1 Department of Nutrition, We studied the effects of transforming growth
factor-
serum-free culture; pit cell lineage; proliferation and
differentiation; mucin synthesis; intracellular signals
GASTRIC FUNDIC GLANDS have a complex organization of
several types of epithelial cells, including pit cells, parietal cells, neck cells, chief cells, and a variety of enteroendocrine cells. These
functionally active cells come from multipotent stem cells that are
found in the isthmus (19), and filiation and kinetics of the matured
cells have been intensively studied in experimental animals. Using the
combined techniques of tritiated thymidine labeling and electron
microscopy, Karam and Leblond (18-22) identified 11 cell types in
the zymogenic zone of the mouse stomach, and they revealed the dynamics
of these cells. Recently, transgenic mice have been introduced to study
the mechanisms of cell lineage-specific and differentiation-dependent
patterns of gene expression in the gastric units (25, 35, 36).
Primary cultures of gastric epithelial cells from rats (40), dogs
(6-8), rabbits (39), and guinea pigs (31, 37, 38) have been used
to study interactions between distinct growth factors and gastric
epithelial cells, and several growth factors, including epidermal
growth factor (EGF), transforming growth factor- The cells maintained in our serum-free culture system did not exhibit
mitotic activity, and electron microscopy showed that a great majority
of the cells were in a pre-pit cell stage. EGF stimulated cell growth,
and at the same time, EGF-treated cells matured into pit cells that
contained large mucus granules positive for galactose oxidase-Schiff
(GOS) reaction, suggesting that this culture system may be an excellent
model to study the processes of maturation of a pit cell lineage. Using
this system, we also studied the effects of TGF- Reagents and media.
DMEM, Ham's F-12, and collagen type I were purchased from Flow
Laboratories (McLean, VA). RPMI 1640 and MEM were obtained from Nissui
Pharmaceutical (Tokyo, Japan). Bovine transferrin and sodium selenite
were purchased from GIBCO BRL (Gaithersburg, MD). EGF from mouse
submaxillary glands (receptor grade) and human recombinant TGF- Preparation and culture of gastric mucosal cells.
Gastric mucosal cells were aseptically isolated from male guinea pigs
weighing ~250 g as described previously (33). The isolated cells were
suspended in DMEM/Ham's F-12 (1:1) mixture, containing 15 mM HEPES,
0.2% BSA, 10 µg/ml transferrin, and 2.5 ng/ml sodium selenite. The
cells were cultured in 24-well culture plates (collagen type I-coated
product from Corning, Corning, NY) or in 35-, 60-, and 100-mm-diameter
culture dishes. These culture dishes were coated with collagen type I
before use.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 (TGF-
1) on guinea pig gastric mucous cells, cultured in
serum-free conditions. Electron microscopy showed that most cells were
pre-pit cells, characterized by the presence of a few secretory
granules scattered in the cytoplasm. Epidermal growth factor (EGF)
stimulated cell growth,
[3H]glucosamine
uptake, and accumulation of mucus granules positive for galactose
oxidase-Schiff reaction. This EGF-induced maturation into pit cells was
confirmed morphologically by the appearance of uniformly dense ovoid or
spherical mucus granules packed in the ectoplasm. Western blotting with
an antiphosphotyrosine antibody showed that TGF-
1 did not inhibit
the EGF-initiated tyrosine phosphorylation of the EGF receptor.
Northern blotting with cDNA probes for
c-fos and
c-myc demonstrated that TGF-
1 did
not affect the EGF-induced expression of the transcripts. However,
TGF-
1-treated cells did not replicate and remained in an immature
stage, even in the presence of EGF, suggesting a potential role of
TGF-
1 in the regulation of proliferation and differentiation of a
pit cell lineage in vivo.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(TGF-
), hepatocyte growth factor, insulin, and insulin-like growth factor I
(IGF-I), have been shown to stimulate proliferation of gastric epithelial cells in those system. Primary cultures of guinea pig gastric epithelial cells usually require the presence of a high concentration of FCS (15, 33, 34). In this case, cultured cells rapidly
formed monolayers within 2-3 days, and a majority of the cells
(90%) were identified as pit cells or surface epithelial cells (pit
top cells) (15). In a previous study (31), we developed a serum-free
culture of guinea pig gastric epithelial cells for studying the
combined actions of EGF and insulin. In the present experiments, we
further characterized this serum-free culture system.
on a pit cell
lineage. TGF-
exerts multiple biological actions, such as inhibition
of epithelial cell proliferation, modulation of cell migration, and
stimulation of extracellular matrix production (4, 26, 28). TGF-
is produced in gastric mucosa (30) and has been suggested to play an
important role in the restitution of injured gastric mucosa (42).
However, the effects of TGF-
on the proliferation and differentiation of gastric epithelial cells have not been studied in
detail. We report here that TGF-
1 completely inhibits the EGF-induced proliferation and maturation of a pit cell lineage cultured
in serum-free conditions.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1
were purchased from Collaborative Research (Bedford, MA) and King
Brewing (Tokyo, Japan), respectively. From American Type Culture
Collection (ATCC) (Rockville, MD), we purchased mouse genomic DNAs for
c-fos (ATCC no. 41041) and
c-myc (ATCC no. 41029) and a cDNA for
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ATCC no.
57090). An enhanced chemiluminescence Western blotting detection
system, [
-32P]dCTP
(>3,000 Ci/mmol), hybridization buffer (Rapid-Hyb buffer), and a
random primer kit were purchased from Amersham (Tokyo, Japan). Monoclonal antibody specific for phosphotyrosine (clone Py54) was
obtained from Oncogene Science (Uniondale, NY). BSA (fraction V) was
from Miles Laboratories (Kankakee, IL).
Histological studies.
Glass coverslips were coated with collagen type I and were placed in
35-mm-diameter culture dishes. Isolated cells from guinea pig fundic
glands were cultured on the collagen-coated coverslips in the culture
dishes for 2 days under the serum-free conditions. Nonadherent cells
were removed by washing with MEM. The attached cells on a glass
coverslip were untreated or treated with 20 nM EGF and/or 1 nM
TGF-1 for 2 days and were subjected to light microscopic
examinations. Pit cells and mucous neck cells were distinguished by the
reactivity of their mucin granules to GOS or paradoxical concanavalin A
(PCS) stainings (23). Parietal cells were identified by
immunocytochemical analysis with a monoclonal antibody against the
-subunit of gastric
H+-K+-ATPase,
in addition to their fine eosinophilic staining after hematoxylin-eosin
staining, as described previously (15).
Detection of phosphotyrosine-containing protein.
Isolated cells (1-1.5 × 106) were cultured for 2 days in
the serum-free medium in collagen-coated 35-mm-diameter culture dishes. Floating cells were removed by washing with MEM, and the attached cells
were incubated for 1 h in the serum-free medium. After exposure of the
cells to 20 nM EGF and/or 1 nM TGF-1 for the indicated times, whole cell proteins were extracted and subjected to Western blot
analysis with a monoclonal antibody against phosphotyrosine, as
previously described (31).
Northern blot analysis.
Isolated cells (1 × 107)
were cultured in the serum-free medium in collagen-coated
100-mm-diameter culture dishes. Attached cells were treated with 20 nM
EGF and/or 1 nM TGF-1 for the indicated times. Total RNA was
prepared from the cells with an acid guanidinium thiocyanate-phenol-chloroform mixture (9). Samples of 20 µg of RNA
were separated on a 1% agarose gel, blotted, and
ultraviolet-crosslinked to a nylon membrane filter (Hybond-N-plus,
Amersham). After prehybridization, the filter was hybridized with a
cDNA probe for c-fos,
c-myc, or GAPDH. A 1.0-kbp
Xba
I-Sst I fragment of the mouse
c-fos genomic DNA and a 1.2-kbp
Sst I-Hind III
fragment of the mouse c-myc DNA were
used to detect the levels of c-fos and
c-myc mRNAs. Northern hybridization
with the cDNA probes of c-fos and
c-myc was performed overnight at
48°C in rapid hybridization buffer (Amersham), containing 40%
formamide for c-fos and GAPDH mRNAs or
20% formamide for c-myc mRNA. The
probes were prelabeled with
[
-32P]dCTP by a
random primer kit. Membranes were washed once in 2× SSC (1×
SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) containing 0.1%
SDS, followed by two washes in 0.2× SSC containing 0.1% SDS.
Washing was carried out at 60-65°C. Membranes were exposed to
Kodak X-ray films for the appropriate times.
Measurement of incorporation of
[3H]glucosamine into cells.
After cells were cultured in the serum-free medium for 2 days in
collagen-coated 35-mm-diameter dishes, floating cells were removed by
washing with PBS, and the remaining cells were incubated overnight in
low-glucose DMEM, containing 15 mM HEPES, 0.2% BSA, 10 µg/ml
transferrin, and 2.5 ng/ml sodium selenite. These cells were stimulated
by 20 nM EGF and/or 1 nM TGF-1.
[3H]glucosamine (1 µCi/ml) was added to the cells at the same time or 24 h later. After
incubation for 24 h with
[3H]glucosamine in the
presence or absence of the factors, the medium was removed and the
cells were washed with PBS. The cells were lysed by adding 500 µl of
50 mM Tris · HCl buffer (pH, 7.2) containing 2%
Triton X-100, and the resultant cell lysate was collected in a
microcentrifuge tube. The lysis was completed by passing the lysate
through a 27-gauge needle several times. After addition of 500 µl of
absolute ethanol, the tubes were placed on ice for 15 min, and
precipitated proteins were pelleted by centrifugation at 10,000 g for 20 min at 4°C. After washing
three times with 70% ethanol, the precipitate was solubilized in 200 µl of 1 N NaOH and neutralized with 200 µl of 1 N acetic acid.
Radioactivity was measured by a liquid scintillation counter and
expressed as dpm per milligram of protein.
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RESULTS |
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Effect of TGF-1 on cell growth.
When isolated cells (2 × 105
cells), suspended in the serum-free medium, were placed in each well of
collagen-coated 24-well culture plates and cultured for 2 days, 0.67 ± 0.9 × 105 cells were
attached to each well. After removal of floating cells by washing with
MEM, the attached cells were used for studying cell growth. In the
serum-free medium, the number of attached cells remained constant for 3 days (Fig.
1A).
EGF at 20 nM caused rapid cell growth, doubling the cell number within
24 h. However, treatment with EGF for a longer period did not
additionally increase cell number. The EGF-induced proliferation was
inhibited by simultaneous addition of TGF-
1 in a dose-dependent
manner, and TGF-
1 at 1 nM completely blocked the growth stimulated
by 20 nM EGF (Fig. 1A). TGF-
1
itself did not significantly change the cell number during the
experimental period (data not shown).
|
Effects of TGF-1 on EGF-induced protein tyrosine
phosphorylation.
To elucidate the mechanism of the inhibitory effect of TGF-
1 on
EGF-stimulated cell growth, we tested whether TGF-
1 affected EGF-dependent intracellular events. As shown in Fig.
2 (lanes 3-5),
EGF caused rapid tyrosine phosphorylation of a protein with a molecular
mass of 170 kDa. This 170-kDa protein was already identified as the
tyrosine-autophosphorylated EGF receptor (31). A strong immunoreactive
band with a molecular mass of 68 kDa was BSA, contaminated from the
serum-free culture medium. Several other protein bands were identical
before stimulation by EGF, but the intensities of these bands were not
changed after addition of EGF. TGF-
1 did not initiate any protein
tyrosine phosphorylation (Fig. 2,
lanes
9-11)
and did not affect the EGF-induced autophosphorylation of the EGF
receptor (Fig. 2, lanes
6-8),
suggesting that TGF-
1 might affect postreceptor signaling pathways
of EGF.
|
Effect of TGF-1 on EGF-induced expression of
c-fos and c-myc
mRNAs.
Low levels of c-fos and
c-myc mRNAs were detected in untreated
control cells (Fig. 3). When cultured cells
were stimulated by EGF, they rapidly and transiently increased the
c-fos mRNA level with a peak at
30-60 min. TGF-
1 alone did not cause the c-fos mRNA expression and did not
change the EGF-stimulated accumulation of
c-fos mRNA (Fig.
3A). EGF also increased
c-myc mRNA with a peak at 2 h (Fig.
3B), while TGF-
1 did not.
Furthermore, TGF-
1 did not alter the magnitude and duration of the
mRNA expression induced by EGF.
|
Cytochemical identification of cultured cells.
After cultivation of isolated cells for 2 days in collagen-coated
plates under complete FCS-free conditions, a majority of the cells
(>80% of total cells) did not contain large mucus granules (Fig.
4A).
About 5% of the cells contained gatherings of large mucus granules
that were visualized by GOS reaction (Fig.
4A), suggesting that these cells are
mature pit cells. Less than 1% of the cells contained
PCS-positive granules, which are relatively specific for mucous neck
cells (data not shown). Parietal cells were characterized by
immunoreactivity for antibody against the -subunit of gastric
H+-K+-ATPase
with 4%-6% of the cultured cells being identified as parietal cells
(data not shown). When gastric epithelial cells were cultured in
FCS-containing media, ~90% of cells possessed large GOS-positive granules (15). Thus the population of mucin-containing cells was
strikingly changed by the presence of FCS, and a majority of the cells,
cultured in serum-free conditions, did not have enough mucus granules
to be detectable by the GOS reaction.
|
Effect of EGF and/or TGF-1 on
[3H]glucosamine uptake.
To confirm the cytochemical data, mucin synthesis in the absence or
presence of EGF and/or TGF-
1 was estimated by measuring the
incorporation of
[3H]glucosamine into
cells (Fig. 5). In untreated control cells, [3H]glucosamine uptake
was constant during the experimental period. TGF-
1 itself did not
change the basal level of the uptake. EGF significantly stimulated the
[3H]glucosamine uptake
by the mucin-less epithelial cells. This increase was again blocked
when TGF-
1 was included, supporting the cytochemical study that
TGF-
1 could block the stimulatory action of EGF on mucin synthesis.
|
Electron microscopy of cultured gastric epithelial cells.
Cytochemical studies with GOS and PCS reactions suggested that EGF
might stimulate the maturation of progenitor cells into pit cells and
that TGF-1 might counterregulate this EGF action. To confirm these
EGF and TGF-
1 actions, we performed electron microscopy, since the
stage of differentiation of a pit cell lineage is well characterized by
the appearance of secretory granules (19, 20).
|
![]() |
DISCUSSION |
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Gastric surface epithelial cells originate from the granule-free progenitor cells located at the isthmus of the gastric unit (19). During upward migration, granule-free pre-pit cell precursors acquire secretory granules at a pre-pit cell stage, and then secretory granules increase in number and size along with maturation into a final surface epithelial cell stage. The matured cells display morphological and biochemical properties, characterized by their large granules, consisting of two major components, GOS-positive mucin and surface-active phospholipids (6, 12).
Guinea pig gastric epithelial cells, dispersed by protease digestion, rapidly attached to plastic culture dishes and formed monolayers within 2-3 days in the presence of 10% FCS. FCS stimulated rapid cell growth within 24 h, and then the cells gradually lost their mitotic activity when the great majority of the cells acquired large GOS-positive granules characteristic of pit cells (31). A great amount of information on the mechanism of proliferation of gastric mucous epithelial cells has been obtained from primary cultures of gastric epithelial cells (7, 8, 33, 37-39). In those experiments, serum-starved cells were used to examine mitogen responses, since the presence of serum may perturb epithelial cell function and differentiation. FCS was required for adherence of cells to plastic culture plates. Chen et al. (8) established a short-term culture of mucosal replicating cells from canine oxyntic glands, and serum effects were minimized by limited exposure to serum only for the first 12-18 h of culture. However, we noticed that a short-term exposure to FCS (for >6 h) could prime the cells for maturing into mucin-containing cells. Therefore, we completely removed FCS for the entire culture period to permit detailed study on proliferation and maturation of a pit cell lineage by individual growth factors.
When isolated cells were suspended in the serum-free medium and placed in collagen type I-coated dishes, immature cells slowly and selectively attached to the plates. These cells did not replicate in serum-free conditions. Cytochemical and electron microscopic studies showed that a majority of cells (~90%) displayed features characteristic of a pre-pit cell stage. In response to EGF, pre-pit cells rapidly proliferated and then acquired large GOS-positive granules on day 2. At that time, these mature cells did not replicate any more. This cessation of cell growth was not due to contact inhibition in our assay conditions. These findings suggest that the immature cells, maintained in our culture conditions, may already be programmed to mature into pit cells and surface epithelial cells, once they are stimulated by a mitogen, such as EGF. Using a novel cell line derived from the gastric epithelial cells of transgenic mice harboring the temperature-sensitive simian virus 40 T antigen, Konda et al. (24) also showed that gastric mucous cells ceased growing when they exhibited periodic acid-Schiff reaction-positive materials. Thus the present culture system might, at least in part, reemerge the process of maturation of a pit cell lineage in vivo.
With use of primary cultures of gastric mucous epithelial cells from
different animals, several growth factors, including EGF, TGF-,
hepatocyte growth factor, insulin, and IGF-I, have been shown to
stimulate proliferation of gastric epithelial cells (7, 8, 33,
37-39). On the contrary, growth-inhibitory factors have not been
studied in detail. We showed that TGF-
1 completely inhibited
EGF-induced proliferation of gastric epithelial cells in culture.
Furthermore, TGF-
1 additionally decreased
[3H]thymidine uptake
below the basal level even in the presence of EGF. TGF-
protein is
expressed in luminal surface epithelial cells and those lining the
gastric pit in normal human stomach (41). It was shown that mature
mucus-producing cells in culture produced TGF-
(2, 7, 24), while
immature pit cells expressed low levels of this factor (24). TGF-
has been suggested to play an important role in density-dependent
growth by autocrine and/or paracrine mechanisms (7). A majority
of the cultured cells in our serum-free conditions were composed of
pre-pit cells, and a small proportion of the cells (<5%) appeared to
be pit cells, containing uniformly dense spherical or ovoid mucus
granules that were detectable by the GOS reaction. Although the cell
number and
[3H]thymidine uptake
by untreated control cells remained constant, our culture system could
not completely eliminate the autocrine and/or paracrine
regulation of cell growth (i.e., through TGF-
). It is possible to
speculate that TGF-
1 might block the autocrine and/or
paracrine control; therefore, it additionally decreased the
[3H]thymidine uptake
below the control level.
It was of interest that TGF-1 also inhibited accumulation of
GOS-positive granules in the cells stimulated by EGF. TGF-
has been
widely viewed as a growth-stimulatory factor for mesenchymal cells and
a growth inhibitor for epithelial cells and is also known as a potent
differentiation factor on several types of cells (4, 26, 28).
Cytochemical and electron microscopic examinations revealed that the
TGF-
1-treated cells remained in an immature pre-pit cell stage,
suggesting that TGF-
1 may act as a potent maturation-inhibitory
factor rather than as a differentiation factor for a pre-pit cell
lineage. In the stomach, expression, localization, and physiological
roles of this factor have not been fully understood. At present, it is
doubtful whether TGF-
actively controls the proliferation and
differentiation of a pit cell lineage in normal gastric mucosa, since
it was reported that fibroblasts and granulocytes sporadically showed
immunoreactivity for proTGF-
, while glandular epithelial cells were
all negative in the normal human stomach (30). However, it was reported
that hyperplastic lesions similar to human gastric cystica profunda developed in the gastric glandular mucosa of TGF-
heterozygous mice
(5). Several lines of evidence have suggested that TGF-
may play an
important role in the restitution and healing of injured gastric mucosa
(10, 42). TGF-
simultaneously exerts multiple biological actions,
such as increases in the synthesis and deposition of extracellular
matrix and modulation of cell migration, and all of these sequential
events contribute to wound repair (1, 3). In fact, transient expression
was also detected in acute phase gastric ulcer (10). The overexpression
of this factor was detected in the mesenchymal cells of fibrous
granulation tissue in the stomach and in diffuse-type gastric carcinoma
(17, 30). In diffuse-type gastric carcinoma, cancer cells as well as
stromal cells (fibroblasts, macrophages, and endothelial cells)
expressed abundant proTGF-
. TGF-
secreted from the cells has been
suggested to promote extensive fibrosis in those lesions, in which
TGF-
may act as a potent growth inhibitor of mucus-secreting cells, as demonstrated in this study.
We also examined the effects of TGF-1 on EGF signaling pathways.
Many lines of evidence have suggested that TGF-
does not inhibit EGF
binding to its receptor and early growth factor-induced events (29).
This was also in the case in gastric epithelial cells; TGF-
1 did not
affect the autophosphorylation of the EGF receptor induced by EGF.
TGF-
induces growth inhibition by upregulating cyclin-dependent
kinase (CDK) inhibitor p15 in certain epithelial cell lines
(13). In addition, the antiproliferative effect of TGF-
is also mediated by repression of the expression of Cdc25A, a CDK
tyrosine phosphatase that activates CDK (16). TGF-
has been shown to
repress c-myc expression, and c-Myc
can transcriptionally induce Cdc25A (11), leading to a possibility that
the suppression of c-myc expression
has been considered to be a central event in the growth-inhibitory
response to TGF-
(32, 16). In our experiments, however, TGF-
1 did
not affect the duration and magnitude of
c-myc mRNA expression after
stimulation of cultured gastric epithelial cells by EGF. Yoshimura et
al. (43) also reported that the degree of
c-fos and
c-myc expressions by EGF, insulin, dibutyryl-cAMP, or TGF-
did not necessary correlate with the effects
of these agents on cell proliferation of rabbit gastric epithelial
cells in culture. TGF-
family members signal through two
transmembrane serine/threonine kinases known as the type I and type II
receptors. Recently, the SMAD [a new word coined from Sma and Mad
(Mothers against dpp)] family of signal transducer proteins has been
identified to be a component in signal transduction pathway downstream
of the serine/threonine kinase receptors (14, 27).
Different members of the SMAD family have different roles in signaling,
and TGF-
signaling has not been studied in gastric epithelial cells.
Elucidation of TGF-
signals and its target genes may help to
understand the regulatory mechanism of proliferation and
differentiation of a pit cell lineage.
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FOOTNOTES |
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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: K. Rokutan, Dept. of Nutrition, School of Medicine, The Univ. of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
Received 11 March 1998; accepted in final form 20 May 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Basson, J. A.,
I. M. Modlin,
S. D. Flynn,
B. P. Jena,
and
J. A. Madri.
Independent modulation of migration and proliferation by growth factors, matrix proteins, and pharmacologic agents in an in vitro model of mucosal healing.
Surgery
112:
299-308,
1992[Medline].
2.
Beauchamp, R. D.,
J. A. Bernard,
C. M. McCutchen,
J. A. Cherner,
and
M. Renewal.
Localization of transforming growth factor and its receptor in gastric mucosal cells.
J. Clin. Invest.
84:
1017-1023,
1989[Medline].
3.
Bennet, N. T.,
and
G. S. Schultz.
Growth factors and wound healing: biochemical properties of growth factors and their receptors.
Am. J. Surg.
165:
728-737,
1993[Medline].
4.
Bernard, J. A.,
G. J. Warwick,
and
I. I. Gold.
The cell biology of transforming growth factor .
Biochim. Biophys. Acta
1032:
79-87,
1990[Medline].
5.
Boivin, G. P.,
J. R. Molina,
I. Ormsby,
G. Stemmermann,
and
T. Doetschman.
Gastric lesions in transforming growth factor -1 heterozygous mice.
Lab. Invest.
74:
513-518,
1996[Medline].
6.
Boland, C. R.,
E. R. Kraus,
J. M. Scheiman,
C. Black,
G. D. Deshmukh,
and
W. O. Dobbins III.
Characterization of mucous cell synthetic functions in a new primary canine gastric mucous cell culture system.
Am J. Physiol.
258 (Gastrointest. Liver Physiol. 21):
G774-G787,
1990
7.
Chen, M. C.,
A. T. Lee,
W. E. Karnes,
D. Avedian,
M. Martin,
J. M. Sorvillo,
and
A. H. Soll.
Paracrine control of gastric epithelial cell growth in culture by transforming growth factor-.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G390-G396,
1993
8.
Chen, M. C.,
A. T. Lee,
and
A. H. Soll.
Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor and insulin-like growth factor I.
J. Clin. Invest.
87:
1716-1723,
1991[Medline].
9.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
10.
Ernst, H.,
P. C. Konturek,
T. Brzozowski,
S. J. Konturek,
and
E. G. Hahn.
Subserosal application of transforming growth factor-1 in rats with chronic gastric ulcers: effect on gastric ulcer healing and blood flow.
J. Physiol. Pharmacol.
47:
443-454,
1996[Medline].
11.
Garaktionov, K.,
X. Chen,
and
D. Beach.
Cdc25 cell-cycle phosphatase as a target of c-Myc.
Nature
282:
511-517,
1996.
12.
Goddard, P. J.,
Y.-C. J. Kao,
and
L. M. Lichtenberger.
Luminal surface hydrophobicity of canine gastric mucosa is dependent on a surface mucous gel.
Gastroenterology
98:
361-370,
1990[Medline].
13.
Hannon, G. J.,
and
D. Beach.
p15INK4B is a potent effector of TGF--induced cell-cycle arrest.
Nature
371:
257-261,
1994[Medline].
14.
Heldin, C.-H.,
K. Miyazono,
and
P. T Dijke.
TGF- signaling from cell membrane to nucleus through SMAD proteins.
Nature
390:
465-471,
1997[Medline].
15.
Hirakawa, T.,
K. Rokutan,
T. Nikawa,
and
K. Kishi.
Geranylgeranylacetone induces heat shock proteins in cultured guinea pig gastric mucosal cells and rat gastric mucosa.
Gastroenterology
111:
345-357,
1996[Medline].
16.
Iavarone, A.,
and
J. Massegue.
Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF- in cells lacking the CDK inhibitor p15.
Nature
387:
412-422,
1997.
17.
Kai, T.,
F. Taketazu,
M. Kawakami,
K. Shimanuki,
S. Yamada,
K. Miyazono,
M. Kato,
and
M. Miyata.
Distribution of transforming growth factor- and its receptors in gastric carcinoma tissue.
Jpn. J. Cancer Res.
87:
296-304,
1996[Medline].
18.
Karam, S. M.
Dynamics of epithelial cells in the corpus of the mouse stomach. IV. Bidirectional migration of parietal cells ending in their gradual degradation and loss.
Anat. Rec.
236:
314-332,
1993[Medline].
19.
Karam, S. M.,
and
C. P. Leblond.
Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cells.
Anat. Rec.
236:
259-279,
1993[Medline].
20.
Karam, S. M.,
and
C. P. Leblond.
Dynamics of epithelial cells in the corpus of the mouse stomach. II. Outward migration of pit cells.
Anat. Rec.
236:
280-296,
1993[Medline].
21.
Karam, S. M.,
and
C. P. Leblond.
Dynamics of epithelial cells in the corpus of the mouse stomach. III. Inward migration of neck cells followed by progressive transformation into zymogenic cells.
Anat. Rec.
236:
297-313,
1993[Medline].
22.
Karam, S. M.,
and
C. P. Leblond.
Dynamics of epithelial cells in the corpus of the mouse stomach. V. Behavior of entero-endocrine and caveolated cells: general conclusions on cell kinetics in the oxyntic epithelium.
Anat. Rec.
236:
333-340,
1993[Medline].
23.
Katsuyama, T.,
and
S. S. Spicer.
Histochemical differentiation of complex carbohydrates with variants of the concanavalin A-horseradish peroxidase method.
J. Histochem. Cytochem.
26:
233-250,
1978[Abstract].
24.
Konda, Y.,
H. Yokota,
T. Kayo,
T. Horiuchi,
N. Sugiyama,
S. Tanaka,
K. Takata,
and
T. Takeuchi.
Proprotein-processing endopeptidase furin controls the growth and differentiation of gastric surface mucous cells.
J. Clin. Invest.
99:
1842-1851,
1997
25.
Lorenz, R. G.,
and
J. I. Gordon.
Use of transgenic mice to study regulation of gene expression in the parietal cell lineage of gastric units.
J. Biol. Chem.
268:
26559-26570,
1993
26.
Massague, J.
Transforming growth factor- family.
Annu. Rev. Cell Biol.
6:
597-641,
1990.
27.
Massague, J.
TGF- signaling: receptors, transducers, and Mad proteins.
Cell
85:
947-950,
1996[Medline].
28.
McCartney-Francis, N. L.,
and
S. M. Wahl.
Transforming growth factor : a matter of life and death.
J. Leukoc. Biol.
55:
401-409,
1994[Abstract].
29.
Moses, H. L.,
E. Y. Yang,
and
J. A. Pietenpol.
TGF- stimulation and inhibition of cell proliferation: new mechanistic insights.
Cell
63:
245-247,
1990[Medline].
30.
Muzoi, T.,
H Ohtani,
K. Miyazono,
M. Miyazawa,
S. Matsuno,
and
H. Nagura.
Immunoelectron microscopic localization of transforming growth factor 1 and latent transforming growth factor
1 binding protein in human gastrointestinal carcinomas: qualitative difference between cancer cells and stromal cells.
Cancer Res.
53:
183-190,
1993[Abstract].
31.
Ogihara, M.,
M. Yamada,
T. Saito,
M. Shono,
and
K. Rokutan.
Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G104-G112,
1996
32.
Pietenpol, J. A.,
R. W. Stein,
E. Moran,
P. Yaciuk,
R. Schlegel,
R. M. Lyons,
M. R. Pittelkow,
K. Munger,
P. M. Howley,
and
H. L. Moses.
TGF-1 inhibition of c-myc transcription and growth in keratinocytes abrogated by viral transforming proteins with pRB binding domains.
Cell
61:
777-785,
1990[Medline].
33.
Rokutan, K.,
T. Hirakawa,
S. Teshima,
S. Honda,
and
K. Kishi.
Glutathione depletion impairs transcriptional activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells.
J. Clin. Invest.
97:
2242-2250,
1996
34.
Rokutan, K.,
R. B. Johnston, Jr.,
and
K. Kawai.
Oxidative stress induces S-thiolation of specific proteins in cultured gastric mucosal cells.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G247-G254,
1994
35.
Roth, K. A.,
S. M. Cohn,
D. C. Rubin,
J. F. Trahair,
M. R. Neutra,
and
J. I. Gordon.
Regulation of gene expression in gastric epithelial cell populations of fetal, neonatal, and adult transgenic mice.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G186-G197,
1992
36.
Roth, K. A.,
J. M. Hertz,
and
J. I. Gordon.
Mapping enteroendocrine cell populations in transgenic mice reveals an unexpected degree of complexity in cellular differentiation within gastrointestinal tract.
J. Cell Biol.
110:
1791-1801,
1990[Abstract].
37.
Rutten, M. J.,
P. J. Dempsey,
T. E. Solomon,
and
R. J. Coffey.
TGF- is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G361-G369,
1993
38.
Rutten, M. J.,
P. Harmon,
and
D. R. Campbell.
Insulin enhances epidermal growth factor- and transforming growth factor--stimulated growth in primary cultures of guinea pig gastric mucous epithelial cells.
Scand. J. Gastroenterol.
26:
965-973,
1991[Medline].
39.
Takahashi, M.,
S. Ota,
T. Shimada,
E. Hamada,
T. Kawabe,
T. Okudaira,
M. Matsumura,
N. Kaneko,
A. Terano,
T. Nakamura,
and
M. Omata.
Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.
J. Clin. Invest.
95:
1994-2003,
1995[Medline].
40.
Terano, A.,
K. J. Ivey,
J. Stachura,
S. Sekhon,
H. Hosojima,
W. N. McKenzie, Jr.,
and
J. H. Wyche.
Cell culture of rat gastric fundic mucosa.
Gastroenterology
83:
1280-1291,
1982[Medline].
41.
Thomas, D. M.,
M. M. Nasim,
W. J. Gullick,
and
M. R. Alison.
Immunoreactivity of transforming growth factor- in the normal adult gastrointestinal tract.
Gut
33:
628-631,
1992[Abstract].
42.
Yanaka, A.,
H. Muto,
H. Fukutomi,
S. Ito,
and
W. Silen.
Role of transforming growth factor- in the restitution of injured guinea pig gastric mucosa in vitro.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G75-G85,
1996
43.
Yoshimura, K.,
S. Ota,
A. Terano,
M. Takahashi,
Y. Hata,
T. Kawabe,
H. Mutoh,
H. Hiraishi,
R. Nakata,
K. Okano,
and
M. Omata.
Growth regulation of rabbit gastric epithelial cells and protooncogene expression.
Dig. Dis. Sci.
39:
1454-1463,
1994[Medline].