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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta ), interleukins (IL-1, IL-6, IL-8), tumor necrosis factor (TNF), and interferons (IFN-gamma ) (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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
ET-1 primer (antisense), bases 675–699:
5′-AAGATCCCAGCCAGCATGGAGAGCG-3′
ET-1 primer (sense), bases 157–181:
5′CGTTGCTCCTGCTCCTCCTTGATGG-3′
ET-3 primer (antisense), bases 479–499:
5′-GCTGGTGGACTTTATCTGTCC-3′
ET-3 primer (sense), bases 23–42:
5′-TTCTCGGGCTCACAGTGACC-3′
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)
ET<SUB>A</SUB> receptor primer (antisense), bases 384–403:
5′-GGAGATCAATGACCACGTAG-3′
ET<SUB>A</SUB> receptor primer (sense), bases −15–5:
5′-CAGATCCACATTAAGATGGG-3′
ET<SUB>B</SUB> receptor primer (antisense), bases 967–986:
5′-AGGACCAGGCAGAATACTGT-3′
ET<SUB>B</SUB> receptor primer (sense), bases 918–937:
5′-GCAGGATTGCCTTGAATGACC-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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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(-).

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.

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.

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.

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.

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(+).

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta , 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-alpha , 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-1beta or TGF-beta . 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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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