Transforming growth factor-{alpha} directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells

Hideaki Kazumori, Shunji Ishihara, Mohammad A. K. Rumi, Cesar F. Ortega-Cava, Yasunori Kadowaki, and Yoshikazu Kinoshita

Second Department of Internal Medicine, Shimane Medical University, Izumo, Shimane 693-8501, Japan

Submitted 24 June 2003 ; accepted in final form 30 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-{alpha} (TGF-{alpha}) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-{alpha} during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-{alpha} and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-{alpha} mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-{alpha} stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-{alpha} in vivo and in vitro. During gastric mucosal injury, TGF-{alpha} was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.

histidine decarboxylase; vesicular monoamine transporter 2; acetic acid-induced ulcer


ENTEROCHROMAFFIN-LIKE (ECL) cells are histamine-containing neuroendocrine cells found in the gastric epithelium that synthesize, store, and release histamine (26). Histamine H2 receptors have been identified in acid secreting parietal cells, and acid secretion is strongly inhibited by histamine H2 receptor antagonists (18). Therefore, histamine secretion from ECL cells is critically important for the control of gastric acid secretion. In ECL cells, histidine decarboxylase (HDC) converts L-histidine to histamine that is transported and stored in secretory vesicles (28). Transport and storage of histamine in secretary granules of ECL cells can be accomplished by vesicular monoamine transporter 2 (VMAT2) (7).

Gastric mucosa is often exposed to aggressive factors, and the resulting mucosal damage is usually followed by acute or chronic inflammation. The process of such wound healing is a complex one that involves filling of the mucosal defect with granulation tissue as well as cell proliferation at the ulcer margin (10). However, for the healing of mucosal damage, gastric acid is known to have deterrent roles, because the inhibition of acid secretion has been shown to accelerate mucosal healing (18).

Several growth factors are known to be secreted during the repair of active mucosal damage (36), and in vivo administration of growth factors has been shown to accelerate ulcer healing by increasing epithelial cell proliferation in the ulcer bed and margin (31). Among these growth factors, transforming growth factor-{alpha} (TGF-{alpha}) is known to contain the potent mitogenic polypeptides of 50 amino acids that show a significant and functional homology with EGF and share the same cell surface receptor, EGF receptor (EGFR) (15). TGF-{alpha} is also reported to inhibit gastric acid secretion by acting on the EGFR on parietal cells (5). EGFR may also be present on gastric ECL cells, because TGF-{alpha} has been reported to stimulate the growth of ECL cells in the Mastomys natalensis (34).

The present study was designed to test the hypothesis that TGF-{alpha} has a regulatory role on ECL cell functions. Counterflow elutriation-enriched ECL cells were used to examine the effects of TGF-{alpha} and proinflammatory cytokines on the functional alterations of ECL cells.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formation of acetic acid-induced chronic ulcers. Seven-week-old male Wistar rats were used for the experiments, with four rats assigned to each group. To induce gastric ulcers with acetic acid, we used the method of Okabe and Pfeiffer (23) with minor modifications, as described previously (12). The rats were killed at 12 h and 1, 2, 3, 5, 7, 10, and 15 days after the induction of ulcers. Excised anterior glandular stomachs with ulcers were rinsed in PBS, snap-frozen in liquid nitrogen, and then stored until extraction of RNA. This experimental protocol was approved by the institutional animal care and experimental committee of Shimane Medical University.

Immunohistochemistry. Immunohistochemical studies were performed to investigate the TGF-{alpha} expression in stomach with acetic acid-induced ulcer. Three days after ulcer induction, rats were killed and the glandular stomachs were removed. Anterior glandular stomachs with ulcers were immunostained for TGF-{alpha}. The opposite side of the gastric tissue without ulcers served as a control. Five-micrometer-thick sections from each specimen were mounted on glass slides and stained with hematoxylin-eosin for light microscopic examination. To identify TGF-{alpha}-producing cells, the tissue sections were incubated with anti-TGF-{alpha} antibody (1:100) (Oncogene Science, Manhesset, NY), followed by incubation with secondary biotinylated anti-mouse immunoglobulin G. The bound antibody was detected using AEC substrate-chromogen system (DAKO, Carpinteria, CA). The sections were counterstained with hematoxylin.

Dispersion of gastric mucosal cells and cell isolation. Seven-week-old male Wistar rats were used for the experiments. Dispersion of gastric mucosal cells was performed as described previously (11, 27). Counterflow elutriation of gastric mucosal cells was performed as described previously (11, 13, 27). Seven different cell fractions (F1-F7) were collected at the following rates: F1, 14 ml/min; F2, 17 ml/min; F3, 20 ml/min; F4, 24 ml/min; F5, 29 ml/min; F6, 37 ml/min; and F7, 80 ml/min. The cells from the F1 fraction, which contained a large number of ECL cells, were further enriched by density gradient centrifugation using Nycodenz (Accurate Chemical, Westbury, NJ). Cells eluted in the F1 fraction were layered on Nycodenz density gradient in Hanks' balanced salt solution containing 0.5% bovine serum albumin and centrifuged at 1,100 rpm for 10 min at room temperature (11, 27). Cells that accumulated at the 1.040 g/ml density interface were mainly ECL cells, with a purity of between 75 and 85% and cell viability >95% by a trypan blue dye exclusion test. Parietal cells were obtained at 80-90% purity from the elutriation of fraction F7 (13).

RT-PCR. Total RNA was extracted by a single-step guanidium thiocyanate-phenol-chloroform method (Isogen; Nippon Gene, Tokyo, Japan), and RT-PCR was performed as described previously (11). In brief, total RNA (5 µg) extracted from isolated ECL and isolated parietal cells was subjected to RT in a First Strand Synthesis Kit (Stratagene Toyobo, Tokyo, Japan) to prepare cDNA. The first-strand cDNA was then amplified directly by a PCR method. PCR was performed with a hot start, then 25 cycles at 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, and a final extension step at 72°C for 7 min. PCR was performed in duplicate experiments, and the correct amplification was confirmed by sequencing the amplified DNA. To standardize the amount and quality of the total RNA, the same samples were analyzed for EGFR, TGF-{alpha}, VMAT2, H+,K+-ATPase, and {beta}-actin. The sense and antisense primers used are summarized in Table 1 (3, 12, 25). RNA extracted from rat total glandular stomachs was used as a positive control, and isolated ECL or parietal cell samples without subjection to RT served as a negative control. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide.


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Table 1. Primer sequences of RT-PCR

 

Effect of TGF-{alpha} on HDC and VMAT2 gene expression in vivo. Seven-week-old male Wistar rats were used for the experiments, with four rats assigned to each group, and each rat was kept from eating for 24 h before treatment. TGF-{alpha} (Sigma, St. Louis, MO) in saline (100 µg/kg body wt) or saline alone was administered subcutaneously. This dose of TGF-{alpha} has been reported to inhibit rat gastric acid secretion down to 10% of the control (9, 30). Three hours after the administration of TGF-{alpha} or saline, the rats were killed and total glandular stomachs were excised. The stomachs were quickly frozen in liquid nitrogen and stored until the extraction of RNA. Blood was obtained from an aorta, and plasma was separated for the measurement of gastrin. Gastrin concentrations were measured using a radioimmuno-assay kit (Gastrin RIA kit II; Dainabot, Tokyo, Japan).

Direct effect of growth factors and proinflammatory cytokines on HDC and VMAT2 gene expression in ECL cells. ECL cells were separated as described above. The density gradient-enriched ECL cells were cultured in 10-cm culture plates in RPMI 1640 supplemented with 5% fetal calf serum and 10 mM HEPES (pH 7.4) at 37°C. Just after the inoculation for culture, various growth factors and proinflammatory cytokines were added to the culture media. After a 3-h incubation, total RNA was isolated from the ECL cells for the measurement of HDC and VMAT2 gene expression by Northern blot analysis. Rat TGF-{alpha} (10-11 to 10-7 M) and mouse EGF (10-11 to 10-7 M; Sigma), 10-9 M mouse IL-1{beta} (Sigma), 10-9 M mouse TNF-{alpha} (Sigma), 10-9 M rat IL-6 (Pepro Tech, London, UK), and 10-9 M human IFN-{gamma} (Pharma Biotechnologies, Hannover, Germany) were used as stimulants. Histamine concentrations in the culture media were measured by a commercially available EIA kit (IBL, Hamburg, Germany).

Effect of TGF-{alpha} on HDC and VMAT2 protein production in ECL cells. Isolated ECL cells were cultured as described above. After 6 h of culture with various concentrations (10-11 to 10-7 M) of rat TGF-{alpha}, the cells were washed with PBS and lysed with 200 µl of lysis buffer (0.1% SDS, 1% Triton X-100, 1% sodiumdeoxycholate, 1 mM phenylmethanesulfonyl fluoride). The samples were boiled for 5 min and then centrifuged at 15,000 g for 30 min. Next, the samples were electrophoresed in polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The membranes were incubated with anti-HDC polyclonal antibody (1:1,000; Euro-Diagnostica, Malmo, Sweden) or anti-VMAT2 polyclonal antibody (1:1,000) (Chemicon International, Temecula, CA) for 2 h, then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (DAKO) for 2 h. Subsequently, the membranes were developed in an enhanced chemiluminescent system (Amersham Biosciences, Chalfont, St. Giles, UK) as described previously (11, 13). Signal densities of HDC and VMAT2 protein product were quantified with a laser densitometer (UltroScan XL; Pharmacia LKB Biotechnology, Uppsala, Sweden). The results were expressed as means ± SE of the percentage of the control.

Northern blot analysis. Total RNA was extracted by a single-step guanidium thiocyanate-phenol-chloroform method (Isogen; Nippon Gene). Total RNA (20 µg) was separated by electrophoresis in a 0.66 mM formaldehyde/1% agarose gel. After transfer to a nitrocellulose membrane (Schlicher & Schuell, Dassel, Germany), the nucleic acids were fixed to the membrane by ultraviolet cross-linking. The probes used for Northern blot analysis were 0.4-kb cDNA of rat TGF-{alpha}, 0.5-kb cDNA of rat EGF, 0.4-kb cDNA of mouse HDC, 0.4-kb cDNA of rat VMAT2, 0.3-kb cDNA of rat H+,K+-ATPase, 0.4-kb of rat gastrin, and 0.7-kb cDNA of rat {beta}-actin (3, 10, 14). Radiolabeling of the probes was performed with [{alpha}32P]deoxycytidine triphosphate using a DNA labeling kit (Multiple DNA labeling systems; Amersham Biosciences). Hybridization was performed at 42°C, and the filters were washed twice for 20 min at 55°C in 0.1x saline-sodium citrate plus 0.1% sodium dodecyl sulfate as described previously (11). The radiolabeled DNA probes were detected with a bioimage analyzer (BAS 2000; Fujix, Tokyo, Japan). The intensity of each signal was measured and standardized by the intensity of {beta}-actin. The results are expressed as means ± SE of the percentage of control values.

Secretion of TGF-{alpha} from cultured gastric cells. The secretion of TGF-{alpha} from parietal cells and ECL cells was measured separately. Unfractionated cells, isolated ECL cells, and isolated parietal cells were cultured for 24 h. TGF-{alpha} concentrations in culture media were measured by a commercially available RIA kit (Peninsula Laboratories, Belmont, CA).

Effect of tyrphostin AG1478 on TGF-{alpha}-induced gene expression in ECL cells in vitro. To assess the role of TGF-{alpha} as a stimulant of HDC and VMAT2 gene expression in vitro, the selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 (Carbiochem, San Diego, CA) was used (17). Isolated ECL cells were preincubated with the absence or presence of AG1478 (10-6 M) in DMSO for 30 min and stimulated with TGF-{alpha} (10-9 M) for 3 h. This concentration of AG1478 can almost completely inhibit the TGF-{alpha}-induced tyrosine phosphorylation of EGFR (2). After the culture, total RNA (10 µg) was extracted and was subjected to Northern blot analysis.

Effect of tyrphostin AG1478 on HDC and VMAT2 expression in vivo. To assess the role of TGF-{alpha} as a stimulant of HDC and VMAT2 gene expression in vivo, acetic acid-induced ulcers were prepared identically as described above. Immediately after ulcer induction, treatment was initiated with intraperitoneal injections of vehicle (0.01% DMSO) or at the dose of 300 µg AG1478 /kg body wt in 0.01% DMSO twice daily and continued for 10 days. The tyrphostin dose was chosen according to a previous study (20, 24), which showed that this dose significantly attenuates EGFR-associated tyrosine kinase activity in rats. The rats were killed 10 days after the ulcer induction. The anterior wall of the stomachs were rinsed in PBS, snap-frozen in liquid nitrogen, and then stored until extraction of RNA.

Statistical analysis. All data are expressed as means ± SE. Statistical comparisons between two groups were done with a Mann-Whitney U-test. Multiple comparisons were done with ANOVA followed by a Dunnett's test. P values of <0.05 were considered statistically significant.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Time course changes of TGF-{alpha} mRNA expression in gastric tissue after the induction of ulcers. After exposure of the stomach to acetic acid, the gastric walls showed characteristic sequential morphological changes, as described previously (10). Within two days, ulcer craters without necrotic tissue had developed, and on the third day, transition from acute to chronic ulcers was observed. By the tenth day, most of the gastric ulcers had healed. Time course changes of TGF-{alpha} gene expression in rat glandular stomachs are shown in Fig. 1. Augmentation of TGF-{alpha} gene expression was observed on the second and third experimental days. No EGF gene expression was detected during the healing of acetic acid-induced ulcers (data not shown).



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Fig. 1. Time course changes of transforming growth factor (TGF)-{alpha} mRNA expression in rat stomachs with acetic acid-induced ulcers. Total RNA (20 µg) was extracted from rat glandular stomachs at the indicated times after induction of ulcers and was subjected to Northern blot analysis with TGF-{alpha} cDNA probe. Blots are representative of 4 separate experiments (A). The relative signal intensities of TGF-{alpha} mRNA to {beta}-actin mRNA levels were quantified with an image analyzer (B). Results are expressed as mean ± SE of 4 rats. *P < 0.05 vs. control.

 

Immunohistochemistry of TGF-{alpha} in gastric mucosa with acetic acid-induced ulcers. In the control stomach, relatively strong TGF-{alpha} immunoreactivity was observed in the parietal cells and in the surface epithelial cells, whereas mucous neck cells were weakly positive as already reported (1, 22). In the acetic acid-induced ulcers, a marked increase in TGF-{alpha} immunoreactivity in the parietal cells predominantly at the ulcer margin was observed (Fig. 2).



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Fig. 2. Immunohistochemistry of TGF-{alpha} in gastric fundic mucosa with acetic acid-induced ulcers. A marked increase in TGF-{alpha} immunoreactivity in the parietal cells at the ulcer margin was observed 3 days after the ulcer induction. A: control fundic gland mucosa. B: gastric mucosa at the edge of acetic acid-induced ulcers. C: higher magnification of gastric mucosa at the edge of acetic acid-induced ulcers.

 

Identification of EGFR on ECL cells. In view of the regulating effect of TGF-{alpha} on ECL cells, we investigated the presence of EGFR on the isolated ECL cells. EGFR mRNA expression in purified ECL and parietal cells is shown in Fig. 3. VMAT2 was found only in ECL cells, whereas H+,K+- ATPase was present only in parietal cells, suggesting the effective separation of gastric mucosal cells. In contrast, EGFR mRNA was found not only in parietal cell fractions but also in enriched ECL cells. TGF-{alpha} mRNA expression was highest in parietal cells, whereas it was weaker in other cells.



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Fig. 3. RT-PCR analyses of vesicular monoamine transporter 2 (VMAT2), H+,K+-ATPase, EGF receptor (EGFR), TGF-{alpha}, and {beta}-actin expressions. Electrophoretic results are representative of 4 separate experiments. Identical volumes of the PCR reaction mixtures without subjection to RT. M, molecular size marker; NC, negative control.

 

Effect of TGF-{alpha} on HDC and VMAT2 gene expression in vivo. Because rat ECL cells possess EGFR that can respond to TGF-{alpha}, we investigated the effects of a subcutaneous TGF-{alpha} injection on gastric ECL cells in vivo. Because HDC and VMAT2 are specific markers of gastric ECL cells and important components for the production and storage of histamine, HDC and VMAT2 gene expressions in gastric tissue in response to TGF-{alpha} were investigated. We found that TGF-{alpha} significantly increased HDC and VMAT2 mRNA expression (Fig. 4). Gastrin mRNA and H+,K+-ATPase mRNA expression in gastric tissue were not changed at 3 h after the administration of 100 µg TGF-{alpha} /kg body wt (Fig. 4). Moreover, there were also no changes in the plasma gastrin concentrations (85.0 ± 35.4 pg/ml in controls vs. 102.0 ± 32.8 pg/ml in TGF-{alpha}-administered rats, n = 4). These results suggested that TGF-{alpha} influences ECL cell function by augmenting histamine metabolism independent of the gastrin system.



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Fig. 4. Effects of TGF-{alpha} on histidine decarboxylase (HDC), VMAT2, H+,K+-ATPase, and gastrin mRNA expressions in vivo. Total RNA (20 µg) was extracted from rat glandular stomachs 3 h after the administration of TGF-{alpha} and then subjected to Northern blot analyses with HDC, VMAT2, H+,K+-ATPase, and gastrin cDNA probes. Blots are representative of 4 separate experiments (A). Relative signal intensities of HDC, VMAT2, H+,K+-ATPase, and gastrin mRNA to {beta}-actin mRNA levels were quantified with an image analyzer (B). Results are expressed as means ± SE of 4 rats. *P < 0.05 vs. control.

 

Direct effects of growth factors and proinflammatory cytokines on HDC and VMAT2 expression in isolated ECL cells. We then examined the effects of TGF-{alpha} on isolated ECL cells in vitro. TGF-{alpha} directly stimulated the expression of HDC and VMAT2 mRNA in a dose-dependent manner from 10-11 to 10-7 M at 3 h after addition (Fig. 5). The effect of TGF-{alpha} on HDC and VMAT2 protein production in isolated ECL cells was investigated next. HDC is produced as an inactive 74-kDa proenzyme, which is then processed into the enzymatically active 53 kDa form (6, 33). TGF-{alpha} stimulated the production of both forms of HDC protein in cultured ECL cells at 6 h after addition. VMAT2 protein production was also stimulated by TGF-{alpha} (Fig. 6). As a result of these changes, TGF-{alpha} augmented the release of histamine from ECL cells in culture media in a dose-dependent manner to over 130% of the control with 10-9 M TGF-{alpha}. In a separate set of experiments, ECL cell counts were determined 6 h after the addition of TGF-{alpha}. With the use of a Neubauer chamber, the number of living cells was counted after trypan blue dye exclusion. No significant change in the viable cell count was observed 6 h after TGF-{alpha} stimulation (data not shown). A radioimmunoassay for gastrin (sensitivity: >16 pg/ml) failed to detect any gastrin in culture media. EGF also directly stimulated the expression of HDC and VMAT2 mRNA in a dose-dependent manner, from 10-11 to 10-7 M (Fig. 7). An inhibition of the EGFR tyrosine kinase AG1478 almost completely inhibited TGF-{alpha}-induced augmentation of HDC and VMAT2 gene expression. Treatment of ECL cells in culture with 10-6 M AG1478 did not change HDC and VMAT2 mRNA expression. AG1478, however, completely eliminated the TGF-{alpha}-induced augmentation of HDC and VMAT2 expression.



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Fig. 5. Effects of TGF-{alpha} on HDC and VMAT2 mRNA expressions in isolated cultured ECL cells. After incubation with various concentrations of TGF-{alpha} (10-11 to 10-7 M) for 3 h, total RNA (10 µg) was extracted and subjected to Northern blot analysis with HDC and VMAT2 cDNA probes. Blots are representative of 4 separate experiments (A). Relative signal intensities of HDC and VMAT2 mRNA to {beta}-actin mRNA levels were quantified with an image analyzer (B). Results are expressed as means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 


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Fig. 6. Effects of TGF-{alpha} on HDC and VMAT2 protein production in isolated cultured ECL cells. After incubation with various concentrations of TGF-{alpha} (10-11 to 10-7 M) for 6 h, protein (30 µg) was extracted and subjected to Western blot analysis. Blots are representative of 4 separate experiments (A). Relative signal densities of HDC and VMAT2 protein production were quantified by laser densitometry (B). Results are expressed as means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 


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Fig. 7. Effects of EGF on HDC and VMAT2 mRNA expressions in isolated cultured ECL cells. After incubation with 10-9 M of EGF for 3 h, total RNA (10 µg) was extracted and subjected to Northern blot analysis with HDC and VMAT2 cDNA probes. Blots are representative of 4 separate experiments (A). Relative signal intensities of HDC and VMAT2 mRNA to {beta}-actin mRNA levels were quantified with an image analyzer (B). Results are expressed as means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 

Several proinflammatory cytokines at concentrations of 10-9 M were also investigated as possible stimulants of HDC and VMAT2 mRNA expression in isolated enriched ECL cells in vitro. Of those investigated, IL-1{beta} and TNF-{alpha} were the only cytokines that could significantly inhibit VMAT2 gene expression. On the other hand, no proinflammatory cytokine influenced HDC gene expression, as shown in Fig. 8. Furthermore, none of them changed the viability of the cultured ECL cells.



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Fig. 8. Effects of several proinflammatory cytokines on HDC and VMAT2 mRNA expressions in isolated cultured ECL cells. After incubation with 10-9 M of various proinflammatory cytokines for 3 h, total RNA (10 µg) was extracted and subjected to Northern blot analysis with HDC and VMAT2 cDNA probes. Blots are representative of 4 separate experiments (A). Relative signal intensities of HDC and VMAT2 mRNA to {beta}-actin mRNA levels were quantified with an image analyzer (B). Results are expressed as means ± SE of 4 separate experiments. *P < 0.05 vs. control.

 

Secretion of TGF-{alpha} from cultured gastric cells. To determine whether TGF-{alpha} acts on ECL cells in a paracrine or autocrine mannner, the concentrations of TGF-{alpha} in culture media were examined. No TGF-{alpha} was detected in the starting culture media nor in the culture media sampled after 6 h culture (sensitivity: >10 pg/ml). TGF-{alpha} was detected in the media after 24-h culture, and its concentration was highest in the culture media of parietal cells (unfractionated cells, 2.6 ± 0.2 x 10-11 M/106 cells; isolated ECL cells, 2.7 ± 0.1 x 10-11 M/106 cells; isolated parietal cells, 3.2 ± 0.2 x 10-11 M/106 cells; n = 4).

Effect of tyrphostin AG1478 on HDC and VMAT2 expression in vivo. To assess the role of increased TGF-{alpha} during healing of acetic acid-induced ulcers, AG1478 was used as an inhibitor of EGFR tyrosine kinase in vivo. Because downregulated HDC and VMAT2 gene expressions returned to the control values 10 days after the induction of the ulcer (10), the rats were treated with AG1478 for 10 days. As expected, AG1478 significantly inhibited the recovery of HDC and VMAT2 gene expression. In the presence of AG1478, mRNA expression of HDC and VMAT2 was <50% of the control 10 days after the induction of acetic acid-induced ulcers.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Gastric acid, which has a major impact on the healing of ulcers, is reported to be suppressed during ulcer healing (10, 14, 16). As a possible mechanism of the inhibition of gastric acid secretion, we and others have reported local and transient downregulations of HDC, VMAT2, and chromogranin A (CgA) mRNA as well as a transient decrease in the number of HDC-, VMAT2-, and CgA-positive cells at the ulcer margins (10, 21). After the transient decreases of HDC and VMAT2, these factors returned to the control levels gradually with the recovery of gastric acid secretion (10, 21). Furthermore, plasma gastrin concentrations transiently increased from days 1 to 5 after the induction of acetic acid-induced ulcers, which may have been caused by a reactive response to the inhibited gastric acid secretion (10, 21, 36). In contrast, increased plasma gastrin is known to augment HDC and VMAT2 expressions in rats (7, 32). Therefore, increased plasma gastrin may be an important regulator for the return of ECL cells to their original functional activities during the late stage of gastric ulcer healing. Notably, the presence of a gastrin response element in HDC promoter explains the stimulation of HDC gene transcription by gastrin. The promoter region of HDC contains Ca2+-, cAMP-, and protein kinase C-responsive elements (29). So far, there appears to be no such specific sequences in the promoter region of the VMAT2 gene (8, 35).

In parallel to gastrin, TGF-{alpha} may also influence gastric acid secretion in addition to its growth-promoting effect (5). Because the expression of TGF-{alpha} mRNA was significantly increased on day 2 of our experiment, it may have an influence not only on gastric mucosal proliferation but also on the control of gastric acid secretion during the active phase of gastric ulcers. TGF-{alpha} shows a significant and functional homology with EGF and shares the same cellular surface receptor, EGFR (15). Furthermore, EGFR immunoreactivity was previously detected in foveolar cells in proliferative zones as well as in parietal cells (1, 22). The effects of TGF-{alpha} on acid secretion have also been characterized in parietal cells, and both TGF-{alpha} and EGF have been shown to act on parietal cells via EGFR and inhibit gastric acid secretion (5).

Mahr et al. (19) recently reported that ECL cells express EGFR and TGF-{alpha} and may be regarded as a growth factor for ECL cells. In the present study, we also detected EGFR expression in isolated ECL cells by RT-PCR, confirming the results reported by Mahr et al. (19). As a ligand of EGFR, TGF-{alpha} was believed to be the most important one during the gastric ulcer healing, although there is a report indicating that EGF is also upregulated in experimental ulcer (14). During ulcer healing, a marked increase in TGF-{alpha} immunoreactivity in the parietal cells at the ulcer margin was observed in this study. We then investigated the effect of TGF-{alpha} on ECL cells in vivo and found that it stimulates HDC and VMAT2 mRNA expression.

As a next step, we examined the effects of TGF-{alpha} on isolated ECL cells in vitro and found that it directly stimulated the expressions of HDC and VMAT2 mRNA in a dose-dependent manner. We then investigated the effects of TGF-{alpha} on HDC and VMAT2 protein production in isolated ECL cells and found that both the inactive 74-kDa proenzyme and enzymatically active 53-kDa form of HDC were increased by the addition of TGF-{alpha}. In ECL cells, the process of conversion of the inactive 74-kDa form to the active 53-kDa form of HDC has not been fully understood. In a rat basophilic/mast cell line (RB-2H3), it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the endoplasmic reticulum, where it is converted to the 53-kDa form (33). Accordingly, TGF-{alpha} may stimulate the production of proenzyme and its conversion to the active form in ECL cells. VMAT2 protein, which is important as a transporter of histamine to secretary vesicles, was also increased in a dose-dependent manner. In addition, histamine release in the culture medium of isolated ECL cells was augmented in a dose-dependent manner by the addition of TGF-{alpha}. EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-{alpha}. All of these findings together suggest that TGF-{alpha} directly stimulates histamine synthesis and secretion in ECL cells and may also stimulate gastric acid secretion indirectly. The intracellular signal-transduction system connecting EGFR and HDC/ VMAT2 expression in ECL cells is unclear. Endogenously produced TGF-{alpha} binds to EGFR and phospholipase C-{gamma}1 (20). These signals may stimulate transcription of HDC and VMAT2 genes via the activation of ERK1 and ERK2 (24). Further study, however, will be necessary to elucidate the precise mechanism of TGF-{alpha}-induced activation of HDC/VMAT2 gene transcription.

It has been reported (1, 4, 22) that TGF-{alpha} is localized to parietal cells by immunohistochemistry and cell separation. In this study, TGF-{alpha} released from parietal cells could be detected after 24 h of culture. Because TGF-{alpha} immunoreactivity in the parietal cells at the ulcer margin was increased in acetic acid-induced ulcers, TGF-{alpha} released from parietal cells may act on ECL cells mainly in a paracrine manner in addition to its autocrine effect. Additionally, we have found that ECL cells secrete smaller amounts of TGF-{alpha} than parietal cells, suggesting that TGF-{alpha} from ECL cells may also act on ECL cells partially in an autocrine manner. Even after 24-h culture, however, the TGF-{alpha} concentration was far lower compared with the concentration of TGF-{alpha} that was used in this study to stimulate ECL cells. Although none of the proinflammatory cytokines tested had any effects on HDC mRNA expression, IL-1{beta} and TNF-{alpha} were confirmed to have an inhibitory effect on VMAT2 mRNA expression. IL-1{beta} has been shown to inhibit gastric acid secretion, and its receptors have also been demonstrated in cultured rat ECL cells (27). Furthermore, short-term exposure of ECL cells to IL-1{beta} is reported to result in sustained functional impairment and to inhibit histamine secretion (27). This functional change of ECL cells may be explained by our observation that IL-1{beta} and TNF-{alpha} strongly inhibited VMAT2 production. The reason why these proinflammatory cytokines inhibit the VMAT2 expression without altering HDC expression is not clear. These cytokines, produced during the inflammation, however, may inhibit the secretion of histamine from ECL cells by inhibiting the storage of histamine in the secretory vesicles. Accordingly, the results of our study may also explain the impaired gastric acid secretion seen during the active stage of gastric ulcers with strong inflammation. With the resolution of inflammation, the TGF-{alpha}-induced stimulation of HDC and VMAT2 expressions in ECL cells may have some role in the recovery of gastric acid secretion in the late stage of gastric mucosal repair.

Taken together, we conclude that TGF-{alpha} should be regarded as an important gastric acid-regulating factor during mucosal injury and healing because of its direct inhibitory effects on parietal cells. Whereas proinflammatory cytokines IL-1{beta} and TNF-{alpha} decrease VMAT2 expression in ECL cells, TGF-{alpha} exhibits its ability to stimulate the expression of both HDC and VMAT2 to restore the functional status of ECL cells.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Ishihara, Second Dept. of Internal Medicine, Shimane Medical Univ., 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan (E-mail: si360405{at}shimane-med.ac.jp).

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.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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