Gastrin stimulates the growth of gastric pit with
less-differentiated features
Yoshitaka
Konda1,
Hitoshi
Kamimura1,
Hiromi
Yokota1,
Naoki
Hayashi1,
Kentaro
Sugano2, and
Toshiyuki
Takeuchi1
1 Department of Molecular
Medicine, Institute for Molecular and Cellular Regulation, Gunma
University, Maebashi 371-8512; and
2 Gastrointestinal Division,
Department of Medicine, Jichi Medical School, Tochigi 329-0498, Japan
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ABSTRACT |
Gastrin stimulates
the growth of gastric mucosa by increasing mostly its glandular region
but is not known to induce the growth of a pit region where its major
constituent cells, gastric surface mucous (GSM) cells, turn over
rapidly. To investigate the effect of gastrin on GSM cells, we
generated hypergastrinemic mice by expressing a human gastrin
transgene. We obtained a hypergastrinemic mouse line whose average
serum gastrin level is 671 ± 252 pg/ml (normal level <150 pg/ml).
Gastrin-positive cells were found in the fundic mucosa. The gastric
mucosa exhibited hypertrophic growth, which was characterized by an
elongated pit with an active proliferative zone, but the glandular
region containing parietal cells was normal or reduced in size. The GSM
cells contained fewer mucous granules than those of control littermates
and lost reactivity to the GSM cell-specific cholera toxin
-subunit
lectin. GSM cells along the foveolar region and many mucous neck cells
became Alcian blue positive, suggesting the appearance of sialomucin in
these cells. We suggest that gastrin stimulates the growth of the
proliferative zone of gastric glands, which results in the elongation
of the pit region whose GSM cells exhibit less-differentiated features.
gastric mucosa; gastric surface mucous cells; gastrin
binding
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INTRODUCTION |
THE GASTRIC GLAND OF THE stomach forms a pit on its
luminal side, followed by an isthmus and a narrow glandular tube deep down to the layer of the muscularis mucosae. The unit is composed of at
least 11 cell types, including mucus-secreting gastric surface mucous
(GSM) cells (also called pit cells), acid-secreting parietal cells,
pepsinogen-secreting chief cells, somatostatin-producing endocrine D
cells, and histamine-producing enterochromaffin-like (ECL) cells (17,
27, 29). These diverse cell types are thought to originate from the
same progenitor cells in the proliferative zone located at the isthmus
and are thought to move upward, downward, or both to their final
specific location.
The GSM cells mature from granule-free progenitor cells devoid of
mucous granules at the proliferative zone and move upward with a short
lifespan of ~3 days in rodents (28). Maturation, characterized by an
increase in mucous granules, is well regulated by growth factors, gut
hormones, and cell-to-cell and/or cell-to-matrix interaction signals
(26, 50). Growth factors include epidermal growth factor (EGF),
transforming growth factor-
(TGF-
), heparin-binding EGF-like
growth factor, and hepatocyte growth factor (6, 37, 47). Gut hormones
include gastrin, CCK, bombesin, and somatostatin. Among these, gastrin
has been extensively studied and is known to promote gastric mucosal
growth (26, 51), which is typically exemplified by the thickened mucosa
frequently observed in patients with gastrin-producing
Zollinger-Ellison (ZE) tumors (9, 15). Increase of mucosal mass has
been reproduced experimentally in rats infused with gastrin for 28 days, whose mucosa displayed an increased number of ECL cells and a
hypertrophied glandular region composed mostly of parietal cells (40).
Thus gastrin stimulates the secretory function as well as the
proliferation of parietal cells and ECL cells, both of which are known
to express a gastrin receptor (2).
Hypergastrinemia is also found frequently in patients with atrophic
gastritis, although the gastric mucosa grows thin in these cases (15).
In type A atrophic gastritis, parietal cells are often impaired by
autoimmune mechanisms. In type B atrophic gastritis, gastric fluid is
neutralized, possibly by ammonia produced from Helicobacter pylori (12, 33). With the
lack of acidity in gastric fluid, gastrin production is upregulated in
the antral endocrine G cells. Several investigators have hypothesized
that atrophied mucosa is a precancerous base and the elevated gastrin may induce the initiation of a gastric cancer (22, 44). This hypothesis
is favored by the fact that the GSM cells of atrophic gastritis exhibit
more mitotic activity than those in healthy individuals (34).
Gastrin is synthesized in G cells as the precursor preprogastrin and
then is processed by proteolysis and amidation reactions to amidated
gastrin (17 amino acids long) (50). In the amidation reaction, the
glycine residue at the carboxy-terminal end serves as the substrate for
the amidation enzymes. Gastrin thus formed exhibits gastric
acid-secreting activity three orders of magnitude higher than does
glycine-extended gastrin (G-Gly) (36). In contrast, both gastrin and
G-Gly appear to have similar strong growth-promoting activity via their
distinct receptor-mediated signaling (41).
Hypergastrinemic animal models have been useful for exploring the
physiological roles of gastrin in mucosal growth. Such transgenic mice
were produced by Wang et al. (53). The mice in their model expressed
gastrin under the control of an insulin promoter, which resulted in
gastrin production in the pancreatic
-cells, and expressed gastrin
under the control of a human gastrin promoter, which resulted in the
production of a noncleaved gastrin precursor in the liver. Wang et al.
(53) demonstrated that gastrin is more potent for gastric mucosal
growth, whereas progastrin is more potent for colonic mucosal growth.
In their gastrin-producing transgenic model, the serum gastrin level
increased twofold compared with the level of littermate controls
(~130 vs. 70 pg/ml). In hypergastrinemia, however, due
to either ZE tumor or atrophic gastritis, serum gastrin levels are
often elevated 10-fold or more (9). For producing such hypergastrinemic
mice, gastrin expression is desirable not only in endocrine cells but
also in nonendocrine cells. We were successful in making such a gastrin expression vector by utilizing the consensus cleavage site of the
proprotein-processing endoprotease furin, Arg-X-(Lys/Arg)-Arg (18).
Amidation enzyme is distributed widely in almost every tissue including
nonneuroendocrine cells, which are able to produce amidated peptides
when their genes were expressed (11, 18, 25). We expressed a gastrin
cDNA under the control of a
-actin promoter, which exhibits strong
expression in a variety of tissues (1, 21). Thus gastrin should be
highly produced in mice expressing this mutated gastrin precursor.
The present study analyzed the hypertrophic gastric mucosa of
hypergastrinemic mice. The hypertrophic mucosa was comprised of an
elongated pit region with an active proliferative zone. The GSM cells
consisting of the elongated pit exhibited less differentiated features
by immunocytochemical and electron microscopy analyses.
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MATERIALS AND METHODS |
Generation of gastrin-expressing transgenic mice.
We used a human gastrin cDNA, for which the peptide product contains
two mutations (18). One mutation is a processing site at the amino
terminus of gastrin:
-Asp-Pro
4-Ser
3-Lys
2-Lys
1
(native) was changed to
-Asp-Arg
4-Arg
3-Lys
2-Arg
1
(mutant). This tetrabasic site was efficiently cleaved by furin, which
is distributed in many cell types (55). The other mutation is at the
carboxy terminus of gastrin after glycine: the progastrin sequence was
terminated after the glycine position by inserting a stop codon. With
this modification, the mutated progastrin was efficiently cleaved and
amidated even in nonneuroendocrine cells (18). The mutated progastrin
cDNA was inserted into the Xho I site
of the pCXN2 vector (39), whose expression is based on the chicken
-actin promoter. We excised the gastrin expression unit with
Sal I from the vector and
microinjected the excised DNA into oocytes from ICR mice
(Nippon Clea, Osaka, Japan). The oocytes were transferred to
pseudopregnant ICR female mice according to standard procedures (19).
Neonatal mice were screened for the presence of the human gastrin
transgene and the endogenous mouse gastrin gene by a PCR method using
oligonucleotides that bracket the 190-bp DNA on the human gastrin cDNA
(5'-AACAGGGACCTGGAGCTACCC-3' and
5'-GTTCTCATCCTCAGCACTGCG-3') and the 300-bp mouse gastrin genomic DNA including the 110-bp intron II
(5'-AATGAGGACCTGGAACAGCGC-3' and
5'-CTGGTCTTCCTCAGCACTGCG-3'), respectively (16). We
obtained five mice with the gastrin transgene. Transgenic lines were
mated and propagated to obtain hypergastrinemic mice.
Morphological studies.
For periodic acid-Schiff (PAS) staining, stomach tissue sections were
fixed in 10% formaldehyde for 3 h at 4°C and stained with PAS,
using the standard method after diastase digestion (31). Proliferation
of the mouse gastric mucosa was examined by two methods, staining of
proliferating cell nuclear antigen (PCNA) and incorporation of the
thymidine analog bromodeoxyuridine (BrdU) (31). BrdU (80 mg/kg body wt;
Sigma Chemical, St. Louis, MO) was injected intraperitoneally into
20-wk-old mice 2 h before killing. Stomach tissues were removed and
fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Small
pieces of the sample underwent saccharose replacement and then were
frozen for microtome sectioning. The following antibodies were used as
a first antibody for immunostaining: rabbit anti-gastrin polyclonal
antibody (Zymed Laboratory, South San Francisco, CA), mouse monoclonal
anti-PCNA antibody (PC10, DAKO, Glostrup, Denmark), mouse anti-BrdU
monoclonal antibody (BioMeda, Foster City, CA), rabbit anti-histamine
polyclonal antibody (Chemicon International, Temecula, CA), and rabbit
anti-somatostatin polyclonal antibody (Peninsula Laboratory, Belmont,
CA). Monoclonal antibody to H-K-ATPase was prepared by injecting
purified rabbit gastric microsome fractions with enriched H-K-ATPase
activity into mice; this antibody recognizes the tertiary
structure of H-K-ATPase made of
- and
-subunits. An
LSAB2/horseradish peroxidase staining kit (DAKO) was used as the
secondary antibody reaction system.
For lectin binding studies, FITC-labeled cholera toxin
-subunit
(CTB) and Dolichos biflorus (DBA)
(Sigma) were used to identify a gastric epithelial cell lineage (13,
14). Characteristics of mucous cells were examined by Alcian blue
staining (at pH 2.5 for acidic mucin including sialomucin and
sulfomucin and at pH 1.0 for sulfomucin) and paradoxical concanavalin A
staining (PCS) for mucous neck cells (23, 46).
For examination via electron microscopy, gastric tissues were fixed in
2.5% glutaraldehyde-2.0% formaldehyde in 0.1% sodium cacodylate
buffer. They were postfixed in 1%
OsO4, treated with 0.5% uranyl
acetate, and embedded in Epon. Ultrathin sections were stained with
lead citrate and uranyl acetate and examined with an H-800 electron
microscope (Hitachi, Tokyo, Japan).
RIAs.
RIA for amidated gastrin was performed using a gastrin assay kit
(gastrin RIA kit II, Dainabot, Tokyo, Japan). This antibody is specific
for gastrin with an amide moiety. The assay for G-Gly was performed as
described previously, using the antibody 8237 (18). This antibody does
not cross-react with amidated forms of gastrin but cross-reacts 100%
with CCK-Gly (8).
Measurement of acid secretion.
Gastric acid secretion was measured according to the method described
previously (38). Briefly, control and transgenic mice (~20 wk old)
were fasted for 3 h and then anesthetized with ether. After the
abdominal wall was incised, the pylorus was ligated, and the incision
was sutured. The gastric fluid in the stomach was collected 4 h after
the pylorus ligation. For maximal acid output, acid secretion was
stimulated by injecting pentagastrin (250 µg/kg body wt) at the
pylorus ligation. The gastric fluid was titrated with 0.1 N NaOH to pH
7.0 using a microtitrator.
RNA analysis.
Isolated total RNA was treated with DNase I (GIBCO BRL) for RT-PCR.
Expression was assessed by RT-PCR using
5'-AACAGGGACCCTGGAGCTACC-3' and
5'-GAAGGAGGTCGGTACCA-3' for human gastrin mRNA (134 bp) and 5'-AATGAGGACCCTGGAACAGCG-3' and
5'-AGAAGGAGGTAGGCACC-3' for mouse gastrin mRNA (135 bp).
 |
RESULTS |
Generation of transgenic mice.
We selected transgenic mice with the 190-bp human gastrin DNA
fragment using PCR and then mated them to propagate a transgenic line.
We deduced the genotype of human gastrin DNA again using PCR and then
classified the mice into one of three genotypes: those with 300-bp
bands and without 190-bp bands, nontransgenic (genotype
/
); those with 300-bp and 190-bp bands (genotype
+/
); and those with 300-bp bands and roughly two times thicker
190-bp bands (genotype +/+) (Fig.
1A).
After classification, plasma gastrin levels of mice fasted overnight
were measured by RIA (Fig. 1B). The
values from the
/
mice averaged 113 ± 46 pg/ml with a
maximum of 204 pg/ml, those from the +/
mice averaged 278 ± 62 pg/ml, and those from +/+ mice were distributed from 317 pg/ml to
1,207 pg/ml with an average of 671 ± 252 pg/ml (Fig.
1B). Although the classification of
genotype, depending on the thickness of the 190-bp bands, is not
absolute, we were able to select a hypergastrinemic mouse group. We
used mice from the +/+ group whose gastrin levels were over the average
for the following experiments. We also measured G-Gly in several mice
of each genotype group. Although the antibody to G-Gly (antibody 8237)
recognizes G-Gly as well as CCK-Gly (8), plasma G-Gly levels were not
elevated in both the +/+ and +/
hypergastrinemic mouse groups
and remained in the same range as those in the
/
control
mouse group (Fig. 1C), suggesting
that the mutated gastrin expressed from the transgene was efficiently processed to amidated gastrin.



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Fig. 1.
Selection of human gastrin transgene-expressing mice.
A: PCR analysis of transgenic mouse
DNA. Top: schema of 300-bp mouse
gastrin genomic DNA and 190-bp human mutant gastrin cDNA.
Oligonucleotides used for PCR are drawn on each DNA. By agarose gel
electrophoresis (bottom), mice with
190-bp DNA bands were selected as transgenic. Then, after comparison of
the intensity of this band with an endogenous 300-bp mouse gastrin
genomic band, transgene genotypes +/ or +/+ were determined.
Electrophoresis patterns are shown for each genotype. M, DNA bands
amplified by the mouse gastrin-specific oligonucleotides. H, DNA bands
amplified by the human gastrin-specific oligonucleotides.
/ , Nontransgenic (control) mice.
B: plasma gastrin levels in each
genotype group. Highest 3 in the +/+ group are hypergastrinemic mice
with over 1,000 pg/ml plasma gastrin. Vertical bars indicate the
average gastrin value in each group.
C: plasma glycine-extended gastrin
(G-Gly) levels in each genotype group.
X-axis is amidated gastrin, and
y-axis is G-Gly. Positive correlation
was not obtained between gastrin and G-Gly levels.
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Expression of gastrin.
Gastrin content was evaluated in a variety of tissues. Compared with
gastrin levels of control mice, those of the transgenic mice were
strikingly high in the corpus of the stomach. The levels were
noticeably high in the small intestine and detectable in the lung,
heart, foregut, liver, and kidney (Fig.
2A). The
gastrin content in the corpus was comparable to that in the antrum
where gastrin is originally produced. This marked expression of the human gastrin transgene was also confirmed by RT-PCR (Fig.
2B). Gastrin was immunostained in
many epithelial cells in the fundic mucosa of HG mice (Fig.
2C, b
and c) but not at all in the control fundic mucosa (Fig. 2Ca).
Gastrin-positive cells looked smaller than parietal cells. In the pit
region, they appeared to be GSM cells (Fig.
2Cc). In the glandular region, they
may be mucous neck cells by their small size. GSM cells and mucous neck
cells are exocrine cells and secrete mucus into gastric lumen. Because gastrin is a secretory peptide, it may be stored in mucous granules. Exocrine cells release secretory proteins into an exocrine duct as well
as into a blood stream, as exemplified by serum amylase and pepsinogen.
We think that gastrin is produced in GSM-type small cells in the fundic
mucosa.

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Fig. 2.
Expression of human gastrin. A:
gastrin content in a variety of tissues. Gastrin was measured after
boiling a 10% homogenate of each tissue. Open columns, gastrin content
in tissues of control mice. Filled columns, gastrin content in tissues
of transgenic mice. B: RT-PCR analysis
of gastrin mRNA levels. Human and mouse gastrin mRNAs were amplified
with species-specific oligonucleotides. F, Foregut; C, corpus; A,
antrum; D, duodenum. C: immunostaining
of gastrin in the gastric mucosa (a
shows control mucosa, b shows mucosa
of transgenic mice, and c shows
enlargement of squared area in b).
Brown-colored gastrin-positive cells are scattered in clusters in the
gastric mucosa of HG mice but not in the mucosa of control mice. Scale
for a and
b = 100 µm; scale for
c = 50 µm.
D: gel filtration of gastrin on a
Sephadex G-50 column. a: Extract from
the control gastric corpus. b: Extract
from the control gastric antrum. c:
Extract from the transgenic mouse gastric corpus.
d: Extract from the transgenic mouse
gastric antrum. Columns were calibrated with blue dextran (Vo),
potassium ferricyanide (Vt), standard gastrin-17 (G17), and standard
gastrin-34 (G34). Similar chromatograms were obtained in at least 3 other experiments for each extract.
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We then analyzed a molecular form of gastrin by Sephadex G-50 gel
filtration. Gastrin from the antrum of both control and transgenic mice
exhibited a major peak at the gastrin-17 (Gly-17) position with a minor
peak at the Gly-34 position (Fig. 2D,
b and
d). In contrast, gastrin from the
corpus of transgenic mice was eluted as a single peak at the G-17
position without a distinct peak at the Gly-34 position (Fig.
2Dc), indicating that the gastrin contained in the corpus is derived from the gastrin transgene.
Overgrowth of gastric pit relative to a reduced parietal cell mass.
The stomachs from the +/+ group hypergastrinemic mice at 7-8 mo of
age were ~30-50% heavier in weight, and their mucosa was markedly thicker than that of controls, although the mucosa was higher
in some parts and lower in other parts (Table
1, higher part = 1.03 ± 0.32 mm, lower
part = 0.48 ± 0.15 mm). The gastric pits with PAS-positive staining
were highly elongated and displayed an orderly structure (Fig.
3, A and
B). This finding is in contrast to
the gastric pit of TGF-
-overexpressing transgenic mice, which display disorderly growth with cystic distensions (10, 46). Both the
higher and lower parts of the pit were longer than the control pit
(Table 1). The gastric mucosa of normal mice was full of
H-K-ATPase-positive parietal cells, with a relatively short pit region
(Fig. 3C). In contrast, in the
mucosa of transgenic mice, the H-K-ATPase-positive glandular region was
normal to reduced in height. Overgrowth of gastric pit relative to a
parietal cell mass in the transgenic mouse mucosa was also confirmed by
using parietal cell-specific lectin DBA (Fig. 3,
E and
F). To examine the actual decrease
in the absolute number of parietal cells, we counted the number of
parietal cells together with ECL cells per gastric gland unit (Table
2). However, we could not obtain the
absolute number of parietal cells because the section of the transgenic
mouse gastric mucosa was thicker in some parts and thinner in other
parts. We presume that total parietal cell mass may not be increased
because the transgenic mouse parietal cell region was normal to reduced
in height.

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Fig. 3.
Overgrowth of gastric mucosa in HG mice.
A, C,
and E show gastric mucosa of normal
mice; B,
D, and
F show gastric mucosa of transgenic
mice. A and
B: diastase-resistant periodic
acid-Schiff staining. Pit region was elongated in the gastric mucosa of
transgenic mice. C and
D: horseradish peroxidase (HRP)
reaction of H-K-ATPase. Brown-colored H-K-ATPase-positive parietal
cells were distributed in the whole mucosa, except in the upper fourth
of gastric glands in normal mice. In contrast, H-K-ATPase-positive
cells were confined in the lower third of the mucosa in transgenic
mice. E and
F: staining with
Dolichos biflorus (DBA) lectin. DBA is
specific for parietal cells. DBA staining confirms H-K-ATPase staining
in C and
D. Scale for
A, B,
C, and
D = 100 µm; scale for
E and
F = 100 µm.
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We then measured acid secretion capacity in control and transgenic
mice. Basal acid output levels were not elevated in transgenic mice,
although their basal acid output was presumed to be stimulated by high
levels of gastrin (Fig. 4). Maximal acid
output levels were also similar between the two groups by gastrin
stimulation. Likewise, maximal acid output levels by carbachol (60 µg/kg body wt) were also similar between the two groups. Because
maximal acid output reflects total parietal cell mass, the mass may be similar between the control and transgenic mouse gastric mucosae. Acid
secretion in the transgenic mice may be well balanced to a normal range
between high levels of gastrin and somatostatin because somatostatin
cells were increased as described in Endocrine-type cells.

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Fig. 4.
Basal and maximal acid output. Open columns, basal acid output. Solid
columns, maximal acid output. Control, acid output in control mice. TG,
acid output in transgenic mice. Maximal acid output was measured after
stimulation with 250 µg/kg pentagastrin. Values are means ± SE of
at least 4 mice.
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Increase of proliferative zone.
The isthmus at the base of the pit region is known as the proliferative
zone (17, 27, 29), which was confirmed by PCNA staining and the
incorporation of BrdU. In normal mucosa, PCNA-positive cells were
scattered from the middle pit region to the upper glandular region
(Fig.
5A). In
the mucosa of transgenic mice, PCNA-positive cells were clustered
heavily at the isthmus region and also scattered to the upper pit
region (Fig. 5B). The distribution
of BrdU-positive cells is consistent with that of the PCNA data, but
the BrdU-positive cell number is limited. In the normal mucosa, only a
few positive cells were scattered at the upper third zone of the
gastric mucosa (Fig. 5C), as in the
rat gastric mucosa (31). In the transgenic mouse mucosa, a higher
number of BrdU-positive cells were distributed in the same zone (Fig.
5D). The labeling index of the cells
with BrdU in the whole gastric gland unit was 0.71 ± 0.24% in the
normal fundic gland; in contrast, it was 5.5 ± 1.0% in the
transgenic mouse gland. PCNA facilitates DNA replication by polymerase
and remains in the nucleus for a few days after cell division (43,
54) so that many more PCNA-positive cells are observed than
BrdU-positive cells.

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Fig. 5.
Increase of proliferative zone in transgenic mice.
A and
C show gastric mucosa of normal mice.
B and
D show gastric mucosa of transgenic
mice. A and
B: HRP reaction of proliferating cell
nuclear antigen (PCNA). Brown-colored nuclei were localized in the
upper fourth of the glands, except in the luminal epithelial cell area
in normal mucosa. In the mucosa of transgenic mice, PCNA-positive cells
were thickly localized at the base of the pit region.
C and
D: HRP reaction of bromodeoxyuridine.
Distribution pattern was similar but less dense than that of PCNA in
both normal and HG mice. Scale for
A-D = 100 µm.
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Less-differentiated features of GSM cells.
We assessed differentiated features of GSM cells using GSM
cell-specific lectin CTB, electron microscopy, and mucus staining. CTB
was positively stained along the foveola-facing membranes of GSM cells
in the pit region of control mice (Fig.
6A). In
contrast, CTB was not positive in the pit region of transgenic mice
(Fig. 6B). Thus surface mucous cells
in the transgenic mice did not express CTB-specific carbohydrate
moieties. In electron microscopy, mucous granules were rich in
luminal-side GSM cells of control mucosa (Fig.
7, A and
C). Granules are composed of at
least two types: small, dense-cored ones and large gray ones (Fig.
7C). Mucous granules in the
transgenic mouse mucosa were reduced in number (Fig. 7,
B and
D). The cytoplasm of GSM cells was
full of enlarged rough endoplasmic reticulums (ERs) (Fig.
7D). In the middle portion of
transgenic mouse pits, GSM cells were aligned in an orderly fashion
(Fig. 7E) and contained various
sizes of granules from small to much larger ones (Fig.
7F), which were larger than those in
control GSM cells. Large, gray granules were also
surrounded by enlarged ERs, such as those in Fig.
7D. ER is often enlarged
in cells with actively producing secretory proteins. Gastrin is
reported to stimulate mucin biosynthesis in the rat gastric corpus
mucosa (20). These types of GSM cells were observed along the elongated
pit of transgenic mice.

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Fig. 6.
Staining with cholera toxin -subunit (CTB) lectin.
A: gastric pit of control mice.
B: gastric pit of transgenic mice. CTB
is specific for gastric surface mucous (GSM) cells located in the upper
gastric pit. Scale for A and
B = 100 µm.
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Fig. 7.
Electron micrograph of GSM cells. A
and C: upper gastric pit of normal
mice. B and
D: upper gastric pit of transgenic
mice. E and
F: middle region of gastric pit of
transgenic mice. Arrowheads in A
indicate rich amounts of mucous granules. PC, parietal cell. Arrowhead
in B indicates the fewer number of
mucous granules. Mitochondria (Mt) are shown in
C. Arrowhead in
F indicates a large gray granule.
Bottom arrow and top arrow indicate a small gray
granule and a small, dense-cored granule, respectively. Scale for
A, B,
and E = 10 µm; scale for
C, D,
and F = 2 µm.
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We then stained the mucosa with Alcian blue, at pH 2.5 for acidic mucin
including sialomucin and sulfomucin (45) and at pH 1.0 for sulfomucin
(23). No Alcian blue staining at pH 2.5 was observed in the control
mucosa (Fig.
8A). In
the mucosa of transgenic mice, Alcian blue at pH 2.5 stained positively
along the luminal and foveolar surfaces of GSM cells in the upper pit region and staining was also positive in many cells inside the glandular mucosa (Fig. 8B). Alcian
blue-positive cells in the glandular mucosa were smaller in size than
parietal cells (Fig. 8C). To
identify this Alcian blue-positive cell type, we then stained the
mucosa with PCS, specific for mucous neck cells (45), and with
anti-PCNA antibody as shown in Fig. 5,
A and
B. PCS-positive neck cells were
scattered among parietal cells in the lower two-thirds of the control
mucosa (Fig. 8E). In contrast, they
were localized in the lower one-fourth to one-third of the transgenic
mouse mucosa (Fig. 8F). By size and
distribution, Alcian blue-positive cells appeared to be mucous neck
cells that were surrounded by parietal cells (Fig. 8,
C and
G). The Alcian blue-positive cell
layer was lower than the PCNA-positive proliferative zone (Fig. 8,
F and H). In contrast to Alcian blue
staining at pH 2.5, Alcian blue staining at pH 1.0 was not positive in
the transgenic mouse mucosa (Fig.
8D).

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Fig. 8.
Staining of mucous cells.
A-C:
Alcian blue staining at pH 2.5, specific to acidic mucin.
D: Alcian blue staining at pH 1.0, specific to sulfomucin.
E-G:
paradoxical concanavalin A staining (PCS), specific to mucous neck
cells. H: PCNA staining.
A and
E show gastric mucosa of control mice;
B-D
and
F-H
show gastric mucosa of transgenic mice.
C: enlargement of squared area in
B. G:
enlargement of squared area in F.
Alcian blue-positive cells look similar to PCS-positive mucous neck
cells in G. Scale for
A, B,
D-F,
and H = 100 µm; scale for
C and
G = 20 µm.
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Endocrine-type cells.
Histamine-producing ECL cells, another gastrin-target cell type, were
stained for histamine. They were almost similar in number between the
control and transgenic mice (Fig. 9,
A and
B). This finding is different from
the report by Wang et al. (53), who demonstrated an increased number of
ECL cells by argyrophil staining. In contrast, somatostatin-producing D
cells were increased in the mucosa of transgenic mice (Fig.
10, A
and B). Only a few
somatostatin-positive cells were present in the normal fundic mucosa,
whereas a number of small D cells were scattered in the transgenic
mouse mucosa [Fig. 10, B and
C (enlargement)]. Thus, although
gastrin receptors are present in parietal cells, ECL cells, and D cells
(7, 35), only somatostatin-producing D cells increased in number in the transgenic mice.

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Fig. 9.
HRP reaction of histamine. A: gastric
mucosa of normal mice. B: gastric
mucosa of transgenic mice. Brown-colored histamine-positive
enterochromaffin-like cells were scattered in the glandular mucosa in
both normal and transgenic mice (arrowheads). Scale for
A and
B = 50 µm.
|
|

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|
Fig. 10.
HRP reaction of somatostatin. A:
gastric mucosa of normal mice. B:
gastric mucosa of transgenic mice. C:
enlargement of squared area in B.
Brown-colored somatostatin-positive D cells were not seen in the
control glandular mucosa but were seen scattered in the glandular
mucosa of transgenic mice (arrowheads). Scale for
A and
B = 50 µm; scale for
C = 10 µm.
|
|
 |
DISCUSSION |
In this study, we generated hypergastrinemic mice, whose gastric mucosa
was overgrown by the elongation of its pit region. Although we used a
chicken
-actin promoter that has been utilized for transgene
expression (1, 21), we unexpectedly found that high expression of the
gastrin transgene was limited to the mucosa of the gastric corpus (Fig.
2). The expression of TGF-
under the control of a ubiquitously
active metallothionein promoter was observed in the gastric mucosa in
some mouse lines and in the liver in other lines (24, 46). Thus,
even if we use a ubiquitously active promoter, the site of gene
expression appears to be affected by many factors, such as gene
products and chromosomal integrated sites. Although the gastrin
gene was not widely expressed in our transgenic mouse line, we obtained
a transgenic mouse line with an average of 671 ± 252 pg/ml plasma
gastrin levels.
The thickened mucosa of our hypergastrinemic mice resulted from the
elongation of its pit region, unlike the hypertrophic gastric mucosa
observed in ZE syndrome individuals, which is caused by the expansion
of the glandular region (15), and that observed in a rat model infused
with gastrin for 28 days (40). In ZE patients and gastrin-infused rat
models, hypergastrinemia induces hypertrophy of the glandular mucosa
and an increase in ECL cells. Our hypergastrinemic mouse model
presented an increase in somatostatin-producing D cells; thus it might
not exhibit an increase in parietal cells and ECL cells. Another
possibility is that the gastrin transgenic mice were exposed to high
levels of gastrin for prolonged periods of time; thus the sensitivity
to gastrin may be decreased and the parietal and ECL cell number may be
also decreased to normal levels. In the gastrin-expressing models by
Wang et al. (53), the glandular region appeared thick in the mice that
produced a noncleaved gastrin precursor from the liver, whereas the pit region appeared elongated in those mice that produced gastrin from the
pancreatic
-cells. The thickened mucosa of our model appeared
similar to that of their gastrin-producing model but not to their
progastrin-producing model. It remains unclear, however, whether
gastrin induces the growth of the pit region and whether progastrin
induces the growth of the glandular region.
The elongated gastric pit of the transgenic mouse model exhibited
less-differentiated features, determined by the following observations.
First, cell proliferation was highly active, as shown by BrdU
incorporation and PCNA-positive staining. Second, there were virtually
no parietal cells over the proliferative zone, which were limited to
the glandular region. Third, GSM cell-specific staining by CTB lectin
was not observed in the transgenic mouse mucosa. Fourth, mucous
granules in the GSM cells of the top pit region were decreased in
number and larger in size, and those in the GSM cells of the middle pit
region appeared large and gray and were surrounded by enlarged ERs.
Finally, Alcian blue-stained cells appeared along the luminal and
foveolar surface of the pit. Furthermore, some of mucous neck cells
were transformed to Alcian blue-positive cells. Because Alcian
blue-positive cells also appeared in the gastric mucosa of the
TGF-
-overexpressing mouse, resembling that of
Ménétrier's disease (45, 46), appearance of Alcian blue-positive cells suggests a premalignant change of gastric mucosa
(23). Thus the elongated pit of the transgenic mouse model is composed
of less-differentiated GSM cells, which are generated from the
extensively active proliferative zone.
Gastrin induces extensive cell mitosis in atrophic gastritis (34). In
type A gastritis, a loss of parietal cells occurs due to autoimmune
mechanisms. Recently, a mouse model lacking parietal cells was made by
using herpes simplex virus thymidine kinase DNA or diphtheria toxin
fragment A DNA as a transgene (4, 32). Parietal cell ablation resulted
in a marked increase of undifferentiated granule-free progenitor cells
in the proliferative zone and an increase of GSM/pit cell and preneck
cell populations. These cells increased in a disorderly manner and
looked similar to the mucosa of type A atrophic gastritis (15),
although plasma gastrin levels were not reported in these models. A
disorderly growth of gastric mucosa was also observed in
TGF-
-overexpressing transgenic mice (10, 46). TGF-
is known to
stimulate gastrin gene expression (3), although plasma gastrin levels
were not described again in the TGF-
-overexpressing mouse models
(10, 46). TGF-
and its EGF receptor are expressed in GSM cells (37, 42, 48). When GSM cells originate from the proliferative zone, we
suggest that gastrin is instrumental in inducing the proliferation of
precursor cells. Then, when they move up and contact TGF-
-expressing GSM cells, they may receive signals via EGF receptors for their proliferation and maturation. Indeed, Chen et al. (5) demonstrated paracrine control of GSM cell growth by TGF-
. These concerted signals might lead to the formation of an orderly arrayed gastric gland
unit. This concerted signaling of gastrin and TGF-
was demonstrated
in the neogenesis of islet
-cells from pancreatic duct cells (52).
Overexpression or underexpression of one of these factors, however, may
lead to the abnormal development of gastric mucosa. Recently, Koh et
al. (30) demonstrated an atrophic change of gastric mucosa in gastrin
gene-disrupted mice, characterized by a decrease of parietal cells and
ECL cells and an increase of mucous neck cells. By overexpressing
gastrin, we showed abnormal elongation of gastric pits composed of
less-differentiated GSM cells in this study. The thickened gastric
mucosa with an elongated GSM/pit cell region will serve as an important
model for studying the role of gastrin in the growth and maturation of
a GSM/pit cell lineage.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Chris Dickinson and Susan Schonlaw-Finnis,
Department of Pediatrics, University of Michigan, for assaying plasma
G-Gly, Dr. Kuniaki Takata, Department of Cell Biology, Institute for
Molecular and Cellular Regulation, Gunma University, for discussing
morphological data, and Reiko Uchida for secretarial assistance.
 |
FOOTNOTES |
This work was supported by grants-in-aid from the Ministry of
Education, Science, Culture, and Sports of Japan.
Present address of Y. Konda: Department of Gastroenterology and
Hepatology, Kyoto University Graduate School of Medicine, 54, Shogoin-kawara-machi, Sakyoku, Kyoto 606-8507, Japan.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. Takeuchi,
Dept. of Molecular Medicine, Institute for Molecular and Cellular
Regulation, Gunma Univ., 3-39-15, Showa-machi, Maebashi 371-8512, Japan
(E-mail: tstake{at}news.sb.gunma-u.ac.jp).
Received 4 December 1998; accepted in final form 17 July 1999.
 |
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