Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom
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ABSTRACT |
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Epithelial organization is maintained by cell proliferation, migration, and differentiation. In the case of the gastric epithelium, at least some of these events are regulated by the hormone gastrin. In addition, gastric epithelial cells are organized into characteristic tubular structures (the gastric glands), but the cellular mechanisms regulating the organization of tubular structures (sometimes called branching morphogenesis) are uncertain. In the present study, we examined the role of the gastrin-cholecystokininB receptor in promoting branching morphogenesis of gastric epithelial cells. When gastric cancer AGS-GR cells were cultured on plastic, gastrin and PMA stimulated cell adhesion, formation of lamellipodia, and extension of long processes in part by activation of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase. Branching morphogenesis was not observed in these circumstances. However, when cells were cultured on artificial basement membrane, the same stimuli increased the formation of organized multicellular arrays, exhibiting branching morphogenesis. These effects were reversed by inhibitors of PKC but not of PI-3 kinase. We conclude that, in the presence of basement membrane, activation of PKC by gastrin stimulates branching morphogenesis.
stomach; gastric glands; extracellular matrix; migration; epithelium
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INTRODUCTION |
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THE ORGANIZATION OF THE EPITHELIUM throughout the gut is maintained by cell proliferation followed by migration and differentiation. Proliferating cells in the gastric epithelium are located in the isthmus region of the glands (12). Cells that leave the cell cycle may migrate either toward the surface, adopting a mucus-secreting phenotype, or toward the base of the gland, where the main differentiated cell types are parietal, chief, and endocrine cells. The latter cell types arrange and maintain themselves in characteristic tubular ensembles that constitute the gastric glands. The assembly of tubular structures is thought to depend on cell-cell and cell-matrix interactions and is modulated and regulated by growth and morphogenetic factors. Loss of gastric glands and reduction in gland length are features of the premalignant condition of gastric atrophy (4). Cells may also be arranged in characteristic tubularlike ribbons in some gastric cancers, so that the control of tubulogenesis may be of pathological as well as physiological relevance.
In kidney, mammary gland, and liver cells, hepatocyte growth factor
(HGF) is known to promote the process of tubule formation, or branching
morphogenesis (1, 11, 15, 21, 23, 27). The mechanisms by
which gastric epithelial cells form tubulelike arrays are less well
studied. It has, however, been reported that heregulin (also known as
neuregulin), which belongs to the epidermal growth factor (EGF) family
(24), promotes formation of tubulelike structures via
activation of erb-B2 and erb-B3 members of the EGF receptor group in a
gastric cell line (3). Moreover, another EGF-like growth
factor, transforming growth factor (TGF)-, appears to stimulate
branching morphogenesis in RGM-1 cells (derived from rat gastric mucus
cells), probably via induction of cyclooxygenase-2 (28).
In addition, the gastric trefoil factor TFF-2 has been reported to
stimulate branching morphogenesis in MCF-7 cells (derived from human
mammary carcinoma) (17). Interestingly, expression of the
mucin MUC-1 is also linked to branching morphogenesis in mammary and
kidney cell lines (9).
It is well recognized that gastrin influences the organization of the gastric epithelium, as well as acutely regulating acid secretion (33). Elevated plasma gastrin concentrations are associated with increased parietal cell mass (25) and hyperplasia of histamine-producing enterochromaffinlike (ECL) cells (2, 18, 19). In transgenic mice with hypergastrinemia, there are initially increased parietal cell numbers and increased gland length (16, 34), indicating that gastrin influences the organization of the gastric mucosa (20). In the present study, we used an assay of branching morphogenesis to study the response of gastric AGS cells to activation of the gastrin-CCKB receptor. The data indicate that gastrin stimulates branching morphogenesis when cells are cultured on basement membrane via activation of protein kinase C (PKC).
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MATERIALS AND METHODS |
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Cells and materials.
The gastric cancer cell line AGS was obtained from the American Type
Culture Collection. In contrast to previous reports (10), in our hands these cells did not express the gastrin-CCKB
receptor as determined by Northern blot, binding of
125I-labeled heptadecapeptide gastrin (G-17), or responses
to incubation with G-17 (up to 10 nM, 92 h). The cells
were stably transfected with the gastrin-CCKB receptor as
previously described (35). Cells were cultured in Ham's
F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1%
wt/vol penicillin/streptomycin (Life Technologies, Paisley, UK) as
described (35). G-17 was obtained from Bachem (St. Helens,
UK); the gastrin-CCKB receptor antagonist L-740093 was a
gift from Merck, Sharpe & Dohme (Rathaway, NJ). Tetramethylrhodamine B
isothiocyanate (TRITC)-conjugated phalloidin, phorbol 12-myristate
13-acetate (PMA), TGF-, lysophosphatidic acid (LPA; oleoyl), and
actinomycin D were obtained from Sigma (Poole, UK). Pertussis toxin
(PTX), PD-98059, PD-153035, AG-1478, LY-294002, wortmannin, and
Ro-32-0432 were obtained from CN Biosciences (Beeston, UK); HGF
was obtained from Genentech (San Francisco, CA). Artificial basement
membrane was obtained from either Sigma or Becton Dickinson (Bedford, UK).
Morphological studies. Cells (104/well) were plated on six-well dishes and incubated for 2 days in full medium and then serum-free medium containing G-17 (30 pM-3 nM), PMA, or other drugs. Cells extending long processes in response to stimuli were scored after 6 h as a proportion of total cells by counting duplicate fields in each of triplicate wells. In addition, cells were cultured on coverslips and F-actin was stained using TRITC-conjugated phalloidin. Images were captured from a Zeiss Axiovert 25 microscope (Carl Zeiss, Welwyn Garden City, UK) using Intellicam software (Matox Electronic Systems).
Adhesion assays. Confluent AGS cells expressing the gastrin-CCKB receptor (AGS-GR cells) were recovered in trypsin-EDTA, plated (2.5 × 105/well, 24-well plates), and incubated with or without G-17 and drugs for 30 min at 37°C. Media and nonadherent cells were then removed, cells were washed three times with PBS, and adherent cells were stained with 0.02% crystal violet. Adherent cells were then washed and solubilized with 2 mM Na2HPO4-50% ethanol, and absorbance was measured at 550 nm by using a SpectraCount plate reader (Packard BioScience, Pangbourne, UK).
Branching morphogenesis assays. Cells (5 × 104) were plated on 24-well plates that had been coated with artificial basement membrane according to the manufacturer's instructions. Cells were cultured in serum-free Ham's F-12 medium, and treatments were applied at the time of plating. In some experiments, branching morphogenesis was studied by video time-lapse microscopy. For this purpose, cells were plated on coverslips previously coated with artificial basement membrane and mounted on the heated stage of a Zeiss Axiovert 100 microscope in a humidified chamber. Images were captured at 5-min intervals with a Hamamatsu 480-80 charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and AQM-2001 software (Kinetic Imaging, Liverpool, UK). In routine assays of branching morphogenesis, cells were examined after 3 h by using a Zeiss Axiovert 25 inverted microscope and were scored by using a scale from 1 to 5 based on the following descriptors: 1, single cells or amorphous groups (<5 cells); 2, linear arrays of cells, without branches; 3, linear and branching arrays of cells; 4, the presence of one or more complete rings of cells (together with linear and branching arrays); and 5, extensive networks of cellular assemblies forming multiple-ringed structures. Experiments were conducted in triplicate wells, and five fields from each well were scored.
Statistics. Data are presented as means ± SE. Sample groups for multiple-treatment experiments were analyzed by using Kruskal-Wallis ANOVA followed by pairwise comparisons of treatments. Where two independent sample groups were compared, data analysis was performed by using a Mann-Whitney U-test. Differences were considered significant when P < 0.05.
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RESULTS |
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G-17 and PMA induce morphological changes and scattering.
Subconfluent AGS-GR cells grew in colonies with
epithelial-like morphology when cultured on plastic dishes (Fig.
1A). There was no difference
between the parental cell line (AGS) and cells expressing the
gastrin-CCKB receptor (AGS-GR). Incubation of
AGS-GR cells with G-17 induced cell scattering and the
extension of long processes, which was maximal at ~6 h
(Fig. 1B). Phalloidin staining of F-actin revealed
predominantly cortical actin in unstimulated cells (Fig.
1C). In response to G-17, phalloidin staining revealed cell
spreading and membrane ruffling within 30 min (Fig. 1D), and
by 6 h there was elaborate remodeling of the actin cytoskeleton supporting lamellipodia and the extension of long processes (Fig. 1E). Similar responses were produced by PMA (100 nM; Fig.
1F).
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G-17 and PMA increase cell adhesion.
The morphological changes in AGS-GR cells in response to
G-17 were associated with increased cell adhesion. Thus in an adhesion assay, G-17 produced a significant fourfold increase in cell adhesion. This response was inhibited by the gastrin-CCKB receptor
antagonist L-740093. Interestingly, G-17-stimulated adhesion was
completely abolished by inhibition of PI-3 kinase by LY-294002
(20-50 µM; Fig. 3) and wortmannin
(100 nM; not shown) and was reduced but not abolished by the PKC
inhibitor Ro-32-0432. PMA (100 nM) produced a small but
significant increase in cell adhesion that was also inhibited by
Ro-32-032 (1 µM), LY-294002 (20-50 µM), and wortmannin (1 µM; not shown) (Fig. 3).
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Branching morphogenesis on artificial basement membrane.
When cells were cultured on the surface of artificial basement membrane
(5 × 104 cells/well, 24-well dishes) and in
appropriate conditions (see below), they assembled into multicellular,
linear, and branching complex structures, i.e., exhibited branching
morphogenesis. Video time-lapse microscopy indicated a progression from
initially dispersed populations of single cells, which through the
extension of processes and migration led to the assembly of elaborate
multicellular assemblies (Fig. 4). During
this phase, the extension of processes appeared to provide a scaffold
along which more complex structures formed. Tracking of individual
cells by video time-lapse microscopy indicated that the assembly of
these cellular complexes was dependent almost exclusively on migration
and not on cell proliferation.
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Assembly of multicellular complexes is stimulated by G-17, serum,
PMA, and LPA.
Branching morphogenesis of AGS-GR cells was stimulated by
G-17 in concentrations from 30 to 1,000 pM when cells were cultured in
serum-free medium on artificial basement membrane. It was possible to
classify the degree of assembly into organized multicellular structures
and so score the effects of different treatments (Fig. 5). Similar effects to those of gastrin
were produced by addition to serum-free medium of FBS (Fig.
6). Moreover, PMA (100 nM) and LPA (50 µM) in serum-free medium also stimulated the formation of
multicellular assemblies (Fig. 6). There was no effect of HGF and
TGF- (not shown) on the branching morphogenesis exhibited by
AGS-GR cells.
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G-17 stimulates branching morphogenesis via the
gastrin-CCKB receptor.
We then examined the effect of gastrin on branching morphogenesis in
AGS-GR cells treated with the gastrin-CCKB
receptor antagonist L-740093. The formation of multicellular assemblies
was inhibited by L-740093 (Fig. 7). To
establish the specificity of this response, we showed that L-740093 had
no effect on LPA, PMA, or serum-induced branching morphogenesis (Fig.
7). PTX (which inhibits signaling through Gi and
Go
) had no effect on responses to either gastrin or LPA
(data not shown), consistent with signaling through the PTX-insensitive
Gq/11
pathway.
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Branching morphogenesis in AGS-GR cells is mediated by
PKC.
To determine the role of PKC in branching morphogenesis in
AGS-GR cells, we studied the effects of the PKC inhibitor
Ro-32-0432. The latter fully inhibited the effect of PMA and
substantially inhibited the effect of G-17 (Fig.
8). Interestingly,
Ro-32-0432 had no significant effect on LPA-induced
branching morphogenesis (Fig. 8). Since PI-3 kinase was downstream of
PKC for the morphological transformation of AGS cells and was important
for gastrin-stimulated cell adhesion, we examined the effect of
wortmannin and LY-294002 on gastrin-stimulated branching morphogenesis.
Neither compound influenced the effects of gastrin on these responses
(not shown). Similarly, the EGF receptor appeared unlikely to mediate
the effects of gastrin since the EGF receptor kinase inhibitors
PD-153035 and AG-1478 had no effect on responses to gastrin, and
neither did the inhibitor of MEK activation PD-98059 (not shown).
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DISCUSSION |
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The results presented here show that gastrin acts via PKC to stimulate remodeling of the actin cytoskeleton in AGS-GR cells, increased cell adhesion, and the extension of long processes. In the presence of basement membrane, the extension of processes appears to provide a scaffold for the arrangement of cells into complex multicellular assemblies consistent with branching morphogenesis. This type of response may contribute to the formation of gastric glandular cells into tubules.
In complex epithelia, proliferating and differentiated cells are frequently localized to morphologically distinct domains that are maintained by cell migration following exit from the cell cycle. In the case of the gastric epithelium, a common class of stem cell is localized in the isthmus region of the gland (13). Migration of cells toward the mucosal surface is associated with differentiation to mucus-secreting phenotypes, whereas cells migrating toward the base of the gland may become parietal, ECL, or chief cells (12). The mechanisms regulating the assembly of cells into gastric glands are poorly understood. Disruption of the processes controlling migration may, however, be a feature in gastric atrophy and in any case is likely to underlie tumor cell invasion. Moreover, the capacity for tubulogenesis may account for the organization of some gastric tumors in which cells assemble into columns or tubulelike structures. We suggest that stimulation of PKC by gastrin is one of the mechanisms that influence these processes.
Assays of branching morphogenesis similar to that used here have been widely employed to study the assembly into organized multicellular structures of endothelial cells (21) and MDCK cells (1, 15, 26). In both endothelial and MDCK cells, HGF strongly stimulates branching morphogenesis in the presence of extracellular matrix (1, 15, 26, 27). Although AGS-GR cells express the HGF receptor c-Met, activation of this receptor in these cells is not associated with branching morphogenesis. Moreover, the branching morphogenesis stimulated by HGF in MDCK cells is mediated by PI-3 kinase. In AGS-GR cells, inhibition of PI-3 kinase blocks cell adhesion and reduces gastrin-stimulated extension of processes by cells cultured on plastic, but, interestingly, this has no effect on branching morphogenesis when cells were cultured on artificial basement membrane. The signaling pathways responsible for the latter phenotype in AGS-GR cells therefore appear to be different from those in MDCK cells. Together, the data suggest that the extension of processes that characterizes the formation of organized multicellular structures on basement membrane appears to reflect the PKC-dependent, PI-3 kinase-independent pathway responsible for remodeling of the actin cytoskeleton. Further work will be needed to identify downstream targets of PKC involved in branching morphogenesis in AGS-GR cells.
Previous studies have shown that gastrin stimulates pathways involving PKC, activation of MAP kinase, and activation of PI-3 kinase (5, 6, 30, 31). For the most part, the activation of these pathways has been linked to control of proliferation and apoptosis. There have been few direct studies of the way that gastrin might regulate events leading to migration or the formation of complex assemblies of cells. We did not find evidence for an involvement of the MAP kinase pathway in these events. The present data do, however, suggest differences in the relative importance of PKC and PI-3 kinase in mediating the remodeling of the actin cytoskeleton and in control of the cell-cell interactions required for adhesion, the extension of processes, and branching morphogenesis. The data imply that PI-3 kinase might in some circumstances be activated by PKC in AGS-GR cells, but this is unlikely to account for all of the present data, and further work on the relationship between, and the activation of, the relevant signaling pathways is needed.
The gastrin precursor progastrin yields the amidated gastrin via intermediates with a COOH-terminal glycine residue (the Gly-gastrins) (7). The latter peptides have low affinity for gastrin-CCKB receptors. Interestingly, however, recent reports suggest that Gly-gastrins may regulate epithelial migration (8) and expression of matrix metalloproteinases MMP-2 and -9 (14). If reproduced in vivo, these effects might be expected to influence epithelial organization, although their relationship to branching morphogenesis induced by stimulation of the gastrin-CCKB receptor remains unclear. Normally, the gastrin-CCKB receptor is expressed by parietal and ECL cells (7). It is, however, worth noting that in prolonged hypergastrinemia the receptor appears to be expressed by mucus neck cells (22), and after damage to the mucosa it is also expressed by surface epithelial cells (29). The role of this receptor in regulating branching morphogenesis may therefore include both the maintenance of normal epithelial organization and adaptive responses to damage.
Previous studies of the mechanisms of branching morphogenesis in other gastric cell lines have identified heregulin (3), cyclooxygenase-2 (28), TFF-2 (17), and MUC-1 (9) as potential regulatory agents. Gastrin is the first neuroendocrine peptide acting through G protein-coupled receptors to be shown to be capable of stimulating branching morphogenesis. Since observations in genetically modified mice suggest that gastrin also regulates gastric mucosal morphology in vivo (34), we suggest that gastrin may be one factor controlling the capacity of gastric epithelial cells to organize into glandular structures. Gastrin regulates the maturation of some gastric epithelial cells (7); it will now be interesting to determine the extent to which the stimulation of tubulogenesis by gastrin is linked to the differentiation of gastric epithelial cells. Either way, it is reasonable to assume that other growth and morphogenic factors are also involved in maintaining the organization of gastric glands, and elucidation of the interactions and relative importance of these is now required.
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ACKNOWLEDGEMENTS |
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We thank Geraint Wilde for help with video time-lapse microscopy and Barry J. Campbell for statistical advice.
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FOOTNOTES |
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This work was supported by grants from the Medical Research Council and the Wellcome Trust.
Address for reprint requests and other correspondence: A. Varro, Physiological Laboratory, Univ. of Liverpool, Crown St., P. O. Box 147, Liverpool L69 3BX, UK (E-mail: avarro{at}liverpool.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 28, 2002;10.1152/ajpgi.00056.2002
Received 8 February 2002; accepted in final form 18 March 2002.
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