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Reg gene expression is increased in rat gastric enterochromaffin-like cells following water immersion stress
GASTROENTEROLOGY 1996;111:45–55
Reg Gene Expression Is Increased in Rat Gastric Enterochromaffin-like Cells Following Water Immersion Stress MASAKYO ASAHARA,* SOTARO MUSHIAKE,‡ SHOICHI SHIMADA,§ HIROKAZU FUKUI,* YOSHIKAZU KINOSHITA,* CHIHARU KAWANAMI,* TSUYOSHI WATANABE,x SATOSHI TANAKA,Ø ATSUSHI ICHIKAWA,Ø YASUO UCHIYAMA,x YOICHI NARUSHIMA,# SHIN TAKASAWA,# HIROSHI OKAMOTO,# MASAYA TOHYAMA,§ and TSUTOMU CHIBA* *Division of Gerontology, Department of Internal Medicine, Kobe University School of Medicine, Kobe; Departments of ‡Pediatrics, §Anatomy and Neuroscience, and xCell Biology and Anatomy I, Osaka University Medical School, Osaka; ØDepartment of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto; and #Department of Biochemistry, Tohoku University School of Medicine, Sendai, Japan
Background & Aims: Reg gene has been isolated from regenerating rat pancreatic islets, and subsequent studies have shown a trophic effect of Reg protein on islet cells. However, little is known about the role of Reg protein in the stomach. The aim of this study was to clarify the localization of Reg messenger RNA (mRNA) and its product in the stomach and to examine changes in the level of their expression during regeneration of gastric mucosal cells. Methods: Gastric lesions were experimentally induced in Sprague–Dawley rats by water immersion stress. Northern blot analysis and in situ hybridization studies were performed to examine changes in mRNA levels. Immunohistochemical studies were performed to identify the cellular localization and to investigate the change in Reg protein level. Results: Reg mRNA and its product were distributed in the basal part of the oxyntic mucosa and were expressed mainly in enterochromaffin-like cells. Levels of both Reg mRNA and its product were markedly increased in the water immersion–induced gastric lesions. Conclusions: Reg mRNA and its product are expressed in gastric enterochromaffin-like cells, and their levels are increased during the healing process of water immersion–induced gastric lesions.
n 1984, Yonemura et al.1 found that administration of poly(ADP-ribose) synthetase inhibitors, such as nicotinamide, to rats subjected to 90% depancreatomy induced regeneration of pancreatic islets. Subsequent screening of the regenerating rat islet-derived complementary DNA (cDNA) library showed a novel gene that encodes a 165–amino acid protein with a 21–amino acid signal peptide.2 The gene has been termed Reg and is abundantly expressed in regenerating islets but not in normal ones.2 Further studies showed that enhanced Reg gene expression is linked to DNA synthesis in pancreatic islets both in vitro3 and in vivo.4 Moreover, it has been shown recently that Reg protein is present in the secre-
I
tory granules of beta cells5 and that administration of Reg protein not only enhances DNA synthesis in isolated islets but also ameliorates diabetes in rats subjected to 90% depancreatomy, suggesting a trophic role for the Reg gene in pancreatic beta cells.6 Interestingly, the predicted three-dimensional structure of the protein shows homology with that of the lectins, a family of growth-promoting molecules.7 – 9 REG gene has also been found in the stomach.10 Although the precise localization and the role of the REG gene in the stomach remains undetermined, it is interesting to speculate that it is involved in the process of gastric mucosal repair, as has been suggested to occur in the pancreas. In the pancreas, REG protein is primarily found in the exocrine gland under normal conditions,11 whereas it is mainly present in the islets during regeneration.5 The present study was therefore undertaken to clarify what type of cells in the stomach express Reg gene and its product and to investigate changes in the level of their expressions in water immersion–induced gastric lesions.
Materials and Methods Animals Male Sprague–Dawley rats (250–300 g) were deprived of food but allowed free access to water for 24 hours before the experiments. Each study was performed with 5 animals in each group. To induce gastric mucosal lesions, the animals were placed in strain cages and immersed in water (23⬚C) for 6 hours as described previously.12 Water-immersed rats were killed 0, 6, 12, 24, 48, and 72 hours after the beginning of Abbreviations used in this paper: ECL cell, enterochromaffin-like cell; GSH, glutathione-S-transferase; HDC, histidine decarboxylase; ISH, in situ hybridization; RT-PCR, reverse-transcription polymerase chain reaction. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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the stress period for Northern blot analysis and 0 and 24 hours after the beginning of the stress period for in situ hybridization (ISH) and immunohistochemical studies. All experiments were reviewed and approved by the Kobe University Ethical Committee for Animal Experiments.
Tissue Preparation The rats were anesthetized with sodium pentobarbital (Nembutal, 55 mg/kg; Abott Laboratories, North Chicago, IL) and perfused transcardinally with ice-cold Zamboni’s solution (4% paraformaldehyde and 0.2% picrinic acid in 0.1 mol/L phosphate buffer, pH 7.4; 2 mL/g body wt). The stomachs were excised, immersed in fixative for 2–3 hours, and incubated in phosphate-buffered saline (PBS) containing 30% (wt/vol) sucrose at 4⬚C until they sank. The glandular stomachs were divided into fundus (body) and antrum. They were then quickly frozen in a dry ice plus 2-methylbutane mixture, and 5-mm sections were cut on a cryostat and thaw-mounted on 3aminopropyl-triethoxysilane–coated slides. The sections were rinsed with 0.1 mol/L PBS and then treated with 10 mg/mL proteinase K (Boehringer Mannheim, Mannheim, Germany) in 50 mmol/L Tris-HCl and 5 mmol/L ethylenediaminetetraacetic acid (EDTA) (pH 8.0) for 10 minutes. After treatment with 4% paraformaldehyde, they were rinsed with PBS, acetylated with 0.25% (vol/vol) acetic anhydride in 0.1 mol/L triethanolamine, rinsed with PBS, dehydrated in an ethanol series, and air dried.
Preparation of Reg RNA Probes The rat Reg cDNA used for ISH study and Northern blot analysis was obtained by reverse-transcription polymerase chain reaction (RT-PCR) amplification of the total RNA extracted from rat pancreas2 using the synthetic oligonucleotide primers 5ⴕ-TGCCAGAACATGAATTC-3ⴕ and 5ⴕ-TTGAACTTGCAGACAAA-3ⴕ under the following conditions: 94⬚C for 30 seconds, 60⬚C for 60 seconds, and 72⬚C for 60 seconds for 35 cycles. The PCR product was subcloned into the pCRII vector using a TA cloning kit (Invitrogen, San Diego, CA). The recombinant plasmid was linearized by cutting at a single site with a restriction enzyme (Xho I for the antisense probe and with BamHI for the sense probe). In vitro transcription was performed using the appropriate RNA polymerase (SP6 RNA polymerase for the antisense probe, T7 RNA polymerase for the sense probe) and [a-35S]uridine triphosphate (Amersham, Little Chalfont, Buckinghamshire, England). The 35Slabeled antisense riboprobe was complementary to part of the cloned Reg gene (nucleotide positions 187–492).2
Preparation of Anti–histidine decarboxylase, Anti-somatostatin, and Anti–chromogranin A Antibodies A glutathione-S-transferase (GST)–histidine decarboxylase (HDC) fusion protein was produced by inserting a 630– base pair fragment of mouse HDC cDNA (nucleotides 1–630) into the expression vector pGEX-2T (Pharmacia LKB Biotech, Piscataway, NJ). This fragment was prepared by PCR with
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oligonucleotide A (5ⴕ-GTGGATCCATGATGGAGCCCTGTGAATAC-3ⴕ) and oligonucleotide B (5ⴕ-GTGGATCCTCAAATCAAGCCAGCCTTCT-3ⴕ), which contain BamHI sites at their 5ⴕ ends. Amplified fragments were digested with BamHI and then subcloned into the BamHI site of pGEX-2T. The resulting fusion protein combined the 210 amino acids of the N-terminal region of mouse HDC with the C terminus of GST. Overnight cultures of Escherichia coli strain HB101, transformed with the expression vector, were diluted 1:10, and the cells were grown for 1.5 hours at 37⬚C before adding isopropyl1-thio-b-D-galactoside (0.1 mmol/L). After an additional 3hour growth period, bacteria were plated, resuspended, and sonicated in PBS containing 1% sarcosyl and 0.2 mmol/L phenylmethylsulfonyl fluoride and then centrifuged at 10,000g for 5 minutes at 4⬚C. The fusion protein in the lysate was then purified by glutathione-coupled Sepharose 4B (Pharmacia) affinity chromatography in 50 mmol/L Tris-HCl (pH 8.0) containing 20 mmol/L reduced glutathione. To raise antisera, male Japanese white rabbits were immunized by intradermal injections of 200 mg purified GST-HDC fusion protein mixed with complete Freund’s adjuvant (1:1) at 2-week intervals. After the fourth booster, the rabbits were bled and antiserum was obtained. To purify anti–GST-HDC antibody, the antiserum was precipitated with ammonium sulfate (0%–50%) and then adsorbed to Sepharose 4B gel coupled with purified GST-HDC fusion protein. The antibody was eluted with glycine-HCl (pH 2.5) and desalted using a PD10 column (Pharmacia) preequilibrated with PBS. The specificity of the anti-HDC antibody was checked by Western blotting and immunohistological study. An absorption test was also performed, in which absorption of the antiserum by excess GST-HDC fusion protein completely eliminated the immunostaining. The antiserum against somatostatin was obtained from Japan Immunoresearch Laboratory Co. Ltd. (Tokyo, Japan). The characterization of this antiserum has been described elsewhere.13 In the present study, absorption of this antiserum with synthetic somatostatin completely eliminated the immunostaining in the stomach. For the generation of anti–chromogranin A antiserum, a 14–amino acid residue peptide corresponding to position 57– 70 of the rat chromogranin A deduced from the cDNA sequence14 was synthesized by a peptide synthesizer (430A; Perkin–Elmer, Foster City, CA). This peptide was conjugated with keyhole limpet hemocyanin (Sigma Chemical Co., St. Louis, MO), and a rabbit anti–chromogranin A polyclonal antiserum was raised against it. The characteristics of the antiserum were examined by immunoblotting and immunocytochemistry: the anti–chromogranin A antiserum properly recognized an 85-kilodalton band of native chromogranin A protein in the blot of rat adrenal gland extract and stained the secretory granules of adrenal chromaffin cells (unpublished data). In addition, the immunostaining in the stomach in the present study was completely eliminated when the antiserum was absorbed with the synthetic peptide described above before use.
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ISH Histochemistry Two protocols were used for the ISH studies. One provided a single label using ISH histochemistry, and the other protocol combined ISH and immunohistochemistry. The procedure used for ISH and detection have been described previously in detail.15 In brief, 200 mL of hybridization mixture containing 50% formamide, 10% dextran sulfate, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, 5 mmol/L EDTA, 0.1 mol/ L NaPO4 , 0.2% sarcosyl, 200 mg/mL salmon sperm DNA, 11 Denhardt’s solution, 500 mg/mL yeast transfer RNA, and 100 mmol/L dithiothreitol and 6.01 105 cpm of 35S-labeled probes were denatured for 2 minutes at 80⬚C, applied to each section, and covered with a siliconized coverslip. Hybridization was performed in a sealed humid chamber for 18 hours at 55⬚C. After hybridization, the slides were immersed in a mixture of 51 SSC (11 SSC is 150 mmol/L NaCl and 15 mmol/L sodium citrate) and 1% mercaptoethanol at room temperature, and the coverslips were allowed to fall off. The sections were incubated in 50% formamide, 21 SSC, and 10% mercaptoethanol at 65⬚C for 30 minutes and equilibrated with ribonuclease buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 0.5 mmol/L NaCl) three times for 10 minutes each at 37⬚C. They were then treated with ribonuclease A (1 mg/mL) in the same buffer for 30 minutes; incubated in 50% formamide, 21 SSC, and 10% mercaptoethanol at 65⬚C for 30 minutes; and rinsed in 21 SSC and in 0.21 SSC at room temperature for 10 minutes each. After washing, the sections were dehydrated in a graded alcohol series and air dried. For autoradiography, the tissue sections were coated with Kodak NTB3 emulsion (diluted 1:1 with distilled water at 42⬚C; Eastman Kodak, Rochester, NY) and exposed for 3 weeks in light-tight boxes at 4⬚C. After development in Kodak D-19 and fixing in Fujifix (Fuji Film, Tokyo, Japan), the sections were rinsed in distilled water, counterstained with H&E, rinsed in tap water, dehydrated in a graded alcohol series, cleared in xylene, and coverslipped. In the second protocol, tissue sections were first stained immunohistochemically using the avidin-biotin complex method following a hybridization procedure described previously.16,17 After pretreatment with 1% normal goat serum in PBS, the tissue sections were incubated with rabbit antimouse HDC antibody (1:600) or rabbit anti-somatostatin antibody (1:600) overnight at 4⬚C in a humidified environment. Endogenous peroxidase activity was blocked with 1.5% H2O2 in 99% methanol for 30 minutes. The slides were washed, and biotinylated goat anti-rabbit immunoglobulin (Gibco– Bethesda Research Laboratories, Gaithersburg, MD; 1:400) was applied overnight at 4⬚C, followed by washing with PBS. Bound antibody was detected using the avidin-biotin peroxidase method (ABC Elite Kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was subsequently revealed by incubating with 3,3ⴕ-diaminobenzidine (Sigma Chemical Co.) in 0.05 mol/L Tris-HCl for 20 minutes at room temperature. After dehydration in a graded alcohol series, the sections were covered with emulsion for autoradiography as described above. The colocalization of HDC or somatostatin immunoreactiv-
Reg EXPRESSION IN GASTRIC ECL CELLS 47
ity and Reg messenger RNA (mRNA) was quantified by counting two randomly selected visual fields for each region in two sections from 5 animals under a light microscope (201 objective, magnification 2001). In the present study, we defined erosions deeper than half of the mucosal height as the erosive lesions and defined an area located more than 1 cm distant from the erosion as the nonerosive region. For each rat, the total number of Reg mRNA–positive cells, HDC- or somatostatin-immunoreactive cells, and double-labeled cells were recorded. Using the ISH plus immunohistochemistry method, cells with grain densities at least five times higher than the background densities were considered to be positively labeled for Reg mRNA, and a distinctive brown chromogen in the cell cytoplasm indicated HDC or somatostatin immunoreactivity. Data are expressed as means { SE. A nonpaired Student’s t test was used for comparison of the data. A P value of õ0.05 was considered significantly different.
Northern Blot Analysis Total RNAs were obtained from the excised glandular stomachs by extraction with guanidine thiocyanate, followed by cesium chloride centrifugation as described previously.18 The total RNAs were separated using electrophoresis on 0.66 mol/L formaldehyde plus 1% agarose gel with 0.4 mol/L 3(N-monopolyno)-propane sulfate, 0.1 mol/L sodium acetate, and 0.02 mol/L EDTA. The nucleic acid was transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and was fixed to the membrane using UV cross-linking. The rat Reg cDNA probe used for the Northern blot analysis was obtained by RT-PCR amplification of the total RNA extracted from rat pancreas (see section Preparation of Reg RNA Probes), and the HDC cDNA probe was also obtained by RT-PCR of the total RNA from the stomach using oligonucleotide primers 5ⴕ-CTGATGCCATCAACTGCTTG-3ⴕ and 5ⴕ-CCCTAAGGTTGCACAGACAA-3ⴕ.19 The plasmids were digested by EcoRI, and 32P-radiolabeled DNA probes were synthesized using a random primer labeling kit (Boehringer Mannheim). Hybridization was performed at 42⬚C for 16 hours, and the filters were washed twice for 20 minutes each at 55⬚C in 0.11 SSC/0.1% sodium dodecyl sulfate, as described previously.18 The radiolabeled DNA probes were detected by exposure to Kodak XAR-5 film at 080⬚C for 24 hours. The signal intensity of the gene expression was quantified using the bioimaging analyzer BAS2000 (Fujix, Tokyo, Japan).20
Immunohistochemistry for Reg Protein A recombinant DNA corresponding to amino acid positions 22–165 of the deduced rat Reg protein2 was synthesized in Saccharomyces cerevisae strain AH22, and a mouse anti– Reg protein monoclonal antibody was raised against it. The antibody was found to be monospecific for Reg protein by Western blot analysis.5 Three micrometers of paraffin-embedded sections was prepared from each animal. The tissue sections were stained immunohistochemically using the avidin-biotin complex method. After pretreatment with 1% normal goat serum in
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PBS, the tissue sections were incubated with mouse anti-rat Reg monoclonal antibody (1:500) for overnight at 4⬚C in a humidified environment. Endogenous peroxidase activity was blocked with 1.5% H2O2 in 99% methanol for 30 minutes. The slides were washed, and biotinylated goat anti-mouse immunoglobulin (ABC Elite Kit, diluted 1:200; Vector Laboratories) was applied for 2 hours at room temperature, followed by washing with PBS. Bound antibody was detected using the avidin-biotin peroxidase method (ABC Elite Kit; Vector Laboratories). Peroxidase activity was subsequently shown by incubating with 3,3ⴕ-diaminobenzidine (Sigma Chemical Co.) in 0.05 mol/L Tris-HCl for 10 minutes at room temperature, followed by hematoxylin staining. After dehydration in a graded alcohol series, the sections were cleared in xylene.
Immunohistochemical Double Staining Immunohistochemical double staining for Reg protein and chromogranin A in the stomachs was performed 24 hours after the beginning of the stress. The sections were incubated with mouse anti-Reg monoclonal antibody (1:500) and rabbit anti–chromogranin A polyclonal antibody (1:500). Then, fluorescein isothiocyanate–conjugated anti-mouse immunoglobulin (Silenus Laboratories, Hawthorn, Australia; dilution, 1:200) and biotinylated sheep anti-rabbit immunoglobulin (ABC Elite Kit, Vector Laboratories) were applied for 2 hours at room temperature, followed by incubation with streptoavidin–Texas Red (Amersham) for 2 hours. After washing with PBS, the sections were observed by confocal laser microscopy (Olympus, Tokyo, Japan).
Results Identification of Cells Expressing Reg Gene ISH with a 35S-labeled Reg riboprobe detected positive cells in the oxyntic mucosa, but not in the pyloric gland mucosa, both in normal and water-immersed rat stomachs (data not shown). Under low-power magnification, most of the cells expressing Reg mRNA, identified by silver grain accumulation, were localized in the basal part of the oxyntic gland in normal rats (Figure 1A and B). Under higher magnification, distinct hybridization signals of Reg mRNA could be detected in normal stomach neither in parietal cells, easily identified by their large size and clear or acidophilic cytoplasm, nor in chief cells, which were identified by the basal localization of their nuclei and their basophilic cytoplasm. Instead, accumulations of silver grains were found over relatively small cells localized primarily in the basal part of the oxyntic gland (Figure 2A). In contrast, no hybridization signals were detectable above background for radiolabeled sense probe (data not shown). To clarify the type of cells expressing Reg mRNA, ISH combined with immunohistochemistry was performed using anti-HDC or anti-somatostatin antibodies. The
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cells positive for HDC immunoreactivity had a brown reaction product in the cytoplasm and were present in the basal part of the oxyntic gland both in normal and water-immersed rat stomachs. Under lower-power magnification, the cells positive for HDC immunoreactivity in the basal part of the oxyntic gland were clearly visible in the bright-field image (Figure 3A) and the Reg mRNA–positive cells showed a similar distribution in dark-field images (Figure 3B), suggesting colocalization of HDC immunoreactivity and Reg mRNA. Under highpower magnification, most of the cells expressing Reg mRNA were positive for HDC immunoreactivity (Figure 3C); 42.6% { 3.2% of HDC-immunoreactive cells expressed the Reg gene and 68.9% { 4.8% of Reg mRNA–positive cells showed HDC immunoreactivity in the normal stomach (Table 1). In contrast, somatostatin-immunoreactive cells were completely devoid of silver grain accumulations, indicating an absence of Reg gene expression in D cells (Figure 4). Changes of Reg Gene Expression in Water-Immersed Rat Stomachs Northern blot analysis showed that Reg gene expression was relatively low in the normal stomach. However, Reg mRNA signals in the stomachs with water immersion–induced gastric lesions gradually increased up to 24 hours and returned to almost normal levels at 72 hours (Figure 5A). When the signal intensities were quantified by BAS 2000, a bioimaging analyzer, Reg gene expression increased to approximately three times control 6 hours after the start of the stress period, reaching its maximum at 24 hours, with a 10-fold increase in its signal intensity compared with normal levels. In agreement with the Northern blot data, ISH with a 35Slabeled Reg riboprobe showed that both the number of Reg mRNA-positive cells and signal intensity in each cell were obviously increased throughout the oxyntic mucosa of water-immersed rat stomachs and that the increase was more prominent in the erosive lesions (Figure 1C and D). It is noteworthy that in the water-immersed rat stomach, Reg gene expression was observed not only in the lower segment but also in the upper segment of the oxyntic gland (Figure 1C and D). However, as with normal stomachs, neither parietal cells nor chief cells were positive for any hybridization signals, even in the treated animals (Figure 2B). In contrast to the Reg mRNA–positive cells, the number of HDC-immunoreactive cells was not changed by water immersion stress (Table 1). Consequently, the number of HDC-immunoreactive cells positive for Reg mRNA signal increased from 42.6% { 3.2% to 72.4% { 5.1% in the erosive lesion by water immersion stress (Table 1).
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Reg EXPRESSION IN GASTRIC ECL CELLS 49
Figure 1. ISH for Reg mRNA in normal and water-immersed rat stomachs. Rat Reg cRNA probes were radiolabeled using [a-35S]uridine triphosphate. Hybridization was performed as described in Materials and Methods. For autoradiography, the tissue sections were coated with Kodak NTB3 emulsion and exposed for 3 weeks. After development, the sections were counterstained with H&E. (A ) Low-power bright-field image of normal oxyntic mucosa. A small number of silver grains was detected in the basal third of the gland (bar Å 400 mm). (B ) Dark-field image of the section in A. A small number of hybridization signals was detected in the lower segment of the basal gland, and the cells were devoid of signals in the submucosa and muscle layers. (C ) Low-power bright-field photomicrograph of the water immersion–induced gastric lesion. Prominent accumulations of silver grains were observed around the erosive lesions in the stomach 24 hours after the start of the stress period (bar Å 400 mm). (D ) Dark-field photomicrograph of the section in C showing increased Reg mRNA expression around the gastric erosion compared with normal stomach. Hybridization signals were detected not only in the lower segment but also in the upper segment of the gland.
HDC mRNA Expression The change in HDC mRNA levels was also investigated in normal stomachs and 6, 12, and 24 hours after the beginning of the stress. HDC mRNA expression increased to about five times of control 6 hours after the start of the stress period, followed by gradual decrease toward 24 hours (Figure 5B). Reg Protein–Immunoreactive Cells Similar to Reg mRNA – positive cells, Reg protein – immunoreactive cells were detected in the oxyn-
tic mucosa but not in the pyloric gland in both normal and treated rat stomachs. In the normal stomachs, few Reg protein – immunoreactive cells were detected in the basal gland of the oxyntic mucosa and both parietal cells and chief cells were devoid of immunoreactivity (Figure 6A and D ), showing similar distribution of Reg mRNA and Reg protein. In the stomachs 24 hours after the beginning of the stress, the number of Reg protein – immunoreactive cells was markedly increased in the erosive lesions and Reg protein immunoreactivity was relatively intense (Figure 6C and F ), whereas
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Figure 2. ISH for Reg mRNA in normal and water-immersed rat stomachs. Higher-magnification photomicrographs corresponding to Figure 1A and C. (A ) Normal oxyntic mucosa. Distinct hybridization signals cannot be observed in either parietal cells (easily identified by their large size and clear or acidophilic cytoplasm) or chief cells (identified by basal localization of their nuclei and their basophilic cytoplasm). Accumulations of silver grains were found over the relatively small cells localized primarily in the basal part of the oxyntic gland (bar Å 20 mm). (B ) Water immersion–induced gastric lesions. Although both the number of positive cells expressing Reg mRNA and the signal intensity of Reg mRNA in a single cell were increased, parietal and chief cells were devoid of any hybridization signals (bar Å 20 mm).
only a slight increase was observed in nonerosive regions (Figure 6B and E ). Immunohistochemical double staining was also performed to characterize Reg protein–immunoreactive cells more precisely. As shown in Figure 7, all Reg protein–immunoreactive cells showed chromogranin A immunoreactivity, confirming that Reg protein is present exclusively in endocrine cells.
Discussion Reg mRNA has been shown to be present in the gastric wall,10 but its precise localization in the stomach has not been elucidated. The present study, using ISH and immunohistochemistry, showed that both Reg mRNA and Reg protein are expressed in the basal part of the oxyntic mucosa but not in the pyloric mucosa. Moreover, parietal and zymogenic cells were completely devoid of any Reg mRNA hybridization signals or Reg protein immunoreactivity, and most of the silver grains in ISH were detected over small cells in the basal part of the gland, suggesting that fundic endocrine cells express Reg mRNA and its product. Confirming these data, we also showed that all the Reg protein–immunoreactive
cells were positive for chromogranin A immunoreactivity. We subsequently performed a combined ISH and immunohistochemistry study to identify what type of endocrine cells express the Reg gene using anti-HDC and anti-somatostatin antibodies. Interestingly, we found that approximately 70% of the cells expressing Reg gene were positive for immunoreactive HDC but that no immunoreactive somatostatin cells had hybridization signals. Because we and other investigators have previously shown that HDC is exclusively present in enterochromaffin-like (ECL) cells on the basis of immunohistochemical study and electronmicroscopic examination,21 – 23 our present data indicate that Reg gene is primarily expressed in ECL cells. However, approximately 30% of Reg mRNA–positive cells did not show either HDC or somatostatin immunoreactivity. It is possible that these are also ECL cells, in which the HDC content is too small to be detected by immunohistochemistry. However, it is well known that the oxyntic mucosa in the rat harbors at least three ultrastructurally distinct endocrine cells: ECL cells, which constitute Ç65% of all endocrine cells; A-like (or X) cells, which constitute Ç25%; and somatostatin D cells, which constitute Ç10%.24 Thus, it may be
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Reg EXPRESSION IN GASTRIC ECL CELLS 51
considered that, in addition to ECL cells, other endocrine cells, such as A-like cells, express the Reg gene. In any case, the present observation that Reg gene is present in endocrine cells in the gastric mucosa is consistent with the fact that pancreatic endocrine cells express Reg mRNA.2,5 The significant increase in both Reg gene expression and Reg protein immunoreactivity in water immersion stress–induced gastric mucosal injury are important findings. In Northern blot analysis, Reg gene expression increased by 10-fold 24 hours after the start of the stress. Moreover, the ISH study showed that not only the number of Reg mRNA–positive cells but also the signal intensity of Reg mRNA in each cell was increased. Similarly, HDC mRNA level was also increased by water immersion stress. However, in spite of the increased number of Reg mRNA–positive cells, the number of the cells immunoreactive for HDC was not altered. The number of HDC-immunoreactive cells positive for Reg mRNA signals increased from 42% to 72% by water immersion stress. Thus, it appears that water immersion stress induces enhancement of Reg and HDC mRNA expression in ECL cells without affecting their total number. These data suggest a possible specific role of Reg gene expression in ECL cells in the process of gastric mucosal damage. Whether the enhanced Reg gene expression, with resulting increase of Reg protein, is directly related to water immersion stress or nonspecifically to gastric mucosal damage is an interesting question. In this study, we clearly show that the Reg gene was most prominently expressed in the proximity of the erosive lesions. In addition, we found that the changes of Reg protein immunoreactivity paralleled those of Reg gene expression. Indeed, in good agreement with the ISH study, the immunohistochemical study showed that the number of Reg protein–immunoreactive cells is in䉳 Figure 3. ISH immunohistochemical (HDC) study of the stomach 24 hours after the stress period. After the hybridization procedure, the sections were immunohistochemically stained for HDC with polyclonal antibody and the standard avidin-biotin complex method. (A ) Brightfield photomicrograph of the water-immersed rat stomach showed that HDC-immunoreactive cells were distributed in the lower segment of the oxyntic gland, similar to cells expressing Reg mRNA, as shown in B (bar Å 400 mm). (B ) Dark-field photomicrograph of the section in A. The distribution of cells expressing Reg mRNA is similar to that of HDC-immunoreactive cells. (C ) Under high-power field, the doublelabeled cells (arrowheads) showed a brown reaction product in the cytoplasm, indicating HDC immunoreactivity, and an accumulation of silver grains overlying the cells, indicating the presence of Reg mRNA. Approximately 70% of HDC-immunoreactive cells became positive for Reg mRNA 24 hours after the start of water-immersion stress, whereas 40% of the cells were positive for Reg gene expression in normal stomach (bar Å 15 mm).
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Table 1. Changes in Number of HDC-Immunoreactive Cells and Reg mRNA–Positive Cells by Water Immersion Stress Reg mRNA–positive cells
HDC-positive cells Total Normal rats Water-immersed rats Nonerosive region Erosive lesion
Reg-positive (%)
Total
HDC-positive (%)
48.4 { 4.3
42.6 { 3.2
29.4 { 2.8
68.9 { 4.8
51.3 { 4.1 51.9 { 4.2
52.9 { 4.7 72.4 { 5.1b,c
38.7 { 3.4a 54.7 { 4.1b,d
71.2 { 4.4 68.1 { 4.6
NOTE. Results are expressed as mean { SE. a P õ 0.05, bP õ 0.01 significantly different from respective values in normal rats. c P õ 0.05, dP õ 0.01 significantly different from nonerosive region of the water-immersed rats.
creased in the erosive lesions. Furthermore, Reg gene and its product have been shown to be overexpressed in the gastric mucosa of indomethacin-treated rats and in an acetic acid–induced gastric ulcer model (unpublished data). Therefore, the enhancement of Reg gene expression may be a generalized phenomenon with damaged gastric mucosa. Recently, Francis et al.3 showed that increased islet cell replication is paralleled by enhanced Reg gene expression in isolated pancreatic islets of rats. Miyaura et al.4 showed that an increase in Reg gene expression was associated with an increase of beta cell volume in animal models. Watanabe et al.6 reported that recombinant rat Reg protein not only stimulates [3H]thymidine incorporation into isolated pancreatic islets of rats in vitro but also ameliorates surgically induced diabetes in vivo. On the other hand, studies predicting the three-dimensional
Figure 4. ISH immunohistochemical (somatostatin) study of the stomach 24 hours after the beginning of the stress. After the hybridization procedure, the sections were immunohistochemically stained for somatostatin and were subjected to the standard avidin-biotin complex method. Under high-power field, somatostatin-immunoreactive cells (arrowheads ) did not show any hybridization signal (bar Å 30 mm).
Figure 5. Northern blot analysis in the normal and water-immersed rat stomachs. Total RNAs were hybridized with the 32P-labeled cDNA probes. (A ) Northern blotting for Reg mRNA. The signals for Reg mRNA gradually increased up to 24 hours after the start of water immersion (top). The nitrocellulose membranes were rehybridized with the probe for b-actin mRNA (bottom). Lane 1, normal stomach; lanes 2–6, stomach 6, 12, 24, 48, and 72 hours after the start of water immersion stress, respectively. (B ) Northern blotting for HDC mRNA. The signals were most increased 6 hours after the beginning of the stress and gradually decreased toward 24 hours. Lane 1, normal stomach; lanes 2–4, stomach 6, 12, and 24 hours after the start of water immersion stress, respectively.
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Reg EXPRESSION IN GASTRIC ECL CELLS 53
Figure 6. Immunohistochemical staining for Reg protein in (A and D ) normal stomachs, (C and F ) erosive lesions, and (B and E ) areas 1 cm distant from the erosions 24 hours after the beginning of the stress. (A ) Reg protein immunoreactivity in the normal oxyntic mucosa. A small number of cells are positive for Reg protein, and most of the cells are distributed in the bottom of the fundic gland (bar Å 400 mm). (B ) Reg protein immunoreactivity in the area 1 cm distant from the erosion. Number of Reg protein–immunoreactive cells is slightly increased compared with the mucosa of the normal rats (bar Å 400 mm). (C ) Reg protein immunoreactivity in the erosive lesion. Number of Reg protein–immunoreactive cells is significantly increased (bar Å 400 mm). (D ) High-power view of A (bar Å 30 mm). (E ) High-power view of B (bar Å 30 mm). (F ) Highpower view of C. Arrowheads indicate the margin of the erosion (bar Å 30 mm).
structure of Reg protein suggest homology with lectins, a family of proteins with diverse roles involved in growth promotion.7,8 Based on these data, the Reg gene is considered to play a role in the regeneration of pancreatic islets. It is also interesting to speculate that increased Reg gene expression participates in the healing of gastric mucosal injury through a direct effect of Reg protein on the gastric mucosal cells. Several investigators have shown, using [3H]thymidine or bromodeoxyuridine labeling, that the number of proliferative cells increased around gastric erosions and ulcers in the healing process of gas-
tric mucosal injury.25 – 27 In the present study, the expression of Reg gene and its product were markedly increased in the erosive lesions. The direct effect of Reg protein on the gastric mucosal cell growth should therefore be examined in future studies. On the other hand, it has been proven that the deduced amino acid sequence of human REG protein is identical to that of pancreatic stone protein2,28,29 and that REG protein and pancreatic stone protein are derived from the same gene.10,30 Pancreatic stone protein was originally discovered as a protein that comprises up to 10% of the
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increased expression of Reg gene and its product in ECL cells suggests that ECL cells play important roles in the healing process of gastric mucosal injury.
References
Figure 7. Immunohistochemical double staining for Reg protein and chromogranin A in the erosive lesions of the stomach 24 hours after the stress. The sections were incubated with anti-Reg monoclonal antibody and anti–chromogranin A polyclonal antibody. Fluorescein isothiocyanate–conjugated anti-mouse immunoglobulin and biotinylated anti-rabbit immunoglobulin were applied, followed by incubation with streptavidin–Texas Red. The sections were observed using confocal laser microscopy. Reg protein immunoreactivity was visualized by fluorescence (fluorescein isothiocyanate) (left), and chromogranin A immunoreactivity was visualized by fluorescence (Texas Red) (right). In the middle, yellow staining represents colocalization of Reg protein and chromogranin A immunoreactivity (bar Å 10 mm).
protein in pancreatic juice, maintaining it in a supersaturated state with respect to calcium carbonate.31 It was therefore speculated that pancreatic stone protein has a role in stabilizing pancreatic juice and preventing pancreatic stone formation. Indeed, in addition to the preferential expression of Reg gene in pancreatic islets during regeneration of the pancreas, some investigators have reported that both REG mRNA and its product were localized in the acinar cells of the human adult pancreas,11,32,33 suggesting exocrine secretion of REG protein. Interestingly, the REG gene contains an amino acid sequence corresponding to that of signal peptide, suggesting a characteristic feature of a secretory protein.2 Moreover, REG protein possesses three disulfide bonds in its molecule, indicating that, similar to trefoil peptides, it may be resistant to enzymatic digestion.34 Thus, although neither signal for Reg mRNA nor Reg protein immunoreactivity in gastric exocrine cells, it is an interesting possibility that Reg protein is secreted into the gastric lumen, influencing the stability of the mucous layers. In conclusion, we show that the expressions of Reg gene and its product in rat ECL cells are increased in water immersion–induced gastric lesions. It has recently been reported that nitric oxide synthesis is increased in gastric ECL cells during the healing process of acetic acid–induced gastric ulcers.35 Although the number of ECL cells was not increased in our present study, the
1. Yonemura Y, Takashima T, Miwa K, Miyazaki I, Yamamoto H, Okamoto H. Amelioration of diabetes mellitus in partially depancreatised rats by poly(ADP-ribose) synthetase inhibitors. Diabetes 1984;33:401–404. 2. Terazono K, Yamamoto H, Takasawa S, Shiga K, Yonemura Y, Tochino Y, Okamoto H. A novel gene activated in regenerating islets. J Biol Chem 1988;263:2111–2114. 3. Francis PJ, Southgate JL, Wilkin TJ, Bone AJ. Expression of an islet regenerating (reg) gene in isolated rat islets: effects of nutrient and non-nutrient growth factors. Diabetologia 1992;35:238– 242. 4. Miyaura C, Ling C, Appel M. Expression of reg gene/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass. Mol Endocrinol 1991;5:226–234. 5. Terazono K, Uchiyama Y, Ide M, Watanabe T, Yonekura H, Yamamoto H, Okamoto H. Expression of reg protein in rat regenerating islets and its co-localization with insulin in the beta cell secretory granules. Diabetologia 1990;33:250–252. 6. Watanabe T, Yonemura Y, Yonekura H, Suzuki Y, Miyashita H, Sugiyama K, Moriizumi S, Unno M, Tanaka O, Kondo H, Bone AJ, Takasawa S, Okamoto H. Pancreatic beta-cell replication and amelioration of surgical diabetes by reg protein. Proc Natl Acad Sci USA 1994;91:3589–3592. 7. Patty L. Homology of pancreatic stone protein with animal lectin. Biochem J 1988;253:309–311. 8. Peterson TE. The amino terminal domain of thrombomodulin and PSP are homologous with lectin. FEBS Lett 1988;231:51–53. 9. Lassere C, Simon MT, Ichikawa H, Dirong S, Nguyen VC, Christa L, Vernier P, Brechot C. Structural organization and chromosomal localization of a human gene (HIP/PAP) encoding a C-type lectin overexpressed in primary liver cancer. Eur J Biochem 1994;224: 29–38. 10. Watanabe T, Yonekura H, Terazono K, Yamamoto H, Okamoto H. Complete nucleotide sequence of human reg gene and its expression in normal and tumoral tissues: the reg protein, pancreatic stone protein, and pancreatic thread protein are one and the same product of the gene. J Biol Chem 1990;265:7432– 7439. 11. Kimura N, Yonekura H, Okamoto H, Nagura H. Expression of human regenerating gene mRNA and its product in normal and neoplastic human pancreas. Cancer 1992;70:1857–1863. 12. Takeuchi K, Okabe S. Importance of pepsin and stomach distension in morphological alteration of stress-induced gastric lesion in pylorus-ligated rats. Dig Dis Sci 1988;33:52–59. 13. Tohyama M, Siosaka S, Takagi H, Inagaki S, Takatsuki K, Sakanaka M, Senba E, Kawai Y, Minagawa H. Somatostatin-like immunoreactivity in the facial, glossopharyngeal and vagal lobes of the carp. Neurosci Lett 1981;24:233–236. 14. Iacangelo A, Okayama H, Eiden ED. Primary structure of rat chromogranin A and distribution of its mRNA. FEBS Lett 1988;227: 115–121. 15. Yoshimuara R, Kiyama H, Kimura T, Araki T, Maeno H, Tnaizawa O, Tohyama M. Localization of oxytocin receptor messenger ribonucleic acid in the rat brain. Endocrinology 1993;133:1239– 1246. 16. Noguchi K, Kowalski K, Solodkin A, Iadarola MJ, Ruda MA. Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons. Mol Brain Res 1991;10:227–233. 17. Noguchi K, Dubner R, Ruda A. Preprokephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and
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is co-localized with Fos and Fos-related proteins. Neuroscience 1992;46:561–570. Nakata H, Matsui T, Ito M, Taniguchi T, Naribayashi Y, Arima N, Nakamura A, Kinoshita Y, Chihara K, Hosoda S, Chiba T. Cloning and characterization of gastrin receptor from ECL carcinoid tumor of Mastomys natalensis. Biochem Biophys Res Commun 1992; 187:1151–1157. Joseph DR, Sullivan P, Wang YM, Kozak CA, Fenstermacher DA, Behrendsen ME, Zahnow CA. Characterization and expression of the cDNA encoding rat histidine decarboxylase. Proc Natl Acad Sci USA 1990;87:733–777. Asahara M, Kinoshita Y, Nakata H, Matsushima Y, Naribayashi Y, Nakamura A, Matsuil T, Chihara K, Yamamoto J, Ichikawa A, Chiba T. Gastrin receptor genes are expressed in gastric parietal cells and enterochromaffin-like cells of Mastomys natalensis. Dig Dis Sci 1994;39:2149–2156. Rubin W, Schwartz B. An electron microscopic radioautographic identification of the ECL cell as the histamine-synthesizing endocrine cell in the rat stomach. Gastroenterology 1979;77:458– 4567. Soll AH, Lewin KJ, Beaven MA. Isolation of histamine-containing cells from rat gastric mucosa: biochemical and morphological differences from mast cells. Gastroenterology 1981;80:717– 727. Kubota H, Taguchi Y, Tohyama M, Matsuura N, Shiosaka S, Ishihara T, Watanabe T, Shiotani Y, Wada H. Electron microscopic identification of histidine decarboxylase–containing endocrine cells of the rat gastric mucosa: an immunohistochemical analysis. Gastroenterology 1984;87:496–502. Simonsson M, Eriksson S, Hakanson R, Lind T, Lonroth H, Lundell L, O’Connor DT, Sundler F. Endocrine cells in the human oxyntic mucosa. A histochemical study. Scand J Gastroenterol 1988;3:1089–1099. Yeomans ND, St John DJB, de Boer WGRM. Regeneration of gastric mucosa after aspirin-induced injury in the rat. Am J Dig Dis 1973;18:773–780. Ohning GV, Guth PH. Time course of mucosal cell proliferation following acute aspirin injury in rat stomach. Dig Dis Sci 1995; 40:1351–1356. Helpap B, Hattori T, Gedigk P. Repair of gastric ulcer. Virchow Arch A Pathol Anat Histopathol 1981;392:159–170.
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28. Roquier S, Verdier JM, Iovanna J, Dagorn JC, Giorgi D. Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content and sequence identity with the endocrine Reg transcript. J Biol Chem 1991; 266:786–791. 29. Giorge D, Bernard JP, Roquier S, Iovanna J, Sarles H, Dagorn JC. Secretary pancreatic stone protein messenger RNA: nucleotide sequence and expression in chronic calcifying pancreatitis. J Clin Invest 1989;84:100–106. 30. Stewart TA. The human reg gene encodes pancreatic stone protein. Biochem J 1989;260:622–623. 31. De Caro A, Multigner L, Lafont H, Lombardo D, Sarles H. The molecular characteristics of a human pancreatic acidic phosphoprotein that inhibits calcium carbonate crystal growth. Biochem J 1984;222:669–677. 32. De La Porte PP, De Caro A, Lafont H, Sarles H. Immunocytochemical localization of pancreatic stone protein in the human digestive tract. Pancreas 1986;1:301–308. 33. Hoorens A, Lemoine NR, McLellan E, Morohoshi T, Kamisawa T, Heitz PU, Stamm B, Ruschoff J, Wiedemann B, Kloppel G. Pancreatic acinar cell carcinoma. An analysis of cell lineage marker, p53 expression, and Ki-ras mutation. Am J Pathol 1993;143: 685–698. 34. De A, Brown DG, Gorman MA, Carr M, Sanderson MR, Freemont PS. Crystal structure of a disulfide-linked ‘‘trefoil’’ motif found in a large family of putative growth factors. Proc Natl Acad Sci USA 1994;91:1084–1088. 35. Akiba Y, Nakamura M, Oda M, Kimura H, Miura S, Tsuchiya M, Ishii H. Nitric oxide synthesis in activated enterochromaffin-like cell in the healing process of gastric ulcer and hypergastrinemia (abstr). Digestion 1994;55:1–62.
Received August 7, 1995. Accepted March 29, 1996. Address requests for reprints to: Tsutomu Chiba, M.D., Ph.D., Division of Gerontology, Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho 7-5-2, Chuo-ku, Kobe 650, Japan. Fax: (81) 78-361-7524. Supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture and a grant from the Ministry of Health and Welfare, Japan.
Reg Gene Expression Is Increased in Rat Gastric Enterochromaffin-like Cells Following Water Immersion Stress MASAKYO ASAHARA,* SOTARO MUSHIAKE,‡ SHOICHI SHIMADA,§ HIROKAZU FUKUI,* YOSHIKAZU KINOSHITA,* CHIHARU KAWANAMI,* TSUYOSHI WATANABE,x SATOSHI TANAKA,Ø ATSUSHI ICHIKAWA,Ø YASUO UCHIYAMA,x YOICHI NARUSHIMA,# SHIN TAKASAWA,# HIROSHI OKAMOTO,# MASAYA TOHYAMA,§ and TSUTOMU CHIBA* *Division of Gerontology, Department of Internal Medicine, Kobe University School of Medicine, Kobe; Departments of ‡Pediatrics, §Anatomy and Neuroscience, and xCell Biology and Anatomy I, Osaka University Medical School, Osaka; ØDepartment of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto; and #Department of Biochemistry, Tohoku University School of Medicine, Sendai, Japan
Background & Aims: Reg gene has been isolated from regenerating rat pancreatic islets, and subsequent studies have shown a trophic effect of Reg protein on islet cells. However, little is known about the role of Reg protein in the stomach. The aim of this study was to clarify the localization of Reg messenger RNA (mRNA) and its product in the stomach and to examine changes in the level of their expression during regeneration of gastric mucosal cells. Methods: Gastric lesions were experimentally induced in Sprague–Dawley rats by water immersion stress. Northern blot analysis and in situ hybridization studies were performed to examine changes in mRNA levels. Immunohistochemical studies were performed to identify the cellular localization and to investigate the change in Reg protein level. Results: Reg mRNA and its product were distributed in the basal part of the oxyntic mucosa and were expressed mainly in enterochromaffin-like cells. Levels of both Reg mRNA and its product were markedly increased in the water immersion–induced gastric lesions. Conclusions: Reg mRNA and its product are expressed in gastric enterochromaffin-like cells, and their levels are increased during the healing process of water immersion–induced gastric lesions.
n 1984, Yonemura et al.1 found that administration of poly(ADP-ribose) synthetase inhibitors, such as nicotinamide, to rats subjected to 90% depancreatomy induced regeneration of pancreatic islets. Subsequent screening of the regenerating rat islet-derived complementary DNA (cDNA) library showed a novel gene that encodes a 165–amino acid protein with a 21–amino acid signal peptide.2 The gene has been termed Reg and is abundantly expressed in regenerating islets but not in normal ones.2 Further studies showed that enhanced Reg gene expression is linked to DNA synthesis in pancreatic islets both in vitro3 and in vivo.4 Moreover, it has been shown recently that Reg protein is present in the secre-
I
tory granules of beta cells5 and that administration of Reg protein not only enhances DNA synthesis in isolated islets but also ameliorates diabetes in rats subjected to 90% depancreatomy, suggesting a trophic role for the Reg gene in pancreatic beta cells.6 Interestingly, the predicted three-dimensional structure of the protein shows homology with that of the lectins, a family of growth-promoting molecules.7 – 9 REG gene has also been found in the stomach.10 Although the precise localization and the role of the REG gene in the stomach remains undetermined, it is interesting to speculate that it is involved in the process of gastric mucosal repair, as has been suggested to occur in the pancreas. In the pancreas, REG protein is primarily found in the exocrine gland under normal conditions,11 whereas it is mainly present in the islets during regeneration.5 The present study was therefore undertaken to clarify what type of cells in the stomach express Reg gene and its product and to investigate changes in the level of their expressions in water immersion–induced gastric lesions.
Materials and Methods Animals Male Sprague–Dawley rats (250–300 g) were deprived of food but allowed free access to water for 24 hours before the experiments. Each study was performed with 5 animals in each group. To induce gastric mucosal lesions, the animals were placed in strain cages and immersed in water (23⬚C) for 6 hours as described previously.12 Water-immersed rats were killed 0, 6, 12, 24, 48, and 72 hours after the beginning of Abbreviations used in this paper: ECL cell, enterochromaffin-like cell; GSH, glutathione-S-transferase; HDC, histidine decarboxylase; ISH, in situ hybridization; RT-PCR, reverse-transcription polymerase chain reaction. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
46 ASAHARA ET AL.
the stress period for Northern blot analysis and 0 and 24 hours after the beginning of the stress period for in situ hybridization (ISH) and immunohistochemical studies. All experiments were reviewed and approved by the Kobe University Ethical Committee for Animal Experiments.
Tissue Preparation The rats were anesthetized with sodium pentobarbital (Nembutal, 55 mg/kg; Abott Laboratories, North Chicago, IL) and perfused transcardinally with ice-cold Zamboni’s solution (4% paraformaldehyde and 0.2% picrinic acid in 0.1 mol/L phosphate buffer, pH 7.4; 2 mL/g body wt). The stomachs were excised, immersed in fixative for 2–3 hours, and incubated in phosphate-buffered saline (PBS) containing 30% (wt/vol) sucrose at 4⬚C until they sank. The glandular stomachs were divided into fundus (body) and antrum. They were then quickly frozen in a dry ice plus 2-methylbutane mixture, and 5-mm sections were cut on a cryostat and thaw-mounted on 3aminopropyl-triethoxysilane–coated slides. The sections were rinsed with 0.1 mol/L PBS and then treated with 10 mg/mL proteinase K (Boehringer Mannheim, Mannheim, Germany) in 50 mmol/L Tris-HCl and 5 mmol/L ethylenediaminetetraacetic acid (EDTA) (pH 8.0) for 10 minutes. After treatment with 4% paraformaldehyde, they were rinsed with PBS, acetylated with 0.25% (vol/vol) acetic anhydride in 0.1 mol/L triethanolamine, rinsed with PBS, dehydrated in an ethanol series, and air dried.
Preparation of Reg RNA Probes The rat Reg cDNA used for ISH study and Northern blot analysis was obtained by reverse-transcription polymerase chain reaction (RT-PCR) amplification of the total RNA extracted from rat pancreas2 using the synthetic oligonucleotide primers 5ⴕ-TGCCAGAACATGAATTC-3ⴕ and 5ⴕ-TTGAACTTGCAGACAAA-3ⴕ under the following conditions: 94⬚C for 30 seconds, 60⬚C for 60 seconds, and 72⬚C for 60 seconds for 35 cycles. The PCR product was subcloned into the pCRII vector using a TA cloning kit (Invitrogen, San Diego, CA). The recombinant plasmid was linearized by cutting at a single site with a restriction enzyme (Xho I for the antisense probe and with BamHI for the sense probe). In vitro transcription was performed using the appropriate RNA polymerase (SP6 RNA polymerase for the antisense probe, T7 RNA polymerase for the sense probe) and [a-35S]uridine triphosphate (Amersham, Little Chalfont, Buckinghamshire, England). The 35Slabeled antisense riboprobe was complementary to part of the cloned Reg gene (nucleotide positions 187–492).2
Preparation of Anti–histidine decarboxylase, Anti-somatostatin, and Anti–chromogranin A Antibodies A glutathione-S-transferase (GST)–histidine decarboxylase (HDC) fusion protein was produced by inserting a 630– base pair fragment of mouse HDC cDNA (nucleotides 1–630) into the expression vector pGEX-2T (Pharmacia LKB Biotech, Piscataway, NJ). This fragment was prepared by PCR with
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oligonucleotide A (5ⴕ-GTGGATCCATGATGGAGCCCTGTGAATAC-3ⴕ) and oligonucleotide B (5ⴕ-GTGGATCCTCAAATCAAGCCAGCCTTCT-3ⴕ), which contain BamHI sites at their 5ⴕ ends. Amplified fragments were digested with BamHI and then subcloned into the BamHI site of pGEX-2T. The resulting fusion protein combined the 210 amino acids of the N-terminal region of mouse HDC with the C terminus of GST. Overnight cultures of Escherichia coli strain HB101, transformed with the expression vector, were diluted 1:10, and the cells were grown for 1.5 hours at 37⬚C before adding isopropyl1-thio-b-D-galactoside (0.1 mmol/L). After an additional 3hour growth period, bacteria were plated, resuspended, and sonicated in PBS containing 1% sarcosyl and 0.2 mmol/L phenylmethylsulfonyl fluoride and then centrifuged at 10,000g for 5 minutes at 4⬚C. The fusion protein in the lysate was then purified by glutathione-coupled Sepharose 4B (Pharmacia) affinity chromatography in 50 mmol/L Tris-HCl (pH 8.0) containing 20 mmol/L reduced glutathione. To raise antisera, male Japanese white rabbits were immunized by intradermal injections of 200 mg purified GST-HDC fusion protein mixed with complete Freund’s adjuvant (1:1) at 2-week intervals. After the fourth booster, the rabbits were bled and antiserum was obtained. To purify anti–GST-HDC antibody, the antiserum was precipitated with ammonium sulfate (0%–50%) and then adsorbed to Sepharose 4B gel coupled with purified GST-HDC fusion protein. The antibody was eluted with glycine-HCl (pH 2.5) and desalted using a PD10 column (Pharmacia) preequilibrated with PBS. The specificity of the anti-HDC antibody was checked by Western blotting and immunohistological study. An absorption test was also performed, in which absorption of the antiserum by excess GST-HDC fusion protein completely eliminated the immunostaining. The antiserum against somatostatin was obtained from Japan Immunoresearch Laboratory Co. Ltd. (Tokyo, Japan). The characterization of this antiserum has been described elsewhere.13 In the present study, absorption of this antiserum with synthetic somatostatin completely eliminated the immunostaining in the stomach. For the generation of anti–chromogranin A antiserum, a 14–amino acid residue peptide corresponding to position 57– 70 of the rat chromogranin A deduced from the cDNA sequence14 was synthesized by a peptide synthesizer (430A; Perkin–Elmer, Foster City, CA). This peptide was conjugated with keyhole limpet hemocyanin (Sigma Chemical Co., St. Louis, MO), and a rabbit anti–chromogranin A polyclonal antiserum was raised against it. The characteristics of the antiserum were examined by immunoblotting and immunocytochemistry: the anti–chromogranin A antiserum properly recognized an 85-kilodalton band of native chromogranin A protein in the blot of rat adrenal gland extract and stained the secretory granules of adrenal chromaffin cells (unpublished data). In addition, the immunostaining in the stomach in the present study was completely eliminated when the antiserum was absorbed with the synthetic peptide described above before use.
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ISH Histochemistry Two protocols were used for the ISH studies. One provided a single label using ISH histochemistry, and the other protocol combined ISH and immunohistochemistry. The procedure used for ISH and detection have been described previously in detail.15 In brief, 200 mL of hybridization mixture containing 50% formamide, 10% dextran sulfate, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, 5 mmol/L EDTA, 0.1 mol/ L NaPO4 , 0.2% sarcosyl, 200 mg/mL salmon sperm DNA, 11 Denhardt’s solution, 500 mg/mL yeast transfer RNA, and 100 mmol/L dithiothreitol and 6.01 105 cpm of 35S-labeled probes were denatured for 2 minutes at 80⬚C, applied to each section, and covered with a siliconized coverslip. Hybridization was performed in a sealed humid chamber for 18 hours at 55⬚C. After hybridization, the slides were immersed in a mixture of 51 SSC (11 SSC is 150 mmol/L NaCl and 15 mmol/L sodium citrate) and 1% mercaptoethanol at room temperature, and the coverslips were allowed to fall off. The sections were incubated in 50% formamide, 21 SSC, and 10% mercaptoethanol at 65⬚C for 30 minutes and equilibrated with ribonuclease buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 0.5 mmol/L NaCl) three times for 10 minutes each at 37⬚C. They were then treated with ribonuclease A (1 mg/mL) in the same buffer for 30 minutes; incubated in 50% formamide, 21 SSC, and 10% mercaptoethanol at 65⬚C for 30 minutes; and rinsed in 21 SSC and in 0.21 SSC at room temperature for 10 minutes each. After washing, the sections were dehydrated in a graded alcohol series and air dried. For autoradiography, the tissue sections were coated with Kodak NTB3 emulsion (diluted 1:1 with distilled water at 42⬚C; Eastman Kodak, Rochester, NY) and exposed for 3 weeks in light-tight boxes at 4⬚C. After development in Kodak D-19 and fixing in Fujifix (Fuji Film, Tokyo, Japan), the sections were rinsed in distilled water, counterstained with H&E, rinsed in tap water, dehydrated in a graded alcohol series, cleared in xylene, and coverslipped. In the second protocol, tissue sections were first stained immunohistochemically using the avidin-biotin complex method following a hybridization procedure described previously.16,17 After pretreatment with 1% normal goat serum in PBS, the tissue sections were incubated with rabbit antimouse HDC antibody (1:600) or rabbit anti-somatostatin antibody (1:600) overnight at 4⬚C in a humidified environment. Endogenous peroxidase activity was blocked with 1.5% H2O2 in 99% methanol for 30 minutes. The slides were washed, and biotinylated goat anti-rabbit immunoglobulin (Gibco– Bethesda Research Laboratories, Gaithersburg, MD; 1:400) was applied overnight at 4⬚C, followed by washing with PBS. Bound antibody was detected using the avidin-biotin peroxidase method (ABC Elite Kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was subsequently revealed by incubating with 3,3ⴕ-diaminobenzidine (Sigma Chemical Co.) in 0.05 mol/L Tris-HCl for 20 minutes at room temperature. After dehydration in a graded alcohol series, the sections were covered with emulsion for autoradiography as described above. The colocalization of HDC or somatostatin immunoreactiv-
Reg EXPRESSION IN GASTRIC ECL CELLS 47
ity and Reg messenger RNA (mRNA) was quantified by counting two randomly selected visual fields for each region in two sections from 5 animals under a light microscope (201 objective, magnification 2001). In the present study, we defined erosions deeper than half of the mucosal height as the erosive lesions and defined an area located more than 1 cm distant from the erosion as the nonerosive region. For each rat, the total number of Reg mRNA–positive cells, HDC- or somatostatin-immunoreactive cells, and double-labeled cells were recorded. Using the ISH plus immunohistochemistry method, cells with grain densities at least five times higher than the background densities were considered to be positively labeled for Reg mRNA, and a distinctive brown chromogen in the cell cytoplasm indicated HDC or somatostatin immunoreactivity. Data are expressed as means { SE. A nonpaired Student’s t test was used for comparison of the data. A P value of õ0.05 was considered significantly different.
Northern Blot Analysis Total RNAs were obtained from the excised glandular stomachs by extraction with guanidine thiocyanate, followed by cesium chloride centrifugation as described previously.18 The total RNAs were separated using electrophoresis on 0.66 mol/L formaldehyde plus 1% agarose gel with 0.4 mol/L 3(N-monopolyno)-propane sulfate, 0.1 mol/L sodium acetate, and 0.02 mol/L EDTA. The nucleic acid was transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and was fixed to the membrane using UV cross-linking. The rat Reg cDNA probe used for the Northern blot analysis was obtained by RT-PCR amplification of the total RNA extracted from rat pancreas (see section Preparation of Reg RNA Probes), and the HDC cDNA probe was also obtained by RT-PCR of the total RNA from the stomach using oligonucleotide primers 5ⴕ-CTGATGCCATCAACTGCTTG-3ⴕ and 5ⴕ-CCCTAAGGTTGCACAGACAA-3ⴕ.19 The plasmids were digested by EcoRI, and 32P-radiolabeled DNA probes were synthesized using a random primer labeling kit (Boehringer Mannheim). Hybridization was performed at 42⬚C for 16 hours, and the filters were washed twice for 20 minutes each at 55⬚C in 0.11 SSC/0.1% sodium dodecyl sulfate, as described previously.18 The radiolabeled DNA probes were detected by exposure to Kodak XAR-5 film at 080⬚C for 24 hours. The signal intensity of the gene expression was quantified using the bioimaging analyzer BAS2000 (Fujix, Tokyo, Japan).20
Immunohistochemistry for Reg Protein A recombinant DNA corresponding to amino acid positions 22–165 of the deduced rat Reg protein2 was synthesized in Saccharomyces cerevisae strain AH22, and a mouse anti– Reg protein monoclonal antibody was raised against it. The antibody was found to be monospecific for Reg protein by Western blot analysis.5 Three micrometers of paraffin-embedded sections was prepared from each animal. The tissue sections were stained immunohistochemically using the avidin-biotin complex method. After pretreatment with 1% normal goat serum in
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PBS, the tissue sections were incubated with mouse anti-rat Reg monoclonal antibody (1:500) for overnight at 4⬚C in a humidified environment. Endogenous peroxidase activity was blocked with 1.5% H2O2 in 99% methanol for 30 minutes. The slides were washed, and biotinylated goat anti-mouse immunoglobulin (ABC Elite Kit, diluted 1:200; Vector Laboratories) was applied for 2 hours at room temperature, followed by washing with PBS. Bound antibody was detected using the avidin-biotin peroxidase method (ABC Elite Kit; Vector Laboratories). Peroxidase activity was subsequently shown by incubating with 3,3ⴕ-diaminobenzidine (Sigma Chemical Co.) in 0.05 mol/L Tris-HCl for 10 minutes at room temperature, followed by hematoxylin staining. After dehydration in a graded alcohol series, the sections were cleared in xylene.
Immunohistochemical Double Staining Immunohistochemical double staining for Reg protein and chromogranin A in the stomachs was performed 24 hours after the beginning of the stress. The sections were incubated with mouse anti-Reg monoclonal antibody (1:500) and rabbit anti–chromogranin A polyclonal antibody (1:500). Then, fluorescein isothiocyanate–conjugated anti-mouse immunoglobulin (Silenus Laboratories, Hawthorn, Australia; dilution, 1:200) and biotinylated sheep anti-rabbit immunoglobulin (ABC Elite Kit, Vector Laboratories) were applied for 2 hours at room temperature, followed by incubation with streptoavidin–Texas Red (Amersham) for 2 hours. After washing with PBS, the sections were observed by confocal laser microscopy (Olympus, Tokyo, Japan).
Results Identification of Cells Expressing Reg Gene ISH with a 35S-labeled Reg riboprobe detected positive cells in the oxyntic mucosa, but not in the pyloric gland mucosa, both in normal and water-immersed rat stomachs (data not shown). Under low-power magnification, most of the cells expressing Reg mRNA, identified by silver grain accumulation, were localized in the basal part of the oxyntic gland in normal rats (Figure 1A and B). Under higher magnification, distinct hybridization signals of Reg mRNA could be detected in normal stomach neither in parietal cells, easily identified by their large size and clear or acidophilic cytoplasm, nor in chief cells, which were identified by the basal localization of their nuclei and their basophilic cytoplasm. Instead, accumulations of silver grains were found over relatively small cells localized primarily in the basal part of the oxyntic gland (Figure 2A). In contrast, no hybridization signals were detectable above background for radiolabeled sense probe (data not shown). To clarify the type of cells expressing Reg mRNA, ISH combined with immunohistochemistry was performed using anti-HDC or anti-somatostatin antibodies. The
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cells positive for HDC immunoreactivity had a brown reaction product in the cytoplasm and were present in the basal part of the oxyntic gland both in normal and water-immersed rat stomachs. Under lower-power magnification, the cells positive for HDC immunoreactivity in the basal part of the oxyntic gland were clearly visible in the bright-field image (Figure 3A) and the Reg mRNA–positive cells showed a similar distribution in dark-field images (Figure 3B), suggesting colocalization of HDC immunoreactivity and Reg mRNA. Under highpower magnification, most of the cells expressing Reg mRNA were positive for HDC immunoreactivity (Figure 3C); 42.6% { 3.2% of HDC-immunoreactive cells expressed the Reg gene and 68.9% { 4.8% of Reg mRNA–positive cells showed HDC immunoreactivity in the normal stomach (Table 1). In contrast, somatostatin-immunoreactive cells were completely devoid of silver grain accumulations, indicating an absence of Reg gene expression in D cells (Figure 4). Changes of Reg Gene Expression in Water-Immersed Rat Stomachs Northern blot analysis showed that Reg gene expression was relatively low in the normal stomach. However, Reg mRNA signals in the stomachs with water immersion–induced gastric lesions gradually increased up to 24 hours and returned to almost normal levels at 72 hours (Figure 5A). When the signal intensities were quantified by BAS 2000, a bioimaging analyzer, Reg gene expression increased to approximately three times control 6 hours after the start of the stress period, reaching its maximum at 24 hours, with a 10-fold increase in its signal intensity compared with normal levels. In agreement with the Northern blot data, ISH with a 35Slabeled Reg riboprobe showed that both the number of Reg mRNA-positive cells and signal intensity in each cell were obviously increased throughout the oxyntic mucosa of water-immersed rat stomachs and that the increase was more prominent in the erosive lesions (Figure 1C and D). It is noteworthy that in the water-immersed rat stomach, Reg gene expression was observed not only in the lower segment but also in the upper segment of the oxyntic gland (Figure 1C and D). However, as with normal stomachs, neither parietal cells nor chief cells were positive for any hybridization signals, even in the treated animals (Figure 2B). In contrast to the Reg mRNA–positive cells, the number of HDC-immunoreactive cells was not changed by water immersion stress (Table 1). Consequently, the number of HDC-immunoreactive cells positive for Reg mRNA signal increased from 42.6% { 3.2% to 72.4% { 5.1% in the erosive lesion by water immersion stress (Table 1).
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Reg EXPRESSION IN GASTRIC ECL CELLS 49
Figure 1. ISH for Reg mRNA in normal and water-immersed rat stomachs. Rat Reg cRNA probes were radiolabeled using [a-35S]uridine triphosphate. Hybridization was performed as described in Materials and Methods. For autoradiography, the tissue sections were coated with Kodak NTB3 emulsion and exposed for 3 weeks. After development, the sections were counterstained with H&E. (A ) Low-power bright-field image of normal oxyntic mucosa. A small number of silver grains was detected in the basal third of the gland (bar Å 400 mm). (B ) Dark-field image of the section in A. A small number of hybridization signals was detected in the lower segment of the basal gland, and the cells were devoid of signals in the submucosa and muscle layers. (C ) Low-power bright-field photomicrograph of the water immersion–induced gastric lesion. Prominent accumulations of silver grains were observed around the erosive lesions in the stomach 24 hours after the start of the stress period (bar Å 400 mm). (D ) Dark-field photomicrograph of the section in C showing increased Reg mRNA expression around the gastric erosion compared with normal stomach. Hybridization signals were detected not only in the lower segment but also in the upper segment of the gland.
HDC mRNA Expression The change in HDC mRNA levels was also investigated in normal stomachs and 6, 12, and 24 hours after the beginning of the stress. HDC mRNA expression increased to about five times of control 6 hours after the start of the stress period, followed by gradual decrease toward 24 hours (Figure 5B). Reg Protein–Immunoreactive Cells Similar to Reg mRNA – positive cells, Reg protein – immunoreactive cells were detected in the oxyn-
tic mucosa but not in the pyloric gland in both normal and treated rat stomachs. In the normal stomachs, few Reg protein – immunoreactive cells were detected in the basal gland of the oxyntic mucosa and both parietal cells and chief cells were devoid of immunoreactivity (Figure 6A and D ), showing similar distribution of Reg mRNA and Reg protein. In the stomachs 24 hours after the beginning of the stress, the number of Reg protein – immunoreactive cells was markedly increased in the erosive lesions and Reg protein immunoreactivity was relatively intense (Figure 6C and F ), whereas
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Figure 2. ISH for Reg mRNA in normal and water-immersed rat stomachs. Higher-magnification photomicrographs corresponding to Figure 1A and C. (A ) Normal oxyntic mucosa. Distinct hybridization signals cannot be observed in either parietal cells (easily identified by their large size and clear or acidophilic cytoplasm) or chief cells (identified by basal localization of their nuclei and their basophilic cytoplasm). Accumulations of silver grains were found over the relatively small cells localized primarily in the basal part of the oxyntic gland (bar Å 20 mm). (B ) Water immersion–induced gastric lesions. Although both the number of positive cells expressing Reg mRNA and the signal intensity of Reg mRNA in a single cell were increased, parietal and chief cells were devoid of any hybridization signals (bar Å 20 mm).
only a slight increase was observed in nonerosive regions (Figure 6B and E ). Immunohistochemical double staining was also performed to characterize Reg protein–immunoreactive cells more precisely. As shown in Figure 7, all Reg protein–immunoreactive cells showed chromogranin A immunoreactivity, confirming that Reg protein is present exclusively in endocrine cells.
Discussion Reg mRNA has been shown to be present in the gastric wall,10 but its precise localization in the stomach has not been elucidated. The present study, using ISH and immunohistochemistry, showed that both Reg mRNA and Reg protein are expressed in the basal part of the oxyntic mucosa but not in the pyloric mucosa. Moreover, parietal and zymogenic cells were completely devoid of any Reg mRNA hybridization signals or Reg protein immunoreactivity, and most of the silver grains in ISH were detected over small cells in the basal part of the gland, suggesting that fundic endocrine cells express Reg mRNA and its product. Confirming these data, we also showed that all the Reg protein–immunoreactive
cells were positive for chromogranin A immunoreactivity. We subsequently performed a combined ISH and immunohistochemistry study to identify what type of endocrine cells express the Reg gene using anti-HDC and anti-somatostatin antibodies. Interestingly, we found that approximately 70% of the cells expressing Reg gene were positive for immunoreactive HDC but that no immunoreactive somatostatin cells had hybridization signals. Because we and other investigators have previously shown that HDC is exclusively present in enterochromaffin-like (ECL) cells on the basis of immunohistochemical study and electronmicroscopic examination,21 – 23 our present data indicate that Reg gene is primarily expressed in ECL cells. However, approximately 30% of Reg mRNA–positive cells did not show either HDC or somatostatin immunoreactivity. It is possible that these are also ECL cells, in which the HDC content is too small to be detected by immunohistochemistry. However, it is well known that the oxyntic mucosa in the rat harbors at least three ultrastructurally distinct endocrine cells: ECL cells, which constitute Ç65% of all endocrine cells; A-like (or X) cells, which constitute Ç25%; and somatostatin D cells, which constitute Ç10%.24 Thus, it may be
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Reg EXPRESSION IN GASTRIC ECL CELLS 51
considered that, in addition to ECL cells, other endocrine cells, such as A-like cells, express the Reg gene. In any case, the present observation that Reg gene is present in endocrine cells in the gastric mucosa is consistent with the fact that pancreatic endocrine cells express Reg mRNA.2,5 The significant increase in both Reg gene expression and Reg protein immunoreactivity in water immersion stress–induced gastric mucosal injury are important findings. In Northern blot analysis, Reg gene expression increased by 10-fold 24 hours after the start of the stress. Moreover, the ISH study showed that not only the number of Reg mRNA–positive cells but also the signal intensity of Reg mRNA in each cell was increased. Similarly, HDC mRNA level was also increased by water immersion stress. However, in spite of the increased number of Reg mRNA–positive cells, the number of the cells immunoreactive for HDC was not altered. The number of HDC-immunoreactive cells positive for Reg mRNA signals increased from 42% to 72% by water immersion stress. Thus, it appears that water immersion stress induces enhancement of Reg and HDC mRNA expression in ECL cells without affecting their total number. These data suggest a possible specific role of Reg gene expression in ECL cells in the process of gastric mucosal damage. Whether the enhanced Reg gene expression, with resulting increase of Reg protein, is directly related to water immersion stress or nonspecifically to gastric mucosal damage is an interesting question. In this study, we clearly show that the Reg gene was most prominently expressed in the proximity of the erosive lesions. In addition, we found that the changes of Reg protein immunoreactivity paralleled those of Reg gene expression. Indeed, in good agreement with the ISH study, the immunohistochemical study showed that the number of Reg protein–immunoreactive cells is in䉳 Figure 3. ISH immunohistochemical (HDC) study of the stomach 24 hours after the stress period. After the hybridization procedure, the sections were immunohistochemically stained for HDC with polyclonal antibody and the standard avidin-biotin complex method. (A ) Brightfield photomicrograph of the water-immersed rat stomach showed that HDC-immunoreactive cells were distributed in the lower segment of the oxyntic gland, similar to cells expressing Reg mRNA, as shown in B (bar Å 400 mm). (B ) Dark-field photomicrograph of the section in A. The distribution of cells expressing Reg mRNA is similar to that of HDC-immunoreactive cells. (C ) Under high-power field, the doublelabeled cells (arrowheads) showed a brown reaction product in the cytoplasm, indicating HDC immunoreactivity, and an accumulation of silver grains overlying the cells, indicating the presence of Reg mRNA. Approximately 70% of HDC-immunoreactive cells became positive for Reg mRNA 24 hours after the start of water-immersion stress, whereas 40% of the cells were positive for Reg gene expression in normal stomach (bar Å 15 mm).
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Table 1. Changes in Number of HDC-Immunoreactive Cells and Reg mRNA–Positive Cells by Water Immersion Stress Reg mRNA–positive cells
HDC-positive cells Total Normal rats Water-immersed rats Nonerosive region Erosive lesion
Reg-positive (%)
Total
HDC-positive (%)
48.4 { 4.3
42.6 { 3.2
29.4 { 2.8
68.9 { 4.8
51.3 { 4.1 51.9 { 4.2
52.9 { 4.7 72.4 { 5.1b,c
38.7 { 3.4a 54.7 { 4.1b,d
71.2 { 4.4 68.1 { 4.6
NOTE. Results are expressed as mean { SE. a P õ 0.05, bP õ 0.01 significantly different from respective values in normal rats. c P õ 0.05, dP õ 0.01 significantly different from nonerosive region of the water-immersed rats.
creased in the erosive lesions. Furthermore, Reg gene and its product have been shown to be overexpressed in the gastric mucosa of indomethacin-treated rats and in an acetic acid–induced gastric ulcer model (unpublished data). Therefore, the enhancement of Reg gene expression may be a generalized phenomenon with damaged gastric mucosa. Recently, Francis et al.3 showed that increased islet cell replication is paralleled by enhanced Reg gene expression in isolated pancreatic islets of rats. Miyaura et al.4 showed that an increase in Reg gene expression was associated with an increase of beta cell volume in animal models. Watanabe et al.6 reported that recombinant rat Reg protein not only stimulates [3H]thymidine incorporation into isolated pancreatic islets of rats in vitro but also ameliorates surgically induced diabetes in vivo. On the other hand, studies predicting the three-dimensional
Figure 4. ISH immunohistochemical (somatostatin) study of the stomach 24 hours after the beginning of the stress. After the hybridization procedure, the sections were immunohistochemically stained for somatostatin and were subjected to the standard avidin-biotin complex method. Under high-power field, somatostatin-immunoreactive cells (arrowheads ) did not show any hybridization signal (bar Å 30 mm).
Figure 5. Northern blot analysis in the normal and water-immersed rat stomachs. Total RNAs were hybridized with the 32P-labeled cDNA probes. (A ) Northern blotting for Reg mRNA. The signals for Reg mRNA gradually increased up to 24 hours after the start of water immersion (top). The nitrocellulose membranes were rehybridized with the probe for b-actin mRNA (bottom). Lane 1, normal stomach; lanes 2–6, stomach 6, 12, 24, 48, and 72 hours after the start of water immersion stress, respectively. (B ) Northern blotting for HDC mRNA. The signals were most increased 6 hours after the beginning of the stress and gradually decreased toward 24 hours. Lane 1, normal stomach; lanes 2–4, stomach 6, 12, and 24 hours after the start of water immersion stress, respectively.
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Figure 6. Immunohistochemical staining for Reg protein in (A and D ) normal stomachs, (C and F ) erosive lesions, and (B and E ) areas 1 cm distant from the erosions 24 hours after the beginning of the stress. (A ) Reg protein immunoreactivity in the normal oxyntic mucosa. A small number of cells are positive for Reg protein, and most of the cells are distributed in the bottom of the fundic gland (bar Å 400 mm). (B ) Reg protein immunoreactivity in the area 1 cm distant from the erosion. Number of Reg protein–immunoreactive cells is slightly increased compared with the mucosa of the normal rats (bar Å 400 mm). (C ) Reg protein immunoreactivity in the erosive lesion. Number of Reg protein–immunoreactive cells is significantly increased (bar Å 400 mm). (D ) High-power view of A (bar Å 30 mm). (E ) High-power view of B (bar Å 30 mm). (F ) Highpower view of C. Arrowheads indicate the margin of the erosion (bar Å 30 mm).
structure of Reg protein suggest homology with lectins, a family of proteins with diverse roles involved in growth promotion.7,8 Based on these data, the Reg gene is considered to play a role in the regeneration of pancreatic islets. It is also interesting to speculate that increased Reg gene expression participates in the healing of gastric mucosal injury through a direct effect of Reg protein on the gastric mucosal cells. Several investigators have shown, using [3H]thymidine or bromodeoxyuridine labeling, that the number of proliferative cells increased around gastric erosions and ulcers in the healing process of gas-
tric mucosal injury.25 – 27 In the present study, the expression of Reg gene and its product were markedly increased in the erosive lesions. The direct effect of Reg protein on the gastric mucosal cell growth should therefore be examined in future studies. On the other hand, it has been proven that the deduced amino acid sequence of human REG protein is identical to that of pancreatic stone protein2,28,29 and that REG protein and pancreatic stone protein are derived from the same gene.10,30 Pancreatic stone protein was originally discovered as a protein that comprises up to 10% of the
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increased expression of Reg gene and its product in ECL cells suggests that ECL cells play important roles in the healing process of gastric mucosal injury.
References
Figure 7. Immunohistochemical double staining for Reg protein and chromogranin A in the erosive lesions of the stomach 24 hours after the stress. The sections were incubated with anti-Reg monoclonal antibody and anti–chromogranin A polyclonal antibody. Fluorescein isothiocyanate–conjugated anti-mouse immunoglobulin and biotinylated anti-rabbit immunoglobulin were applied, followed by incubation with streptavidin–Texas Red. The sections were observed using confocal laser microscopy. Reg protein immunoreactivity was visualized by fluorescence (fluorescein isothiocyanate) (left), and chromogranin A immunoreactivity was visualized by fluorescence (Texas Red) (right). In the middle, yellow staining represents colocalization of Reg protein and chromogranin A immunoreactivity (bar Å 10 mm).
protein in pancreatic juice, maintaining it in a supersaturated state with respect to calcium carbonate.31 It was therefore speculated that pancreatic stone protein has a role in stabilizing pancreatic juice and preventing pancreatic stone formation. Indeed, in addition to the preferential expression of Reg gene in pancreatic islets during regeneration of the pancreas, some investigators have reported that both REG mRNA and its product were localized in the acinar cells of the human adult pancreas,11,32,33 suggesting exocrine secretion of REG protein. Interestingly, the REG gene contains an amino acid sequence corresponding to that of signal peptide, suggesting a characteristic feature of a secretory protein.2 Moreover, REG protein possesses three disulfide bonds in its molecule, indicating that, similar to trefoil peptides, it may be resistant to enzymatic digestion.34 Thus, although neither signal for Reg mRNA nor Reg protein immunoreactivity in gastric exocrine cells, it is an interesting possibility that Reg protein is secreted into the gastric lumen, influencing the stability of the mucous layers. In conclusion, we show that the expressions of Reg gene and its product in rat ECL cells are increased in water immersion–induced gastric lesions. It has recently been reported that nitric oxide synthesis is increased in gastric ECL cells during the healing process of acetic acid–induced gastric ulcers.35 Although the number of ECL cells was not increased in our present study, the
1. Yonemura Y, Takashima T, Miwa K, Miyazaki I, Yamamoto H, Okamoto H. Amelioration of diabetes mellitus in partially depancreatised rats by poly(ADP-ribose) synthetase inhibitors. Diabetes 1984;33:401–404. 2. Terazono K, Yamamoto H, Takasawa S, Shiga K, Yonemura Y, Tochino Y, Okamoto H. A novel gene activated in regenerating islets. J Biol Chem 1988;263:2111–2114. 3. Francis PJ, Southgate JL, Wilkin TJ, Bone AJ. Expression of an islet regenerating (reg) gene in isolated rat islets: effects of nutrient and non-nutrient growth factors. Diabetologia 1992;35:238– 242. 4. Miyaura C, Ling C, Appel M. Expression of reg gene/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass. Mol Endocrinol 1991;5:226–234. 5. Terazono K, Uchiyama Y, Ide M, Watanabe T, Yonekura H, Yamamoto H, Okamoto H. Expression of reg protein in rat regenerating islets and its co-localization with insulin in the beta cell secretory granules. Diabetologia 1990;33:250–252. 6. Watanabe T, Yonemura Y, Yonekura H, Suzuki Y, Miyashita H, Sugiyama K, Moriizumi S, Unno M, Tanaka O, Kondo H, Bone AJ, Takasawa S, Okamoto H. Pancreatic beta-cell replication and amelioration of surgical diabetes by reg protein. Proc Natl Acad Sci USA 1994;91:3589–3592. 7. Patty L. Homology of pancreatic stone protein with animal lectin. Biochem J 1988;253:309–311. 8. Peterson TE. The amino terminal domain of thrombomodulin and PSP are homologous with lectin. FEBS Lett 1988;231:51–53. 9. Lassere C, Simon MT, Ichikawa H, Dirong S, Nguyen VC, Christa L, Vernier P, Brechot C. Structural organization and chromosomal localization of a human gene (HIP/PAP) encoding a C-type lectin overexpressed in primary liver cancer. Eur J Biochem 1994;224: 29–38. 10. Watanabe T, Yonekura H, Terazono K, Yamamoto H, Okamoto H. Complete nucleotide sequence of human reg gene and its expression in normal and tumoral tissues: the reg protein, pancreatic stone protein, and pancreatic thread protein are one and the same product of the gene. J Biol Chem 1990;265:7432– 7439. 11. Kimura N, Yonekura H, Okamoto H, Nagura H. Expression of human regenerating gene mRNA and its product in normal and neoplastic human pancreas. Cancer 1992;70:1857–1863. 12. Takeuchi K, Okabe S. Importance of pepsin and stomach distension in morphological alteration of stress-induced gastric lesion in pylorus-ligated rats. Dig Dis Sci 1988;33:52–59. 13. Tohyama M, Siosaka S, Takagi H, Inagaki S, Takatsuki K, Sakanaka M, Senba E, Kawai Y, Minagawa H. Somatostatin-like immunoreactivity in the facial, glossopharyngeal and vagal lobes of the carp. Neurosci Lett 1981;24:233–236. 14. Iacangelo A, Okayama H, Eiden ED. Primary structure of rat chromogranin A and distribution of its mRNA. FEBS Lett 1988;227: 115–121. 15. Yoshimuara R, Kiyama H, Kimura T, Araki T, Maeno H, Tnaizawa O, Tohyama M. Localization of oxytocin receptor messenger ribonucleic acid in the rat brain. Endocrinology 1993;133:1239– 1246. 16. Noguchi K, Kowalski K, Solodkin A, Iadarola MJ, Ruda MA. Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons. Mol Brain Res 1991;10:227–233. 17. Noguchi K, Dubner R, Ruda A. Preprokephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and
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28. Roquier S, Verdier JM, Iovanna J, Dagorn JC, Giorgi D. Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content and sequence identity with the endocrine Reg transcript. J Biol Chem 1991; 266:786–791. 29. Giorge D, Bernard JP, Roquier S, Iovanna J, Sarles H, Dagorn JC. Secretary pancreatic stone protein messenger RNA: nucleotide sequence and expression in chronic calcifying pancreatitis. J Clin Invest 1989;84:100–106. 30. Stewart TA. The human reg gene encodes pancreatic stone protein. Biochem J 1989;260:622–623. 31. De Caro A, Multigner L, Lafont H, Lombardo D, Sarles H. The molecular characteristics of a human pancreatic acidic phosphoprotein that inhibits calcium carbonate crystal growth. Biochem J 1984;222:669–677. 32. De La Porte PP, De Caro A, Lafont H, Sarles H. Immunocytochemical localization of pancreatic stone protein in the human digestive tract. Pancreas 1986;1:301–308. 33. Hoorens A, Lemoine NR, McLellan E, Morohoshi T, Kamisawa T, Heitz PU, Stamm B, Ruschoff J, Wiedemann B, Kloppel G. Pancreatic acinar cell carcinoma. An analysis of cell lineage marker, p53 expression, and Ki-ras mutation. Am J Pathol 1993;143: 685–698. 34. De A, Brown DG, Gorman MA, Carr M, Sanderson MR, Freemont PS. Crystal structure of a disulfide-linked ‘‘trefoil’’ motif found in a large family of putative growth factors. Proc Natl Acad Sci USA 1994;91:1084–1088. 35. Akiba Y, Nakamura M, Oda M, Kimura H, Miura S, Tsuchiya M, Ishii H. Nitric oxide synthesis in activated enterochromaffin-like cell in the healing process of gastric ulcer and hypergastrinemia (abstr). Digestion 1994;55:1–62.
Received August 7, 1995. Accepted March 29, 1996. Address requests for reprints to: Tsutomu Chiba, M.D., Ph.D., Division of Gerontology, Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho 7-5-2, Chuo-ku, Kobe 650, Japan. Fax: (81) 78-361-7524. Supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture and a grant from the Ministry of Health and Welfare, Japan.