Induction of endothelin-1 synthesis by IL-2 and its modulation
of rat intestinal epithelial cell growth
Takeharu
Shigematsu1,
Soichiro
Miura2,
Masahiko
Hirokawa1,
Ryota
Hokari1,
Hajime
Higuchi1,
Naoyuki
Watanabe1,
Yoshikazu
Tsuzuki1,
Hiroyuki
Kimura1,
Shinichiro
Tada1,
Ruri C.
Nakatsumi1,
Hidetsugu
Saito1, and
Hiromasa
Ishii1
1 Department of Internal Medicine, School of
Medicine, Keio University, Tokyo 160-8582; and
2 Second Department of Internal Medicine,
National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
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ABSTRACT |
Endothelin (ET), a
vasoconstrictive peptide, is known to have a variety of biological
actions. Although ET is released by vascular endothelial cells, other
cell populations also have been reported to synthesize and release ET.
In this study, we examined whether ET is synthesized by intestinal
epithelial cells and whether it affects induction of epithelial cell
proliferation by interleukin-2 (IL-2). Subconfluent monolayers of
intestinal epithelial cells (IEC-6 and IEC-18) were maintained in
serum-free medium before addition of rat IL-2. Both IEC-6 and IEC-18
cells released ET-1 into the medium under unstimulated conditions, as
determined by a sandwich ELISA. IL-2 significantly enhanced ET-1
release in a time-dependent manner. ET-3 was not detectable in the
culture media of either cell line. Expression of ET-1 and ET-3 mRNA in epithelial cells was assessed by competitive PCR. Both cell lines were
shown to express ET-1 mRNA, but no ET-3 mRNA was detected. IL-2
treatment enhanced ET-1 mRNA expression by both IEC-6 and IEC-18 cells.
Both cell lines also expressed mRNA for
ETA and ETB receptor subtypes. When cell
proliferation was assessed, exogenous ET-1 induced a slight
proliferative response in both types of cells that was consistent and
significant at low ET-1 concentrations; cell growth was inhibited at a
higher concentration (10
7
M). IL-2 produced a significant proliferative response in both cell
lines. However, the addition of ET-1
(10
7 M) to culture media
attenuated the IL-2-induced increase in cell proliferation.
ETA-receptor antagonists
significantly enhanced cellular proliferation, suggesting involvement
of the ETA receptor in modulation
of IL-2-induced intestinal epithelial cell growth.
ETA receptor; mRNA expression
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INTRODUCTION |
ENDOTHELIN (ET) is a potent vasoconstrictor peptide
with 21 amino acid residues that originally was isolated and
characterized from the supernatant of cultured porcine endothelial
cells (44). Three ET isopeptides (ET-1, ET-2, ET-3), identified in
humans, pigs, and rats (14, 15, 44), have different pressor and vasoconstrictor activities. Among these, ET-1 is the strongest pressor
and vasoconstrictor ever isolated. Currently, two distinct ET receptors
with different affinities for ET-1 and ET-3 have been identified. ET-1
is a more potent agonist than ET-3 at the ETA receptor, while these two
peptides have similar potencies at the
ETB receptor (36). These
receptors, originally identified in blood vessels, also have been found
in many other organs, including the heart, kidneys, brain, and liver
(4). In keeping with the wide distribution of ET receptors, ETs elicit
a wide variety of biological effects, including positive cardiac
inotropism and chronotropism, inhibition of sodium reabsorption, and
neuroendocrine functions (21, 36). More importantly, ET-1 promotes the
growth of various cell types, such as fibroblasts, mesangial cells,
endothelial cells, and melanocytes (1).
Several reports (22, 23) have emphasized the gastrointestinal tract as
a major target of the ETs. We have demonstrated that
infusion of ET-1 into the mesenteric artery induced significant ischemia-reperfusion injury in intestinal mucosa (23). More recently, we reported (22) that ET-1 is significantly involved in
endotoxin-induced microcirculatory damage in the rat small intestine.
However, little is known regarding the exact source and target of ETs
in the intestinal mucosa under physiological and pathophysiological
conditions. A broad spectrum of regulatory peptides recently have been
identified within the intestinal mucosa, and intestinal epithelial
cells have been shown to synthesize soluble mediators that may be
important modulators of epithelial cell function and immune cell
interactions. These regulatory peptides include transforming growth
factors (TGF-
), interleukins (IL-1, IL-6, IL-8), tumor necrosis
factor (TNF), and interferons (IFN-
) (9, 16, 19, 31, 35, 38). Nitric
oxide synthase activity and nitric oxide formation also have been
demonstrated in intestinal epithelial cells (8, 41). Recently, IL-2
receptors have been identified in rat intestinal epithelial cell lines,
and their participation in cell restitution and proliferation has been
demonstrated (5, 7). Because IL-2 is pivotal in the generation and
regulation of the immune response among numerous cytokines, intestinal
epithelial cell function should be directly affected by mucosal immune
reactions. This complex network of interacting mediators within the
intestinal mucosa appears important to a range of local biological
processes (26). However, little information is available regarding the details and local consequences of expression of ETs in intestinal epithelial cells.
In the present study, we explore the possibility that ETs are expressed
and released by intestinal epithelial cells, using the in vitro model
of monolayers of two nontransformed rat intestinal epithelial cell
lines, IEC-6 and IEC-18. We found that IL-2, a potent cytokine whose
biological targets have been presumed to be largely limited to
lymphocyte and macrophage populations (17, 43), stimulated ET-1 release
from intestinal cells; ET-1, in turn, may be involved in regulating the
growth of these cells. These findings suggest that ET can facilitate a
coordinated response by epithelial cells and cellular constituents of
the local immune system that is present in the intestinal mucosa.
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MATERIALS AND METHODS |
Cell cultures and IL-2 treatment.
Cell lines IEC-6 and IEC-18, originating from rat intestinal epithelia
(30), were purchased from American Type Culture Collection (Rockville,
MD). The stock cultures were grown in an atmosphere of 5%
CO2 at 37°C in a culture
medium composed of DMEM containing 5% FCS, 10 µg/ml insulin, 50 U/ml
penicillin, 50 U/ml streptomycin, and 4 mM glutamine. IEC-18 from
passages
8 or
9 were used. In experiments to assess
the effects of IL-2 on cells, recombinant rat IL-2 (Sigma Chemical, St.
Louis, MO) was added to subconfluent monolayers at a concentration of
10 U/ml, and the cells were cultured for 48 h.
Determination of ET release.
Subconfluent monolayers were cultured in serum-free DMEM or in DMEM
containing 5% FCS, and culture was maintained for 48 h. Culture media
were collected at 0, 6, 12, 24, and 48 h, and the concentrations of
ET-1 and ET-3 were determined by ELISA using a kit supplied by Takeda
Pharmaceutical (Osaka, Japan) (39). Samples were stored at
70°C in polypropylene tubes containing aprotinin and EDTA at
final concentrations of 300 KIU/ml and 2 mg/ml, respectively. The
sample was extracted using Sep-Pak
C18 cartridges, and the extracts
were dissolved in 250 µl of assay buffer (0.02 M phosphate buffer at
pH 7 containing 10% Block Ace, 0.4 M NaCl, and 2 mM EDTA). Samples to
be tested in 100 µl of the assay buffer were added to each well of
98-well microplates coated with monoclonal antibody against ET-1 and
incubated at 4°C for 24 h. After being washed with PBS, the well
contents were allowed to react with 100 µl of anti-ET (15-21)
Fab'-horseradish peroxidase diluted 1:400 in incubation buffer
(0.02 M phosphate buffer, pH 7, containing 1% BSA, 0.4 M NaCl, and 2 mM EDTA) at 4°C for 24 h. After three washes with PBS, bound enzyme
activity was measured with an ELISA reader (NJ-2000, Inter Med, Tokyo, Japan), using o-phenylenediamine as
the chromogen. Concentrations of protein in samples were measured by
the method of Lowry et al. (18).
RNA extraction and PCR amplification.
Total RNA was isolated from intestinal epithelial cells using RNAzol
(Biotcx, Houston, TX). Cells were lysed with 1.0 ml RNAzol/dish. Isolation and extraction were performed according to the
manufacturer's suggested protocol. Briefly, RNA was extracted with
chloroform and precipitated with isopropanol. The precipitated RNA was
washed with 70% ethanol. The concentration of the extracted RNA was
calculated by measuring the optical density at 260 nm. The ratio of the
optical density at 260 nm to that at 280 nm was always >1.9. The
quality of RNA was assessed by the intactness of 28S and 18S bands and the lack of degradation on agarose-gel electrophoresis.
Aliquots of RNA (5 µg) were reverse-transcribed using an RT-PCR kit
from Stratagene (La Jolla, CA). Briefly, 5 µg of RNA in 38 µl of
diethyl pyrocarbonate-treated water was mixed with 0.3 µg of
oligo(dT), heated at 65°C for 5 min, and then cooled slowly at room
temperature. The following reagents were added to the tubes: 5 µl of
10× concentrated synthesis buffer (final concentration, 10 mM
Tris · HCl, pH 8.3, 50 mM KCl, 1.5 mM
MgCl2), 1 µl of RNase block
inhibitor (40 U/µl), 2 µl of 100 mM dNTPs, and 1 µl of Moloney murine leukemia virus-RT (50 U/µl). The reaction mixture was
incubated for 1 h at 37°C before the reaction was terminated by
incubating the tube at 90°C for 5 min and on ice for 10 min. The
tube was stored at
80°C until PCR was performed using the
Takara Taq kit (recombinant
Taq DNA polymerase; Takara
Biochemicals, Tokyo, Japan), with rat-specific primers prepared on a
DNA synthesizer (Sawady Technology, Tokyo, Japan). The primers were
designed according to cDNA sequences of rat preproET-1 (32) and
preproET-3 (34) and are as follows
The
cDNA amplification products were predicted to be 543 bp in length for
ET-1 and 477 bp for ET-3. To initiate the PCR, we added 2 µl of RT
products to the PCR master mix, including 10× PCR reaction buffer
diluted to final concentrations of 10 mM Tris · HCl,
pH 8.3, 50 mM KCl, and 1.5 mM
MgCl2, and 2.5 U of recombinant
Taq DNA polymerase, 50 pM each of the
primers, and 200 µM dNTPs. Tubes were placed in a Programmed
Tempcontrol system (Applied Biosystems, Tokyo, Japan) that was
programmed as follows: 1) incubation
at 94°C for 3 min (initial denaturation); 2) 30 cycles of the following
sequential steps: 94°C for 1 min (denaturation), 60°C for 1 min
(annealing), and 72°C for 3 min (extension); and
3) incubation at 72°C for 7 min
(final extension). The PCR products were size-fractionated by agarose
gel electrophoresis. After electrophoresis and ethidium bromide
staining, DNA bands were visualized with an ultraviolet
transilluminator. We confirmed the identity of the PCR products by
direct DNA sequencing, using ABI PRISM linkage mapping sets
(Perkin-Elmer Applied Biosystems) on an Applied Biosystems model 377 DNA sequencer.
In the case of ET receptors, PCR was performed with the following
specific primers according to Cai et al. (3)
The cDNA amplification products
were predicted to be 418 bp in length for the
ETA receptor and 900 bp for the
ETB receptor. The following
conditions were used in the Programmed Tempcontrol system. ET
receptors: 1) incubation at 94°C
for 3 min (initial denaturation); 2)
30 cycles of the following sequential steps: 94°C for 1 min
(denaturation), 55°C (ETA
receptor) or 60°C (ETB receptor) for 1.5 min (annealing), and 72°C for 1.5 min
(extension); and 3) incubation was
done at 72°C for 7 min (final extension).
Expression of ET-1 in intestinal epithelial cells after IL-2 treatment
was compared with that in nontreated cells by competitive PCR, which
was performed using the PCR MIMIC Construction kit (Clontech
Laboratories, Palo Alto, CA). First, nonhomologous internal standard
DNA fragments, called PCR MIMICs, were constructed for use in
competitive PCR amplification to quantitate target mRNA levels. A PCR
MIMIC consists of a heterologous DNA fragment with primer templates
that are recognized by a pair of gene-specific primers. In the present
study, we designed the PCR MIMIC for a PCR product 340 bp in size.
Serial dilutions of PCR MIMICs were added to PCR amplification
reactions containing the experimental cDNA samples.
Cell proliferation and DNA synthesis.
Cells were cultured in 96-well multiwell plates in DMEM containing 10%
FCS. After cells had attached to their substrates, the culture medium
was changed to serum-free DMEM or DMEM containing 5% FCS for growth
experiments at 24 h after plating. The number of cells was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay at 24 and 48 h after each treatment. On
termination of incubation, reduction of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (inner salts) to formazan was assessed using a CellTiter 96 AQ nonradioactive cell proliferation assay kit (Promega, Madison, WI).
DNA synthesis was estimated with an immunocytochemical assay kit using
monoclonal bromodeoxyuridine (BrdU) antibody to detect BrdU
incorporation in cellular DNA (RPN 210; Amersham, Tokyo, Japan).
Agents studied.
To assess the effect of IL-2 on cells, we added IL-2 (Sigma Chemical)
at 10 U/ml to the culture media. The selective protein kinase C (PKC)
inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and the
selective calmodulin inhibitor
N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7) also were obtained from Sigma Chemical. These agents were added to culture media at a concentration of 30 µM, and
ET release was determined.
To examine the effect of ET-1 on proliferation of IEC-6 and IEC-18
cells, ET-1 (10
11 to
10
7 M) (American Peptide,
Sunnyvale, CA) was added to serum-free culture medium.
A specific ETA-receptor
antagonist, BQ-123 (donated by Banyu Pharmaceutical, Tokyo) (13), or a
combined ETA- and
ETB-type receptor antagonist,
bosentan (donated by Roche) (6), was added at a concentration of 10 µM to culture media containing 5% FCS. Cell
proliferation was determined at 24 h.
Statistical analysis.
All results are expressed as means ± SE. Differences among groups
were evaluated by one-way ANOVA and Fisher's post hoc test. Any
difference for which P < 0.05 was considered statistically significant.
 |
RESULTS |
Figure 1 depicts changes over time in ET-1
concentration in the culture media of IEC-6 and IEC-18 cells. Both cell
lines were shown to release a small amount of ET-1 (<10 pg/ml, 4 × 10
9 M at 48 h)
under unstimulated conditions. IL-2 significantly enhanced release of
ET-1 into culture media in a time-dependent manner for both IEC-6 and
IEC-18 cells. In serum-free medium, IL-2 administration increased the
release of ET-1 to ~50 pg/ml (2 × 10
8 M) at 48 h. In DMEM
containing 5% FCS, ET-1 release was significantly greater, reaching
400 pg/ml (1.6 × 10
7
M) and 300 pg/ml (1.2 × 10
7 M) under IL-2
stimulation for IEC-6 and IEC-18 cells, respectively. ET-3 was not
detectable in the culture media of IEC-6 or IEC-18 cells either before
or after IL-2 treatment.

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Fig. 1.
Time-course changes in endothelin-1 (ET-1) concentrations in culture
media of IEC-6 (A) and IEC-18 cells
(B). Subconfluent monolayers were
cultured in serum-free DMEM or in DMEM containing 5% FCS, and
concentrations of ET-1 in culture media were determined by enzyme
immunoassay. Recombinant rat interleukin-2 (IL-2) was added to the
subconfluent monolayers at a concentration of 10 U/ml, and the
incubation was continued for 48 h. Values are expressed as means ± SE of 6 experiments. * P < 0.05 vs. IL-2( )FCS( ).
# P < 0.05 vs.
IL-2(+)FCS( ).
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As determined by RT-PCR, both IEC-6 and IEC-18 cell lines specifically
expressed ET-1 mRNA at 543 bp (Fig. 2). In
contrast, no mRNA for ET-3 was detectable in these cell lines.
Expression of ET-1 by IEC-6 and IEC-18 cells 2 h after IL-2 treatment
was compared with that in nontreated cells using competitive PCR (Fig. 3). In both cell lines, a significant
increase in ET-1 mRNA expression was observed after addition of IL-2.

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Fig. 2.
Expression of ET-1 and ET-3 mRNA by IEC-6 and IEC-18 cells as
determined by RT-PCR. Both cell lines express specific ET-1 mRNA at 543 bp. In contrast, there is no detectable ET-3 mRNA in these cell
lines.
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Fig. 3.
Expression of ET-1 by IEC-6 (A) and
IEC-18 cells (B) 2 h after treatment
with IL-2 compared with nontreated cells using competitive PCR.
Recombinant rat IL-2 was added to subconfluent monolayers at the
concentration of 10 U/ml, and culture was continued for 2 h. Both cell
lines showed specific expression of ET-1 mRNA as shown at 543 bp. The
PCR MIMIC (see MATERIALS AND
METHODS) was designed so that the PCR product would
be 340 bp, and serial dilutions of PCR MIMICs were added to PCR
amplification reactions containing the specified amounts of the
experimental cDNA samples. In both cell lines, a significant increase
in ET-1 mRNA expression was observed after IL-2 administration.
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ET receptor mRNA expression by IEC-6 and IEC-18 cells was determined by
RT-PCR (Fig. 4). Both cell lines were shown
to express ETA receptor mRNA at
418 bp and ETB receptor mRNA at
900 bp.

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Fig. 4.
Expression of ET receptor (ETA and
ETB) mRNA by IEC-6 and IEC-18
cells as determined by RT-PCR. Both cell lines were shown to express
ETA receptor mRNA at 418 bp and
ETB receptor mRNA at 900 bp.
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Figure 5 shows the effect of the PKC
inhibitor H-7 and the calmodulin inhibitor W-7 on the release of ET-1
into culture media. Cells were grown in media containing 5% FCS. H-7
treatment significantly attenuated ET-1 release from both IEC-6 and
IEC-18 cell cultures. W-7 similarly inhibited ET-1 release in both cell
lines.

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Fig. 5.
Effect of PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine
(H-7) and calmodulin inhibitor
N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide
hydrochloride (W-7) on ET-1 release into culture media. Cells were
grown in media containing 5% FCS, and time course of changes in ET-1
concentrations in the culture media was determined for IEC-6
(A) and IEC-18 cells (control;
B). H-7 treatment significantly
attenuated the increase in ET-1 release by both IEC-6 and IEC-18 cell
lines. Similarly, W-7 also significantly inhibited the enhancement of
ET-1 release into the media by these cell lines.
* P < 0.05 vs. control.
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We used the MTT assay to determine the effect of ET-1 administration on
proliferation of IEC-6 and IEC-18 cells in serum-free media (Fig.
6). Addition of ET-1 induced a slight but
consistent proliferative response that was significant at 24 h in both
cell types at low concentrations
(10
11-10
9
M, 0.025-2.5 pg/ml); ET-1 inhibited cell growth at a higher
concentration (10
7 M, 250 pg/ml).

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Fig. 6.
Effect of ET-1 administration on proliferation of IEC-6 and IEC-18
cells in serum-free media as determined by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Addition of ET-1 to the culture media induced a slight, but
constant and significant proliferative response within 24 h in both
cell lines at a lower concentration
(10 11-10 9
M) and inhibited their growth at a higher concentration
(10 7 M). Values are
expressed as means ± SE of 6 experiments.
* P < 0.05 vs. control.
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We also used the MTT assay to determine the effect of IL-2 on
proliferation of IEC-6 and IEC-18 cells in serum-free media. In Fig.
7, the rate of proliferation is expressed
as the percent increase from control values without IL-2. IL-2 time
dependently increased cell proliferation up to 48 h in both cell lines.
Addition of ET-1 at a concentration of
10
7 M significantly
attenuated IL-2-induced cell proliferation at 48 h, while ET-1 at a
lower concentration (10
9 M)
did not affect IL-2-induced proliferation. IL-2 did not produce any
significant increase in cell proliferation when either cell type was
cultured in medium containing 5% FCS (data not shown). The effect of
IL-2 on DNA synthesis by IEC-6 and IEC-18 cells was assessed by BrdU
uptake (Fig. 8). In serum-free media, IL-2 significantly promoted DNA synthesis in both cell lines at 24 h. Again,
addition of ET-1 at a concentration of
10
7 M, but not at
10
9 M, significantly
suppressed the IL-2-induced increase in DNA synthesis at 24 h.
Moreover, promotion of DNA synthesis at 24 h also was attenuated when
either cell line was cultured in the medium containing 5% FCS. Cells
were examined by light microscopy using the trypan blue exclusion test
at the end of the experiments and were found to be morphologically
intact. With or without IL-2 treatment, the number of detached cells
was negligible after 48 h.

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Fig. 7.
Effect of IL-2 on IEC-6 (A) and
IEC-18 cell (B) proliferation in
serum-free media as determined by MTT assay; attenuating effect of
ET-1. Rate of proliferation was expressed as %increase in control
values without IL-2. Recombinant rat IL-2 was added to subconfluent
monolayers at the concentration of 10 U/ml, and the culture was
continued up to 48 h. In some experiments, ET-1 at the concentration of
10 7 or
10 9 M was added with IL-2
and cell proliferation was assessed at 48 h. Values are expressed as
means ± SE of 6 experiments.
* P < 0.05 vs. controls.
# P < 0.05 vs. IL-2(+) 48 h.
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Fig. 8.
Effect of IL-2 on DNA synthesis by IEC-6
(A) and IEC-18 cells
(B) as assessed by bromodeoxyuridine
uptake. DNA synthesis in both cell lines was determined at 24 h in
serum-free media. Rate of proliferation was expressed as %increase of
control values without IL-2. Recombinant rat IL-2 was added to the
subconfluent monolayers at the concentration of 10 U/ml, and the
culture continued up to 24 h. In some experiments, ET-1 at the
concentration of 10 7 or
10 9 M was added with IL-2
and cell proliferation was assessed at 24 h. Moreover, the effect on
DNA synthesis by IL-2 was also determined at 24 h when these cells were
cultured in media containing 5% FCS [FCS(+)]. Values are
expressed as means ± SE of 6 experiments.
* P < 0.05 vs.
controls. # P < 0.05 vs.
IL-2(+).
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Figure 9 displays the effect of ET-receptor
antagonists on proliferation of IEC-6 and IEC-18 cells. Treatment with
BQ-123, a selective ETA-receptor
antagonist, significantly induced proliferation of these cells.
Bosentan, an ETA- and
ETB-receptor antagonist, similarly
enhanced cell proliferation.

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Fig. 9.
Effect of ET-receptor antagonists on IEC-6 and IEC-18 cell
proliferation. BQ-123 or bosentan was added at the concentration of 10 µM to the culture media containing 5% FCS, and cell proliferation
was determined at 24 h by the MTT assay.
* P < 0.05 vs. control.
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DISCUSSION |
Our present results revealed that cultured rat intestinal epithelial
cells can synthesize and release ET-1 and that IL-2 significantly stimulates ET-1 production by these cells. In addition, we found that
intestinal epithelial cells showed no ability to produce ET-3. ET
production is not limited to vascular endothelial cells but has been
demonstrated in a wide variety of other cell types. Expression of ET-1
mRNA has been reported in vascular smooth muscle cells, myocytes,
bronchial epithelial cells, mesangium cells, astrocytes, and
macrophages. On the other hand, although ET-3 mRNA has been found in
tissue from the brain, spleen, adrenal gland, and small intestine, ET-3
production has not been demonstrated in cultured cell lines (2), a
negative finding consistent with our own results.
Several factors have been shown to promote synthesis and release of
ET-1 in experiments using vascular endothelial cells. These include
such growth factors as TGF-
, cytokines such as IL-1 or IL-6,
thrombin, vasopressin, and angiotensin II, as well as mechanical shear
stress and hypoxia (11, 29). However, factors regulating ET-1 release
in other cell types have not been investigated extensively.
Nonendothelial cells are regulated not only by the same stimulatory
factors as endothelial cells, but also by cell-specific ones (27). For
example, estrogen and progesterone in uterine endometrial cells,
gonadotropin in corpus luteum cells, and lipopolysaccharide in
macrophages are known to induce ET-1 synthesis (27, 28, 42). In
bronchial epithelial cells, ET-1 production is upregulated by a variety
of cytokines in allergic diseases, including IL-2, IL-1, IL-6, IL-8,
TNF-
, and granulocyte-macrophage colony-stimulating factor, as well
as others (12). ET-1 may be a proinflammatory mediator
with a role in the pathogenesis of asthma, a condition in which IL-2
and other cytokines continuously stimulate respiratory epithelial cells
(4). In the present study, we demonstrated that the synthesis and
secretion of ET-1 by intestinal epithelial cells was enhanced
significantly by addition of IL-2. We focused on this cytokine, because
in our preliminary study IL-2 produced significantly greater ET release
than did other cytokines such as IL-1
or TGF-
. Ciacci et al. (5)
have reported that IEC-6 cells have functional IL-2 receptors involved in the response of intestinal epithelial cells to this cytokine (5).
Because production and actions of IL-2 are closely related to immune
cell populations, our present results suggest that ET synthesis by
intestinal epithelial cells may be closely associated with the
intestinal mucosal immune response. Recently, IL-15, which is produced
predominantly by monocytes, has been found to share many biological
functions with IL-2 due to common receptor components (37). The role of
IL-15 in ET-1 production by intestinal epithelial cells should be
explored further in this context.
Our present results suggest the possibility that activation of PKC is
important in ET-1 synthesis. The finding that ET-1 release also was
significantly inhibited by W-7 in intestinal epithelial cells suggests
that calmodulin is involved in this process. Our results are in accord
with previous reports of ET-1 synthesis stimulation in vascular
endothelial cells by thrombin, angiotensin II, and vasopressin to be
associated with calcium release in response to the phosphatidylinositol
turnover and activation of PKC (10, 11, 25). Further investigation is
necessary to elucidate details of the intracellular mechanism of ET-1
mRNA induction in intestinal epithelial cells.
Recently, growth-promoting activity exerted by ET on various cell types
has drawn attention (1, 33). ET-1 has been shown to regulate DNA
synthesis in various types of vascular smooth muscle cells and
fibroblasts. An increase in DNA synthesis also has been reported in
other types of cells, such as astrocytic glial cells, osteoblastic
cells, melanocytes, and mesangial cells (1). The regulatory signal by
which ET-1 promotes or inhibits cell growth is unknown, although the
participation of ET in mitogenesis involves activation of multiple
transduction pathways, including production of second messengers,
release of intracellular calcium pool, extracellular calcium influx,
and stimulation of immediate early gene expression (1). However, in
some cell lines ETs reportedly have inhibited cell growth or have shown
little effect, particularly at high concentrations. Morbidelli et al.
(24) found that in endothelial cells isolated from bovine adrenal
capillaries and human umbilical veins, high concentrations of ET-1 and
ET-3 failed to further increase cellular proliferation, describing a
bell-shaped dose-response curve. One explanation for these effects could be that the ETA receptor may
inhibit cell proliferation. Our results suggest a dual effect of ET-1
on cell proliferation. At low concentrations, ET-1 exerted a
growth-promoting effect on intestinal epithelial cells under serum-free
unstimulated conditions. In contrast, higher ET-1 concentrations
inhibited cell growth. Moreover, under conditions of cell growth
stimulated by IL-2, ET-1 also was shown to attenuate cell growth.
Because IL-2 demonstrated an ability to enhance ET-1 release from
cultured intestinal epithelial cells, ET-1 is considered a factor that
modulates cell growth of intestinal epithelial cell in combination with
this cytokine. Moreover, inhibition of DNA synthesis in intestinal
epithelial cells by administration of IL-2 in FCS-containing media
suggests that at high concentrations ET-1 may participate, at least in part, in this growth inhibition.
Two cell populations in the liver cell, Ito cells and sinusoidal
endothelial cells, are known to secrete ET-1. Mallet et al. (20) have
shown that binding of ET-1 to ETB receptors causes potent
growth inhibition of human Ito cells, in contrast to other cell types.
Tamamori et al. (40) have reported that ET-3 induces hypertrophy of
neonatal rat cardiac myocytes and that the
ETB receptor mRNA is upregulated
in these hypertrophied cells. Tamamori et al. (40) also suggested that
the ETB receptor in cardiac myocytes may contribute in part to the hypertrophy-promoting action of
ET-3. In the present study, however, either BQ-123 or
bosentan produced a selective and concentration-dependent stimulation
of intestinal epithelial cells; this suggests that the
growth-modulating effect of ET-1 on intestinal epithelial cells is
mediated by the ETA receptor
subtype.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hirokazu Matsumoto (Takeda Chemical Industries,
Tsukuba, Japan) for the kind advice about ET determination. We also
thank Dr. Mitsuo Yano (Tsukuba Research Institute, Banyu Pharmaceutical) for the generous donation of BQ-123 and Roche for the
generous donation of bosentan.
 |
FOOTNOTES |
This study was supported in part by grants from the Ministry of
Education, Science and Culture of Japan and from Keio University.
Address for reprint requests: H. Ishii, Internal Medicine, School of
Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Received 14 August 1997; accepted in final form 24 April 1998.
 |
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