Laminins and TGF-
maintain cell polarity and functionality
of human gastric glandular epithelium
Jean-René
Basque,
Pierre
Chailler, and
Daniel
Ménard
Canadian Institute of Health Research Group on the
Functional Development and Physiopathology of the Digestive Tract,
Department of Anatomy and Cell Biology, Faculty of Medicine,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
J1H 5N4
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ABSTRACT |
The human gastric glandular
epithelium produces a gastric lipase enzyme (HGL) that plays an
important role in digestion of dietary triglycerides. To assess the
involvement of extracellular matrix components and transforming growth
factor-
1 (TGF-
1) in the regulation of this enzymic function,
normal gastric epithelial cells were cultured on collagen type I,
Matrigel, and laminins (LN)-1 and -2 with or without TGF-
1.
Epithelial morphology and HGL expression were evaluated using
microscopy techniques, enzymic assays, Western blot, Northern
hybridization, and RT-PCR. A correlation was observed between the cell
polarity status and the level of HGL expression. TGF-
1 alone or
individual matrix components stimulated cell spreading and caused a
downfall of HGL activity and mRNA. By contrast, Matrigel preserved the
morphological features of differentiated epithelial cells and
maintained HGL expression. The combination of LNs with TGF-
1 (two
constituents of Matrigel) exerted similar beneficial effects on
epithelial cell polarity and evoked a 10-fold increase of HGL levels
that was blunted by a neutralizing antibody against the
2-integrin subunit and by mitogen-activated protein
kinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). This
investigation demonstrates for the first time that a powerful synergism
between a growth factor and basement membrane LNs positively influences
cell polarity and functionality of the human gastric glandular
epithelium through an activation of the
2
1-integrin and effectors of two MAPK pathways.
human stomach; extracellular matrix; integrin; mitogen-activated
protein kinase
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INTRODUCTION |
REGULATION AND
MAINTENANCE of epithelial differentiation at the level of the gut
mucosa is governed by extracellular signals present in the cell
microenvironment. These are represented not only by growth factors but
also by cell-to-cell interactions and their underlying extracellular
matrix (ECM) (9, 32, 35). For epithelial cells, part of
the ECM occurs in the form of a basement membrane that provides
positional information and cues for cell polarity as well as signals
that regulate cell behavior. Laminins (LNs) are among the major
constituents of basement membranes, either native or reconstituted
(Matrigel) (43), and they promote cellular adhesion,
migration, proliferation, and expression of tissue-specific genes in
differentiating epithelial cells and other tissues (13, 20, 33,
49). Furthermore, a number of growth factors including
transforming growth factor-
1 (TGF-
1) are associated with basement
membranes (50). The latter peptide regulates cell-matrix
interactions (40), which implies that TGF-
1 may
function as a regulator of epithelial morphogenesis and
expression of tissue-specific proteins (21, 25, 39). In
the context of gastric epithelial physiology, however, the combinatory
effects of ECM components and TGF-
1 have never been specifically studied.
An original and important aspect of digestive functions attributed to
the human gastric glandular epithelium pertains to hydrolysis of fat,
which is assumed by a lipase enzyme produced by zymogenic chief cells
(14, 30). We have shown that human gastric lipase (HGL) is
expressed early during ontogeny (before midgestation) (31)
and is uniquely colocalized with fundic-type pepsinogen (Pg5) in fetal
chief cells (6). Our studies also revealed different regulatory patterns for HGL and Pg5 during in utero development and in
culture experiments. Therefore, it appears essential to clearly
understand the molecular mechanisms controlling the acquisition and
maintenance of chief cell functions when one considers the physiological importance of gastric lipolysis and the fact that its
role increases in the context of perinatal physiology (23) and pathological conditions (1) associated with pancreatic immaturity or insufficiency. For this purpose, adequate cellular models
must be made available, and a promising tool for identifying the
specific regulators of gastric digestive functions and their underlying
mechanisms of action is a novel primary culture model representative of
the normal human gastric epithelium (5). These cultures
form coherent monolayers in the absence of a biological matrix,
mesenchymal constituents, and hormones. They contain all epithelial
cell types, including functional glandular chief cells that retain
their ability to produce digestive enzymes. Under such minimal
conditions, however, the epithelial phenotype is altered, and the
expression of HGL is spontaneously downregulated (5). Our
new model thus appears unique to determine the appropriate extracellular environment necessary for such complex regulatory events
as the maintenance of epithelial cell polarity and the induction of
gastric zymogen expression.
On a functional basis, growth and differentiation of gut epithelial
cells likely depend on their interaction with ECM components using
cell-surface receptors of the integrin family (9, 10). The
2
1-integrin, which is unique among these
receptors for having a dual specificity for collagen and LNs, has been
shown to play a key role in mammary epithelial morphogenesis in vitro
(12, 27). We recently analyzed the distribution of ECM
proteins and receptors in developing human stomach (16,
46) and provided evidence for the potential involvement of LN-1
(or the closely related LN-10), LN-2, and
2
1-integrin in epithelial glandular differentiation. In the current investigation, we have demonstrated that collagen type I and Matrigel, respectively, exert negative and
positive effects on cell polarity and HGL expression in primary cultures of gastric epithelium. In fact, the maintenance of epithelial morphology and functionality of cultured cells is mediated by a
powerful synergism between LNs and TGF-
1 that involves signaling through the
2
1-integrin and activation of
both p42/p44 and p38 mitogen-activated protein kinase (MAPK) pathways.
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MATERIALS AND METHODS |
Specimens.
Tissues from 47 fetuses varying in age from 17 to 20 wk of gestation
postfertilization were obtained from normal elective pregnancy
terminations (41). No tissues were collected from cases
associated with known fetal abnormality or fetal death. Studies were
approved by the Institutional Human Subject Review Board. The stomach
was brought to the culture room, immersed in dissection medium, i.e.,
Leibovitz L-15 (GIBCO BRL/Life Technologies, Burlington, ON, Canada)
plus gentamicin and nystatin (40 µg/ml each), and prepared within 30 min at room temperature.
Primary culture.
Cardia and pyloric antrum were excised from the stomach, leaving the
body and fundus regions. Tissues were then cut into explants (3 × 3 mm2) and rinsed with dissection medium. The gastric
epithelium was isolated as described previously (5) by
using a nonenzymatic procedure originally designed for primary cultures
of intestinal epithelium (34). Explants were immersed in
Matrisperse (Collaborative Biomedicals/Becton Dickinson Labware,
Bedford, MA) for 16-20 h at 4°C and gently agitated during the
last hour, allowing dissociation of the epithelium as intact sheets or
large aggregates. After centrifugation (100 g, 7 min), the
pellet was resuspended in culture medium, a 1:1 mixture of Dulbecco's
modified essential medium (DMEM) and Ham's F-12 (GIBCO) supplemented
with penicillin/streptomycin and 10% fetal bovine serum (vol/vol,
Cellect Gold FBS; ICN Pharmaceuticals, Montreal, QC, Canada). This
material was fragmented mechanically into small aggregates and seeded
at high density in six-well (5 × 104 cells in 2.5 ml)
or 24-well plates (1.5 × 104 cells in 1 ml) precoated
with different biological matrices: collagen type I (20 µg/ml;
Collaborative Biomedicals), purified LN-1 (10 µg/ml; GIBCO), purified
LN-2 (or Merosin, 10 µg/ml; GIBCO), LN-1/LN-2 mixture (1:1, 10 µg/ml), and Matrigel (3.4 mg/ml; Collaborative Biomedicals). The
effect of TGF-
1 (R&D Systems, Minneapolis, MN) was tested at the 5 ng/ml concentration (in the presence of 0.1% BSA), either without
matrix coating or with LNs. The synthetic agents PD-98059
(2'-amino-3'-methoxyflavone) and SB-203580
[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; Calbiochem, La Jolla, CA], which respectively inhibit MAPK kinase (MKK)-1/2 (activators of p42/p44MAPK) (19) and
p38
- and
-isoforms (28) were added at the 20 µM
concentration in some primary cultures to assess the contribution of
these effectors in the regulatory process. In all cases, cell preparations were left undisturbed for 6 h to allow attachment. Culture medium was discarded and renewed every 48 h. In other experiments, neutralizing antibodies directed against
2-
and
3-integrin subunits (clones P1E6 and P1B5,
respectively; GIBCO) were added after initial attachment (6 h) at the
10 µg/ml concentration.
Microscopy and immunocytochemistry.
The epithelial morphology of primary cultures was examined after
selected time intervals by using bright-field and phase-contrast microscopy. For indirect immunofluorescence, epithelial preparations were seeded on glass coverslips precoated with matrices, and antibody staining was performed as described in our reference study
(5). Briefly, specimens were fixed for 12 min in 3%
formaldehyde or 5 min in acetone:chloroform (1:1) at 4°C and then
permeabilized with 0.1% Triton X-100 for 3 min and incubated with
primary antibodies directed against
2-integrin subunit
(1:400, clone P1E6), E-cadherin (1:800; Transduction Laboratories,
Lexington, CT), and HGL (1:4,000). Fluorescein
isothiocyanate-conjugated goat anti-mouse or goat anti-rabbit
immunoglobulin G (Boehringer Mannheim, Laval, QC, Canada) were used as
secondary antibodies (1:30 and 1:40, respectively) and were added for
45 min. Specimens mounted in glycerol:PBS (9:1) were observed with a
Reichert Polyvar 2 microscope (Leica Canada, St-Laurent, QC, Canada)
equipped for epifluorescence. All appropriate controls where the
primary antibody was omitted were performed and shown negative, as
exemplified in Fig. 7. For ultrastructural studies, specimens were
prefixed for 15 min at room temperature in an equal volume of culture
medium and fresh 2.8% glutaraldehyde diluted in 0.2 M cacodylate
buffer containing 7.5% ultrapure sucrose, fixed for 30 min in 2.8%
glutaraldehyde-7.5% sucrose diluted in 0.1 M cacodylate, and postfixed
in 2% osmium tetroxide diluted in 0.1 M cacodylate for 1 h. They
were dehydrated and covered with a 3-mm layer of Epon 812 resin. After
polymerization (48 h at 60°C), the plastic substratum was detached
and specimens were inverted and reembedded. Thin sections were
visualized on a JEM-100CX electron microscope (JEOL, Peabody, MA).
Gel electrophoresis and immunoblotting.
Total protein samples from three independent experiments were lysed in
2× buffer containing 2%
-mercaptoethanol in 20 mmol Tris · HCl (pH 6.8). As standardized earlier for gastric
primary cultures (5), 60- to 80-µg aliquots were
resolved by SDS-PAGE on 12% acrylamide gels, transferred onto
nitrocellulose membranes, and processed with a Western-Light Plus
chemiluminescence detection system (Tropix, Bedford, MA). After initial
quenching in blocking buffer (1% Blotto-PBS), the following specific
primary antibodies were added: anti-E-cadherin (1:1,000),
anti-
2-integrin subunit (1:400), and anti-cytokeratin-18
(1:5,000, clone CY-90; Sigma-Aldrich, Oakville, ON, Canada). After
being washed in PBS, membranes were incubated with biotinylated goat
anti-rabbit or anti-mouse immunoglobulin G (1:10,000) and with alkaline
phosphatase-conjugated streptavidin (1:20,000). Finally, immunoreactive
proteins were revealed with a solution of ultrapure CSPD (Tropix)
containing Nitro-Block (1:20). Autoradiographs were exposed in a linear
range by using a LKB XL Ultroscan (Pharmacia, Baie d'Urfé, QC, Canada).
Northern hybridization and reverse transcription-polymerase chain
reaction.
Total RNA was isolated from three separate primary cultures by
using TriPure reagent (Boehringer Mannheim) and processed as formerly
described (5, 44). For Northern blot analysis,
equivalent amounts of RNA (20 µg) were separated by electrophoresis
through 1% agarose gels containing 6% formaldehyde and blotted onto
nylon membranes (Hybond-N; Amersham, Oakville, ON, Canada). Equal
loading was confirmed by ethidium bromide staining. Membranes were
hybridized with cDNA probes specific for HGL and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5, 44),
blotted dry, and autoradiographed.
The reverse transcription (RT) reaction was carried out at 37°C
by using Ready-To-Go First-Strand Beads (Amersham) to which was added 1 µg of total RNA mixed with oligo(dT)12-18 and SuperScript RT RNase H
reverse transcriptase. The cDNA
samples prepared were directly used for polymerase chain reaction (PCR)
amplification. PCR primers complementary to the reported cDNA sequences
of HGL and GAPDH were used: 100 pmol of both upstream sense primers
LIPASE-1, 5'-CTGAGGAAACTGCAGGTCCA-3', and GAPDH-1,
5'-CCACCCATGGCAAATTCCATGGCA-3', as well as 100 pmol of downstream
antisense primers LIPASE-2, 5'-AGAAGCACTGCAATGT-3', and GAPDH-2,
5'-TCTAGACGGCAGGTCAGGTCCACC-3', all in the presence of 2.5 units of
Taq polymerase (Roche Molecular Biochemicals, Laval, QC,
Canada). The cDNA was subjected to 25 cycles of denaturation (1 min at
94°C), annealing (1 min at 52-63°C), and primer extension (1 min at 72°C) in a DNA thermal cycler (model 480; Perkin Elmer Applied
Biosystems, Mississauga, ON, Canada). Amplification yielded a
1,311-base pair (bp) fragment for HGL and a 612-bp fragment for GAPDH.
Enzymic assays: lipase and pepsin.
Lipolytic activity was measured by using a long-chain triglyceride
substrate (tri[1-14C]oleic acid; Amersham) and fatty
acid-free BSA (A-6003; Sigma-Aldrich) as carriers of released fatty
acids, and the emulsion was prepared by sonication as described
previously (31). The assay system contained the following
reagents in a final volume of 200 µl: 1.2 µmol of labeled
triglyceride, 10 µmol of citrate-phosphate buffer (pH 6.0), 0.1 µmol of BSA, 2 µmol of Triton X-100, and 100 µl of cell
homogenate. Free [14C]oleic acid produced after a 1-h
incubation was separated by liquid-liquid partition in
chloroform-methanol-heptane (1.41:1.25:1), precipitated with 0.1 M
carbonate-borate buffer (pH 10.5), and quantitated by liquid
scintillation spectrometry (11). Specific lipase activity
was expressed as nanomoles of free fatty acid released per minute per
milligram of protein. Pepsin activity resulting from the activation of
pepsinogen at acid pH was measured in 100 µl of cell homogenate by
adding 1 ml of 2% (wt/vol) dialyzed hemoglobin (Sigma-Aldrich) diluted
in 0.1 M glycine-HCl buffer (pH 3.0). The reaction was carried out at
37°C for 10 min and stopped with 6.2% (wt/vol) trichloroacetic acid.
The resulting free amino acid products were separated by centrifugation
and quantitated by spectrometry using an L-tyrosine
standard. Specific pepsin activity was expressed in international units
(µmol/min) per milligram of protein. Protein content of the
homogenates was measured using Folin reagent. Results are reported as
means ± SE, and the statistical significance of differences in
enzyme activity between culture intervals or treatments was established at 95% and determined by analysis of variance followed by Student's t-test when significance was indicated (number of
experiments is specified in legends of Figs. 1-8).

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Fig. 1.
Optical microscopy of gastric epithelial primary
cultures. By 48 h postplating, cell colonies cultured on collagen
type I had spread intensely (A). In the presence of
Matrigel, compact multicellular clusters (arrows) were visible at the
center of epithelial colonies (B and C). When
seeded on purified laminin (LN)-1 (D), LN-2, or transforming
growth factor (TGF)- 1 alone (E), aggregates spread and
eventually generated monolayers of flattened cells, as shown with
collagen type I. Combination of LNs (LN1/LN2) with TGF- 1
(F) caused the formation of dense multicellular structures
(arrow) similar to those noted on Matrigel. Bar, 50 µm (phase
microscopy, except B under bright field).
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Fig. 2.
Transmission electron microscopy of Epon-embedded gastric
primary cultures after 48 h. On collagen type I (A),
cells grew as coherent flat monolayers displaying sparse microvilli on
their apical membrane (arrows). Matrigel maintained epithelial colonies
as compact organoids where secretory cells were sometimes facing a
central lumen (B). Individual cells displayed a polarized
phenotype and abundant granules in their supranuclear cytoplasm
(C). Gastric cells cultured on LN-1, LN-2 (D), or
TGF- 1 alone (E) exhibited a depolarized appearance and
few electron-luscent granules (asterisks). In the combined presence of
TGF- 1 and LNs (LN1/LN2 mixture is shown) (F), epithelial
cells in multicellular aggregates were characterized by intercellular
junctions (open arrowheads), apical microvilli, basal nuclei, and
secretory granules. Bar, 1 µm.
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Fig. 3.
Effects of collagen type I, LNs (LN-1/LN-2 mixture), and TGF- 1
(5 ng/ml; plus 0.1% BSA) on the expression of human gastric lipase
(HGL) and epithelial junctions. A: specific HGL activity was
measured in cells at 1.5, 3, and 5 days of culture. Coll-I,
collagen type I; d, days. Values represent means ± SE of 3 separate and independent experiments. For each condition, activity
significantly decreased between culture intervals. * Significant
difference between conditions on a specific day (P < 0.05). B and D: representative Northern blot
analyses at 1.5, 3, and 5 days show the effect of collagen type I
(B) and LNs (D) on HGL mRNA compared with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA control. Total
RNA was prepared as described previously (5).
C: Western blot analysis of E-cadherin protein compared with
cytokeratin-18 (K18) control in cells cultured on collagen type I. E-cad, E-cadherin. E: indirect immunofluorescence of
E-cadherin after 5 days of culture with LNs. Arrowheads indicate a
cytoplasmic and diffuse staining. Bar, 50 µm.
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Fig. 4.
Effects of Matrigel on the expression of HGL and
epithelial junctions. A: specific HGL activity measured in
cells at 1.5, 3, and 5 days of culture. Values represent means ± SE of 4 separate and independent experiments. B:
representative Northern blot analysis at 1.5, 3, and 5 days showing HGL
and GAPDH mRNAs. C: Western blot analysis of E-cadherin.
D and E: immunodetection of E-cadherin in
multicellular polarized clusters after 1.5 (D) and 5 days of
culture (E). Bar, 50 µm.
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Fig. 5.
Combinatory effects of LNs (LN-1/LN-2 mixture) and
TGF- 1 on HGL expression and epithelial junctions. A:
specific HGL activity was measured in cells at 1.5, 3, and 5 days and
was found to increase significantly between culture intervals
(P < 0.05). Values represent means ± SE of 5 separate and independent experiments. B: representative
Northern blot analysis at 1.5, 3, and 5 days showing HGL and GAPDH
mRNAs. C: Western blot analysis of E-cadherin. D
and E: immunodetection of E-cadherin in multicellular
polarized clusters after 1.5 (D) and 5 days of culture
(E). Bar, 50 µm.
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Fig. 6.
Combinatory effects of LNs (LN-1/LN-2 mixture)
and TGF- 1 on HGL immunoreactivity and epithelial morphology. With
the use of an anti-HGL antibody, chief cells inside dense and compact
colonies showed a strong immunoreactivity after 1.5 days of culture
(A). Staining was most intense after 5 days of culture
(B). Ultrastructural analyses revealed numerous
secretory vesicles (C) as well as junctional complexes at
apical margins between cells (D) comprising zonula occludens
(ZO), zonula adherens (ZA), and macula adherens (MA). Bars, 50 µm
(A and B); 0.5 µm (C and
D).
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Fig. 7.
Role of 2-integrin subunit in the maintenance of HGL
expression. In 3 different cultures seeded on LN-1/LN-2 with TGF- 1,
immunodetection with the anti- 2 P1E6 antibody revealed
staining at cell-cell boundaries after 1.5 (A) and 5 days
(B). Controls for which the primary antibody was omitted
showed no significant staining (C). D:
representative Western blot analysis at 1.5, 3, and 5 days showing
2-subunit protein and K18 control. E: 2 representative RT-PCR for HGL mRNA in cells cultured 48 h on
LNs/TGF- 1 in the presence of neutralizing antibodies:
anti- 2-integrin (+ 2; inhibitory effect),
anti- 3-integrin (+ 3; no effect), and a nonrelevant
anti-K18 immunoglobulin (+IgG; no effect). GAPDH mRNA probe was used as
an internal control. Bar, 50 µm.
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Fig. 8.
Role of mitogen-activated protein kinase (MAPK)
pathways in induction of HGL expression. A: specific HGL
activity measured in cells after 48 h of culture in the presence
of selective inhibitors of p42/44 [PD-98059 (PD), 20 µM] and
p38 / [SB-203580 (SB), 20 µM] MAPK pathways. Values represent
means ± SE of 3 independent experiments. * Significant
decrease vs. LN/TGF- 1 (P < 0.05). B:
RT-PCR for HGL mRNA and GAPDH control in 3 cultures maintained 48 h on LNs/TGF- 1 with PD and/or SB. C: Western blot
analysis of 2-integrin subunit protein and K18 control
shows no alteration in the presence of inhibitors.
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RESULTS |
Epithelial cell morphology.
Freshly isolated gastric epithelium was resuspended in culture fluid
and then fragmented mechanically into multicellular aggregates, and
this material was plated in dishes precoated with collagen type I or
Matrigel. Primary cultures seeded on collagen type I attached and
spread rapidly, as visualized after 48 h of culture (Fig.
1A). Between this interval and
day 3, they had already formed a confluent monolayer. In the
presence of Matrigel, epithelial suspensions also attached rapidly but,
rather, formed compact multicellular structures or organoids (Fig. 1,
B and C). We next sought to identify the specific
constituents of Matrigel matrix responsible for this striking
morphological feature. When incubated with purified LN-1, LN-2,
LN-1/LN-2 mixture, or 5 ng/ml TGF-
1, epithelial cells attached and
spread rapidly (Fig. 1, D and E), recalling their
behavior on collagen type I. Interestingly, the use of LN-1 and/or LN-2
in combination with TGF-
1 allowed the formation of compact
epithelial cell colonies after 48 h of culture (Fig.
1F). LNs and TGF-
1 thus synergized in the present system to reproduce the beneficial effect of Matrigel.
When viewed by transmission electron microscopy after 48 h,
gastric epithelial cells exhibited a different morphology depending on
the biological matrix used as substratum. Cells cultured on collagen
type I were generally characterized by a relatively less polarized
phenotype (Fig. 2A). They grew
as flat monolayers displaying small and sparse microvilli on their
apical surfaces, and few secretory granules were observed. On the
contrary, addition of Matrigel resulted in the presence of
three-dimensional clusters lined by well-polarized epithelial cells
(Fig. 2, B and C). Their integrity and polarity
were indicated by the adequate intracellular localization of basal
nuclei, well-organized junctional complexes at the apical margins of
cells, and abundant secretory organelles. However, gastric epithelial
cells cultured with purified LN-1, LN-2, or TGF-
1 alone always
exhibited a less polarized appearance (Fig. 2, D and
E), as observed with collagen type I. The combination of
TGF-
1 with LN-1, LN-2, or LN-1/LN-2 mixture (Fig. 2F)
exerted the same beneficial effects as Matrigel on the maintenance of differentiated phenotypes and cell polarity in epithelial cells. Cultured cells displayed a basal nucleus as well as specialized junctions, numerous microvilli, and secretory granules in their apical regions.
Gastric lipase expression in zymogenic chief cells.
To understand the molecular mechanisms involved in the maintenance of
human chief cell functional differentiation, we next verified how HGL
expression would be modulated in the same culture conditions. First,
the use of collagen type I coating induced a very significant decrease
of HGL activity (Fig. 3A), and
this effect correlated with a drastic reduction of HGL mRNA signals during the incubation period (Fig. 3B). The level of
immunoreactive E-cadherin protein also diminished markedly between 1.5 and 5 days of culture (Fig. 3C). By contrast, Matrigel
prevented this downregulation, because cellular lipase activity (Fig.
4A) and HGL mRNA levels (Fig.
4B) remained constant during culture. Of note, HGL activity
levels measured after 1.5 days in the presence of Matrigel were
fourfold higher than those reported on collagen type I. Matrigel also
maintained the expression of E-cadherin protein (Fig. 4C)
and its adequate distribution at sites of cell-to-cell contact in
multicellular polarized clusters after 1.5 (Fig. 4D) and 5 days of culture (Fig. 4E).
To delineate the contribution of individual Matrigel components
affecting HGL gene expression in our primary culture model, we
investigated the potential implication of LN-1, LN-2, and TGF-
1. Seeding on both purified LNs resulted in a decrease of HGL activity and
mRNA levels during culture (Fig. 3, A and D).
Compared with collagen type I however, HGL activity was significantly
higher at each time interval, and the expression of E-cadherin protein remained constant (not shown), although its subcellular localization was altered. After 5 days, the immunoreactive staining appeared cytoplasmic and diffuse in some cultured cells, indicating that E-cadherin was dissociated from intercellular contacts (Fig.
3E). Of note, each purified LN used separately gave
similar results (not shown). TGF-
1 alone did not prevent the
downfall of HGL cellular activity (Fig. 3A) and mRNA levels
during the same culture interval. Most importantly, the combination of
TGF-
1 with LN-1, LN-2, or LN-1/LN-2 (Fig.
5) induced a progressive increase of HGL
activity (Fig. 5A) and mRNA levels (Fig. 5B);
compared with Matrigel and collagen type I, lipase activity was
significantly upregulated 2.4- and 10-fold after 1.5 days. LNs/TGF-
1
also maintained constant E-cadherin protein levels (Fig. 5C)
and preserved its adequate localization at cell-cell boundaries in
epithelial clusters after 1.5 (Fig. 5D) and 5 days of
culture (Fig. 5E). Chief cells present in these
compact and well-polarized structures showed a granule-like
distribution of immunoreactive HGL at 1.5 days postplating (Fig.
6A), and
staining was most intense after 5 days (Fig. 6B).
Ultrastructural analyses also revealed highly polarized secretory cells
reminiscent of those found in gastric glands. A significant proportion
contained supranuclear secretory vesicles with a heterogenous core
(Fig. 6C) characteristic of the zymogen granules found in
fetal chief cells (6). Junctional complexes (Fig.
6D) were visualized at apical margins between cells as
indicated by the presence of zonula occludens (tight junction), zonula
adherens (adherens junction), and macula adherens (desmosome).
Finally, it is noteworthy that pepsin activity resulting from the
activation of cellular pepsinogen (Pg5) was not up- or downmodulated
with biological matrices, as opposed to HGL, except for a slight
stimulation with Matrigel at the end of the incubation period (compared
with all other conditions; see Table
1).
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Table 1.
Specific pepsin activity measured in primary gastric epithelial
cultures: effect of biological matrices and MAPK inhibitors
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Involvement of
2-integrin in the maintenance of HGL
expression.
The recent observation that
2
1-integrin
expression may be relevant to the differentiation and/or maintenance of
human glandular chief cells (16) prompted us to verify its
presence in gastric primary cultures seeded on LNs with TGF-
1. Using
the P1E6 monoclonal antibody that reacts with nonoverlapping epitopes
on
2-subunit, we observed immunoreactivity at cell-cell
boundaries during the initiation of culture (Fig.
7A). After 5 days postplating,
the
2-subunit was properly localized at the periphery of
cells (Fig. 7B), and Western blotting experiments confirmed
the continuous expression of the
2-subunit from 1.5 days
onward (Fig. 7D). To assess the possible role of
2
1-integrin in the upregulation of HGL
expression, we performed neutralization assays with the same antibody.
After initial attachment (6 h) of epithelial cells on LNs/TGF-
1,
addition of 10 µg/ml anti-
2 for 48 h produced a
significant reduction of HGL mRNA levels in culture, as revealed by
RT-PCR experiments (Fig. 7E, left). In another
set of experiments, addition of a neutralizing antibody against the
3-integrin subunit, poorly expressed in gastric
glandular cells (16) as well as the addition of a
nonrelevant anti-keratin-18 immunoglobulin did not modulate HGL (Fig.
7E, right), thus confirming the specificity of
response to anti-
2 antibody.
Implication of MAPK cascades in HGL expression.
To determine whether intracellular MAPK cascades associated with growth
factor and ECM signaling pathways take part in the LNs/TGF-
1-induced
regulation of HGL expression in chief cells, we tested selective
inhibitors of the MKK-1/2
p42/p44 pathway (PD- 98059) and
p38
/
(SB-203580). Compared with gastric primary cultures showing
maximal levels of HGL activity with LNs/TGF-
1, addition of PD-98059
or SB-203580 caused a significant decrease (Fig.
8A) without affecting pepsin
activity (Table 1). RT-PCR experiments also were performed after
48 h of culture, and HGL mRNA signals were analyzed by
semiquantitative densitometry (Fig. 8B). As expected, each
compound induced a strong downregulation of HGL mRNA levels (~50%
with PD-98059 and ~80% with SB-203580 vs. LNs/TGF-
1 without
inhibitors). More interestingly, the simultaneous presence of both
inhibitors (PD-98059 + SB-203580) completely abolished mRNA
expression, thus suggesting that the synergistic response to
LNs/TGF-
1 involves a parallel stimulation of p42/p44 and p38 MAPK
pathways. Their effects seem to be mediated directly through MAPK
function, because PD-98059 and SB-203580 did not modulate the
expression of
2-integrin subunit protein (Fig.
8C) and because we observed no significant alteration of the
cell phenotype during the 48-h incubation period (data not shown).
 |
DISCUSSION |
In recent years, an understanding of the intrinsic mechanisms by
which mucosal growth factors and ECM cooperatively regulate gut
epithelial functions has emerged. One current hypothesis is that
paracrine and autocrine growth factors mainly contribute to the
stimulation of cell proliferation and migration, often leading to
expansion of the mucigenic cell lineage, while repressing the terminal
differentiation of digestive cells (32, 35). We have
demonstrated that members of the epidermal growth factor (EGF) family
stimulate the proliferation of epithelial precursors and the synthesis
of mucus and that they downregulate the expression of HGL in maturing
chief cells (44, 47), supporting the suggestion that this
concept would apply to the human stomach. According to another recent
hypothesis, the spatiotemporal expression of ECM components, and
basement membrane proteins in particular, may play an inductive or
permissive role during the morphogenesis of digestive functional units
and their maintenance in the mature gastrointestinal tract (9,
10).
The present study was conducted to investigate the contribution of ECM
on the maintenance of epithelial morphology and digestive functions in
isolated human gastric cells. To decipher the cellular and molecular
mechanisms involved, we developed a new primary culture model that is
free of mesenchymal/submucosal influences and that is representative of
the intact fetal gastric epithelium (5, 6). The model
allows the growth and maintenance of all gastric epithelial cell types,
including functional glandular chief cells, in the absence of added
biological matrix. No signs of cytoplasmic vacuolization and cell
detachment are visible during the first week of culture. Under these
minimal conditions, however, attachment to the culture substratum is
effective after 1 day, the polarity status of epithelial cells is
modified, and the expression of HGL in chief cells is spontaneously
downregulated (5). Logically, one could suggest that these
phenomena could be attributed to the lack of ECM constituents, as
reported for other epithelial cell types (4, 13).
Our experiments clearly demonstrate that collagen type I allowed the
rapid attachment of epithelial aggregates that spread intensely and
lost their polarized phenotype, while the expression of E-cadherin
protein was downregulated. These effects are consistent with earlier
observations made on cultures of intestinal epithelial cells where this
interstitial ECM component optimizes cell adhesion and migratory
behavior (7, 8). Furthermore, the expression of HGL in
gastric cells was drastically repressed under these conditions. When
Matrigel was used as coating, cell spreading was less stimulated and
morphological parameters were all ameliorated. Epithelial aggregates
adhered and conserved a compact organization in which individual cells
retained the functional characteristics of a polarized gastric
epithelium, as verified at the ultrastructural level and by the
maintenance of E-cadherin in epithelial junctions. Interestingly, this
reconstituted basement membrane exerted a beneficial effect on HGL gene
expression as lipase activity and HGL mRNA levels remained constant.
Such observations are in accordance with those reported for intestinal
epithelial cells (15, 22, 36) and other cell types
(2, 4, 37), illustrating that Matrigel is particularly
effective as a culture substratum for maintaining cell polarity or even
eliciting a spectrum of phenotypic changes toward full differentiation.
Also of note, pepsin enzymic activity remained constant in the presence
of collagen type I and only slightly increased with Matrigel after 5 days, as opposed to HGL. This finding further reinforces the concept
that gastric zymogens are differently regulated in vivo
(31) and in vitro (5, 6, 47).
A logical follow-up to these experiments was to investigate the
implication of LNs and TGF-
1 in the Matrigel-induced response, because it has been shown that they are major constituents of this
reconstituted basement membrane (42, 50). Although the regulatory potential of LN-1 on the expression of brush-border enzymes
in intestinal cells has been verified by a number of investigators (8, 22, 36, 48), we compared herein the effects of two LN
variants on the basis of the differential distribution of LN-1/LN-10 (ubiquitous) and LN-2 (base of gland) along the gastric foveolus-gland axis (16, 46). Also, the TGF-
1 peptide exerts a
biological activity different from other growth factors, because it
generally acts as an antimitogen on cultured epithelial cells and
sometimes promotes their morphological and functional differentiation
(40). Our data indicate that gastric epithelial cells
seeded on LN-1, LN-2, or LN-1/LN-2 mixture or in the presence of
TGF-
1 alone exhibited higher HGL activity compared with collagen
type I, which downregulated HGL more drastically after 5 days. This
variation may be related to the differential regulation of E-cadherin,
because only collagen type I strongly repressed this junctional marker during the first days of incubation. However, cells maintained with LNs
or TGF-
1 formed a flattened monolayer, and a progressive and
significant downregulation of HGL still occurred. In the same cultures,
signs of E-cadherin dissociation from cell-cell contacts were observed
in the long term. In comparison, when LNs and TGF-
1 were added
simultaneously, the primary cultures were composed of isolated and
compact organoid structures and the highly polarized phenotype of
epithelial cells was preserved. These conditions also maintained a
proper distribution of E-cadherin at cell-cell boundaries and the
presence of a functional secretory apparatus in chief cells.
LNs/TGF-
1 even upregulated HGL levels compared with Matrigel,
possibly reflecting the purified nature of the ligands. We also noted
that each LN form as well as the LN-1/LN-2 mixture exerted similar
effects, suggesting that any possible difference in their secondary or
tertiary structure has no significant repercussion on LN bioactivity in
our system, at least on cell morphology and HGL. In summary, the last
results indicate that gastric primary cultures necessarily require both
LN and TGF-
1 supplements for expressing their optimal functional
characteristics, because only this combination is able to maintain
epithelial cell polarity and induce HGL expression. Moreover, such
findings suggest that the regulation of gastric digestive functions
would be coordinated by a delicate balance of inducer and repressor
factors. In our basic reference describing the primary culture
methodology (5), we reported that EGF negatively regulates
HGL mRNA and activity without affecting pepsin, as previously
demonstrated in gastric mucosa explants for EGF itself and other
mitogens like TGF-
, insulin-like growth factor (IGF)-I, and IGF-II
(44, 45).
ECM components such as basement membrane LNs modulate gut epithelial
cell functions by interacting with integrin receptors (9,
10). Our previous study (16) revealed an increased expression and redistribution of
2-integrin subunit at
the basal membrane of epithelial cells in the inferior portion of the
fetal gastric gland. This finding suggested that the
2
1-integrin may play a key role in the
differentiation and/or maintenance of glandular cell types, especially
zymogenic chief cells vs. the parietal cell lineage where expression of
2-subunit was strongly repressed. This view is supported
by the fact that simultaneous addition of a neutralizing antibody
against the
2-subunit with LNs/TGF-
1 almost abrogated
HGL mRNA production in primary cultures after 48 h. In addition,
there is increasing evidence that intracellular signals elicited by
hormones, growth factors, and integrin ligands exert their
cooperativity on cell responses through a convergent regulation of MAPK
cascades (3, 17). Several investigators have indeed
demonstrated that the presence of LNs or TGF-
commonly elicits the
activation of p42/p44 MAPKs (18, 24, 38). This was
specifically verified for intestinal cell lines (29, 52), whereas TGF-
also activates p38
/
MAPKs in gastric cell
cultures (26). Further adding to the relevance of MAPK
function in gastric physiology, we have shown that an acute
deregulation of basal p42/p44 activity by the mitogens EGF and TGF-
was partly responsible for inhibiting HGL expression (44).
As shown here, the specific inhibitor of p42/p44 cascade, PD-98059
(19), significantly reduced the stimulatory effect of
LNs/TGF-
1 on HGL activity and mRNA after 48 h, and this
downregulation was even stronger with SB-203580, an inhibitor of p38
(28). Also, their combination completely abrogated the
inductive effect of LNs/TGF-
1. Therefore, our results suggest that a
parallel activation of two MAPK cascades is necessary for an optimal
regulation of HGL gene expression in chief cells. Such cooperativity
has been observed in myoblasts where both p38 and p42/p44 activities
are required to activate myogenic transcription and hypertrophic growth
of myotubes (51). In conclusion, the present investigation
identifies TGF-
1 and LNs as positive regulators of cell polarity and
HGL expression in gastric epithelial cultures. It is thus the first
demonstration that a powerful synergism between a growth factor and
basement membrane LNs maintains the functionality of human gastric
glandular epithelial cells, including the zymogenic chief cell
population, through an activation of the
2
1-integrin and effectors of two MAPK pathways.
 |
ACKNOWLEDGEMENTS |
We thank Drs. A. De Caro, F. Carrière, and R. Verger (Centre
National de la Recherche Scientifique, Laboratoire de Lipolyse enzymatique, Marseille, France) for generously providing the
anti-gastric lipase (HGL) antibody and Drs. A. Poulin and F. Jacot
(Département de la Santé Communautaire du Centre
Hospitalier Universitaire de Sherbrooke) for excellent cooperation in
providing tissue specimens for this study. We also thank Dr. Nathalie
Rivard for helpful discussion and revision of the manuscript.
 |
FOOTNOTES |
This research was supported by a grant from the Canadian Institutes for
Health Research (to D. Ménard).
Preliminary results were presented at the 101st annual meeting of the
American Gastroenterological Association in San Diego, CA, and
published in abstract form (Gastroenterology 118, Suppl 2: A287, 2000).
Address for reprint requests and other correspondence: D. Ménard, Département d'anatomie et de biologie cellulaire,
Faculté de médecine, Université de Sherbrooke, 3001, 12e ave N, Sherbrooke, Québec, Canada J1H 5N4
(E-mail: dmenard{at}courrier.usherb.ca).
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.
10.1152/ajpcell.00150.2001
Received 22 March 2001; accepted in final form 13 November 2001.
 |
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