basolateral release of prostaglandins in
this cell line (4).
Studies of HCA-7 monolayers have previously demonstrated that the
secretory response to BK is due to both increased cAMP (as a result of
increased endogenous prostaglandin production) and intracellular
Ca2+ (20). Although an additive or
even synergistic effect of BK-stimulated SCC might have been
anticipated, TGF-
preconditioning of HCA-7 cells paradoxically
downregulated BK-induced acute prostaglandin production, and there was
an accompanying reduction in cAMP production and
Cl
secretion. This effect
would appear to be mediated by prostaglandins because long-term (24 h)
exposure of monolayers to exogenous
PGE2 at concentrations similar to
that produced by TGF-
also significantly reduced BK-stimulated cAMP
production and Cl
secretion
in Ussing chambers. The mechanism by which TGF-
reduced the
secretory responses to BK was further investigated by adding AA before
stimulation with BK. In these experiments, BK-stimulated Cl
secretion could not be
restored to control levels in TGF-
-conditioned monolayers,
indicating that depletion of substrate (AA) for the COX enzyme is not
the mechanism by which TGF-
reduces the acute response to BK.
Therefore, chronic exposure of HCA-7 cells to prostaglandins may make
them partially resistant to further stimulation by BK, and this may
form part of a negative-feedback loop to limit the inflammatory and
secretory responses. This is not without precedent, as it has recently
been reported that prostaglandins exert negative-feedback effects on
the expression of COX-2 in macrophages (25) and on cAMP production in
airway smooth muscle cells (24) and may be part of an endogenous
regulatory mechanism. The inhibition of BK-induced increases in cAMP by
EGF in a human epidermoid carcinoma cell line (A431) has also recently
been described (19).
This downregulation of secretory responses, along with the enhancement
of barrier function implied by the increased resistance we have
reported in the present experiments, may contribute to a homeostatic
role for TGF- in modulating secretory and permeability changes that
can be induced by inflammation. Some authors have previously reported
reduced Cl
secretion in an
inflamed colonic mucosa (14). Because TGF-
primarily upregulated
COX-2 expression in a colony of HCA-7 cells similar to that used in our
experiments (4), it is tempting to speculate that
exaggerated PGE2 production as a
result of COX-2 induction is part of the negative-feedback system
exerting a braking effect on the inflammatory response.
Our experiments do not establish the mechanism by which TGF- leads
to downregulation of the responses to BK, which is the subject of
ongoing research. Possibilities include tachyphylaxis as a result of
prostaglandin receptor downregulation and alterations in localization,
making COX enzymes less accessible to substrate released by BK.
Illuminating these mechanisms will require an understanding of the
extent to which COX-1 and COX-2 contribute on the one hand to
TGF-stimulated prostaglandin synthesis and on the other to the
prostaglandin synthesis and secretory responses stimulated by BK.
It is possible that TGF- could have effects on HCA-7 cells apart
from stimulating prostaglandin production to attenuate the secretory
response to BK. The implication that an indirect metabolic mechanism is
involved is further supported by time course experiments showing a lag
of 1 h to the onset, and 12 h to the maximal effect, of TGF-
attenuation of BK-stimulated ion transport. In T84 cells, pretreatment
with EGF, which binds to the same ligand as TGF-
, reduced the
secretory response to carbachol partly by increasing intracellular
D-myo-inositol
3,4,5,6-tetrakisphosphate, which negatively affected
Cl
secretion (36). Whether
TGF-
reduces BK-stimulated anion secretion by a similar mechanism in
HCA-7 cells is not known. Interestingly, in our experiments with HCA-7
cells, TGF-
or prostaglandin preconditioning did not significantly
affect the response to basolateral carbachol compared with that for
control monolayers (Fig. 8), a finding that may reflect inherent
differences in secretory mechanisms between the T84 and HCA-7 cell
lines. In addition, we have shown that both COX-1 and COX-2 are
constitutively expressed in HCA-7 cells, whereas T84 cells show only
low levels of mRNA for COX-1 and none for COX-2 (Beltinger, unpublished
observations) and respond only weakly to BK (1).
Although growth factors are generally recognized for their role in
proliferation and restitution of epithelia and in maintaining mucosal
integrity, they have only recently been recognized to have a role in
regulating intestinal ion transport and inflammation. Previous studies
have shown that TGF- and EGF regulate other secretory functions in
the gastric mucosa (32) and in pancreatic acinar cells (35). EGF has
also been demonstrated to enhance glucose and sodium absorption in the
rat ileum (10, 15, 23). As already mentioned, EGF inhibits
carbachol-stimulated electrogenic Cl
secretion in T84 cells
(36).
Moreover, TGF- is elevated during gut inflammation (8, 9, 29), and
pretreatment with EGF in a rat model of colitis reduced inflammation
(30). Increases in prostaglandin synthesis associated with the
induction of COX are generally regarded as having proinflammatory
effects; our data suggest that they may also play a homeostatic counter
role, which may be compromised during treatment with COX inhibitors.
Regulatory mechanisms in the presence of an activated COX system would
be useful to prevent high-level prostaglandin production (due to
inflammatory mediators such as BK), which may in turn lead to the
stimulation of intestinal secretion and excessive fluid and electrolyte
losses from the body. Finally, a complete understanding of the effect
of TGF-
on colonic mucosal secretory responses will need to take
into account the ability of immunocytes to stimulate, and
myofibroblasts to downregulate,
Cl
secretion.
Hitherto, research on growth factors in the gut has emphasized their role in maintaining normal epithelial integrity and stimulating repair processes, as well as their possible involvement in malignant change. Our data are consistent with the notion that growth factors may also have an important homeostatic role in limiting the secretory responses to BK.
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ACKNOWLEDGEMENTS |
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HCA-7 colony 29 cells were a kind gift from Dr. Susan Kirkland.
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FOOTNOTES |
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J. Beltinger was supported by a grant from the Swiss National Science Foundation and the Ciba-Geigy-Jubilaeums-Stiftung, Switzerland.
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: W. A. Stack, Division of Gastroenterology, Univ. Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK (E-mail: william.stack{at}nottingham.ac.uk).
Received 19 August 1998; accepted in final form 22 January 1999.
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