1 Laboratoire de Signalisation Cellulaire Normale et Tumorale, EA MNRT 2995,
Faculté de Pharmacie, 15 Avenue C. Flahault, 34093 Montpellier,
France
2 University of Melbourne, Department of Surgery, Austin Hospital, Melbourne,
VIC 3084, Australia
3 CNRS UPR 1086, Route de Mende, 34293 Montpellier, France
* Author for correspondence (e-mail: fhollande{at}ww3.pharma.univ-montp1.fr)
Accepted 12 December 2002
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Summary |
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Progastrin induced the dissociation of both tight junction (TJ) and
adherens junction (AJ) complexes in IMGE-5 cells. In progastrin-secreting
DLD-1 human colorectal carcinoma cells, expression of an antisense gastrin
construct restored membrane localisation of zonula occludens-1 (ZO-1),
occludin, ß-catenin and E-cadherin. This restoration was reversed by
treatment with exogenous progastrin. Endogenous or exogenous progastrin also
increased the paracellular flux of mannitol, and induced cell migration of
several gastrointestinal cell lines. In addition, progastrin enhanced Src
tyrosine kinase activity and induced a spatial delocalisation of protein
kinase C. Using dominant-negative mutants and pharmacological
inhibitors, we showed that the stimulation of Src kinase activity was
essential for the regulation of TJs. By contrast, the dissociation of AJs
involved phosphatidylinositol 3-kinase, partly through the formation of a
complex with protein kinase C
. We conclude that separate pathways
mediate the disruption of AJs and TJs by progastrin. Either pathway may
contribute to the co-carcinogenic role of this prohormone in colorectal
carcinoma.
Key words: Tight junctions, ß-catenin, Progastrin, Src, PI3-kinase
![]() |
Introduction |
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The establishment and stability of both AJs and TJs is tightly regulated
in particular, by growth factors, cytokines and hormones
(Boyer et al., 2000;
Nusrat et al., 2000
). Such
regulation, although poorly understood, seems essential for the modulation of
paracellular permeability in various epithelia
(Coyne et al., 2002
;
Nathanson et al., 1992
), for
the epitheliummesenchyme transition (Boyer
et al., 2000
), and for development, morphogenesis and wound
healing (Hellani et al., 2000
;
Jacinto et al., 2001
). The
observations that abnormal expression of ß-catenin promoted tumour
development in adenomatous polyposis coli (APC) mutant mice
(Kongkanuntn et al., 1999
),
that germline mutations of E-cadherin result in familial gastric cancer
(Guilford et al., 1998
), and
that there is a strong correlation between tumour differentiation and the
expression of occludin and ZO-1 along the gastrointestinal tract
(Kimura et al., 1997
), suggest
that modulation of both TJs and AJs can also have a significant impact on
tumour development and metastasis.
Gastrin has long been known as an important hormone for the development and
function of the gastrointestinal tract
(Dockray et al., 2001), and
the role of amidated and nonamidated progastrin-derived peptides as growth
factors (Wang et al., 1996
;
Koh et al., 1999
) and
cocarcinogens (Singh et al.,
2000a
; Aly et al.,
2001
) has been well documented. Recently, various independent
reports have also raised the possibility that progastrin-derived peptides may
regulate epithelial cell adhesion or migration. We have shown that
glycine-extended gastrin17 (Ggly) induces the dissociation of the
E-cadherin/ß-catenin complex, the delocalisation of ß-catenin from
the AJ to the cytoplasm and the migration of gastric epithelial cells
(Hollande et al., 2001a
). Ggly
was also shown to promote the invasiveness of the human colon carcinoma cell
line LoVo (Kermorgant and Lehy,
2001
). Similar dissociating effects of amidated
gastrin17 (Gam) have been described in Madine-Darby canine kidney
(MDCK) cells transfected with the gastrin/cholecystokinin-B (G/CCK-B) receptor
(Bierkamp et al., 2002
). An
important breakthrough concerning the role of progastrin-derived peptides in
cell motility was achieved with the finding, in vivo, that gastrin induced
parietal cell migration in mouse gastric mucosa
(Kirton et al., 2002
).
In this study, we investigated for the first time the direct effect of
recombinant human progastrin6-80
(Baldwin et al., 2001) on
cell-cell adhesion and migration in gastrointestinal epithelial cell lines.
The cell lines chosen were the conditionally immortalised nontumorigenic mouse
gastric line IMGE-5 (Hollande et al.,
2001b
) and the human colorectal carcinoma cell line DLD-1
(Dexter et al., 1981
). IMGE-5
cells produce no detectable progastrin-derived peptides, whereas DLD-1 cells
synthesise and secrete similar amounts of progastrin and Ggly, but little Gam.
We now report that treatment of IMGE-5 cells with progastrin, or reduction of
nonamidated gastrin production by stable transfection of DLD-1 cells with
vectors expressing antisense gastrin (ASG), induced significant changes in the
subcellular localisation and association of AJ and TJ proteins, and profoundly
affected cell adhesion and motility. We also identified the involvement of
Src, phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C
(PKC
) in the two separate signalling pathways that mediate the novel
effects of progastrin on TJs or AJs.
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Materials and Methods |
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Antibodies and cell culture
Polyclonal anti-actin antibody, calphostin C, LY294002 and
phalloïdin-FITC were from Sigma-Aldrich (St Louis, MO), and the Src
kinase inhibitor PP2 was from Calbiochem (La Jolla, CA). Antibodies against
claudin-1, claudin-2, occludin and ZO-1 were from Zymed (San Francisco, CA).
The SYM139 monoclonal antibody against symplekin has been described previously
(Keon et al., 1996).
Antibodies against p125 focal adhesion kinase (p125FAK), E-cadherin,
ß-catenin, PI3-kinase and phosphotyrosine (PY20) were from Transduction
Laboratories (Lexington, KY).
The IMGE-5 (Hollande et al.,
2001b) and young adult mouse colon (YAMC)
(Hollande et al., 1997
) cell
lines were generally grown in Dulbecco's modified Eagle's medium (DMEM)
containing 1 unit/ml
-interferon and 5% fetal calf serum (FCS) at
33°C (permissive conditions). For all experiments, cells were transferred
to 39°C in the same medium without
-interferon (nonpermissive
conditions), where they show differentiated characteristics, such as
expression of functional AJs and TJs. All experiments were performed on cells
between passage number 15 and 35. The DLD-1 colorectal carcinoma cell line
(Dexter, 1981) was from the American Tissue Culture Collection (ATCC;
Manassas, VA).
Preparation of Src kinase dominant-negative mutants and assay of Src
kinase activity
The cDNA encoding a constitutively activated Src mutant with the tyrosine
in position 527 mutated to a phenylalanine was cloned into the EcoRI
site of the eukaryotic expression vector pSGT vector, which had been derived
from the vector pSG5 (Stratagene, La Jolla, CA) by insertion of a new
polylinker containing the following restriction sites: EcoRI,
SpeI, BamHI, EcoRV, XhoI and
BglII. The Src dominant-negative double mutant (Src-/-)
was prepared from the constitutively activated mutant by mutation of lysine
295 in the ATP-binding site to alanine with the Transformer site-directed
mutagenesis kit (Clontech, Palo Alto, CA). The primer used was
5'-GTGGCCATCGCGACTCTGAAGCCC-3'. The double mutant
Src-/- lacks kinase activity but maintains an open conformation and
hence retains the capacity to interact with substrates and regulators.
Kinase activity in lysates of IMGE-5 and DLD-1 stable transfectants was
assessed by measurement of phosphorylation of denatured enolase
(Rodier et al., 1995).
Expression of antisense gastrin, dominant-negative mutants and
GFP-tagged constructs
DLD-1 cells were stably transfected with the full-length ASG construct
(Hollande et al., 1997) in
pcDNA3.1. Three clones each of cells transfected with the ASG construct or
with pcDNA3.1 only were monitored for Gam, Ggly and progastrin production and
secretion, and then used for all experiments. Preparation of cell extracts and
conditioned media, as well as radioimmunoassays, for all three gastrin forms
were performed as described previously
(Hollande et al., 1997
).
DLD-1 and IMGE-5 cells were stably transfected with the SH2 p85
mutant of PI3-kinase (kindly provided by W. Ogawa, University of Kobe, Japan).
All transfections were performed using Lipofectin® (Life Technologies,
Rockville, MD) according to the manufacturer's instructions, and stable
transfectants were selected using 500 µg/ml G418.
PKC-enhanced green fluorescent protein (EGFP) (Clontech, Palo Alto,
CA) was transiently expressed using Lipofectin® (Life Technologies,
Rockville, MD) according to the manufacturer's instructions. Twenty-four hours
after transfection, cells were seeded onto 13 mm glass coverslips and grown
overnight, serum-starved for 180 minutes under nonpermissive conditions,
treated with various agents as described in the figure legends, then washed
once with phosphate-buffered saline (PBS) and fixed for 10 minutes with 2%
paraformaldehyde in PBS at room temperature.
Immunocytochemical detection of junction proteins
Immunocytochemistry and bromodeoxyuridine (BrdU) incorporation experiments
were performed as described previously
(Hollande et al., 2001a).
Briefly, cells were grown under nonpermissive conditions on 14 mm glass
coverslips in DMEM containing 5% FCS as previously described
(Hollande et al., 2001a
).
Cells were then serum-starved for 24 hours and treated with agents to be
tested in DMEM containing 0.1% heat-inactivated FCS for the indicated period
of time. Cells were then fixed in ice-cold methanol for 3 minutes at 4°C
(for BrdU staining), or in 2% paraformaldehyde in PBS for 10 minutes at room
temperature (for immunocytochemistry). After three PBS washes, cells were
incubated for 5 minutes in PBS containing 0.2% Triton X-100 followed by PBS
containing 0.2% gelatine for 10 minutes. Cells were then incubated with
primary antibodies for 2 hours, coverslips were washed three times in PBS, and
the appropriate secondary antibody was incubated for 1 hour. After two PBS
washes and one rinse in water, coverslips were mounted on slides in cytifluor
(Oxford Instruments, Oxford, England). Confocal microscopy was then performed
using a Biorad 1024 CLMS System, as described previously
(Hollande et al., 2001b
).
Immunoprecipitation and western blotting
Cells were grown in 100 mm Petri dishes under permissive conditions until
they reached 90% confluence. Cells were then transferred to nonpermissive
conditions (Hollande et al.,
2001b) and serum-starved for 24 hours, stimulated with or without
the indicated concentration of progastrin6-80 for various time
periods with or without a 1 hour preincubation with either 10 µM LY294002
or 0.3 µM calphostin C, and lysed using the procedure described previously
(Hollande et al., 2001a
). In
the case of ß-catenin/E-cadherin association studies, 100 µg of
protein lysate per sample was immunoprecipitated in Tris/NaCl (pH 7.5)
containing 1% Nonidet P40, 100 µM sodium orthovanadate and 1 mM
dithiothreitol (DTT) (wash-lysis buffer), using 1 µg of anti-ß-catenin
antibody for 2 hours at 4°C, followed by 100 µl of 20% protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech, Piscataway, NJ) overnight.
Samples were washed three times in washlysis buffer and centrifuged for 10
seconds at 10,000 g. The pellet was resuspended in loading
buffer, denatured for 3 minutes at 95°C, centrifuged for 30 seconds at
10,000 g, and proteins in the supernatant were separated on an
8.5% SDS-polyacrylamide gel. Proteins were transferred onto a nitrocellulose
membrane using a semi-dry blotting system (Bio-rad, Hercules, CA). Membranes
were then incubated with the appropriate primary antibodies, and detection was
performed with alkaline phosphatase-coupled anti-rabbit or anti-mouse
immunoglobulin G followed by incubation with a
5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium solution, pH 9.2
(Sigma, St Louis, MO). Membranes were scanned using a HP ScanJet 5200C and
protein bands were analysed densitometrically with Fuji BAS software
(Berthold, Bundoora, Australia).
Migration experiments
Wound-healing experiments were performed to assess the effects of
progastrin on cell migration. Cells were grown in 12-well plates at 33°C
under permissive conditions until they reached 80% confluence; they were then
transferred into a 39°C incubator and were serumstarved for 24 hours. The
confluent monolayer was then wounded linearly using a pipette tip, washed
three times with PBS and treated with or without agents to be tested for the
indicated length of time, in the presence of 0.1% FCS. Morphology and
migration of cells was then observed and photographed at regular intervals for
24 hours.
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Results |
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|
Reduction of progastrin expression induces an epithelial-like
morphology
In contrast to IMGE-5 cells, DLD-1 colorectal carcinoma cells were found to
produce and secrete significant amounts of progastrin and Ggly
(Fig. 2A), but negligible
amounts of Gam. Expression of an ASG construct was found to reduce
significantly the production (Fig.
2A, top panel) and secretion
(Fig. 2A, bottom panel) of
progastrin and Ggly by DLD-1 cells. When observed by bright-field microscopy
the morphology of ASG-expressing cells was much more epithelial-like than that
of cells transfected with vector only (VO)
(Fig. 2B). In addition,
expression of ASG resulted in a delocalisation of actin, with much more
cortical actin detectable in confluent DLD-1/ASG cells than in DLD-1/VO
controls (Fig. 2C). Treatment
of DLD-1/ASG cells with 5 nM progastrin6-80 for 4 hours modified
their morphology, and resulted in a slight decrease in cortical actin
expression on confluent cells (Fig.
2C) and the extension of membrane processes in nonconfluent cells
(Fig. 2B).
|
Reduction of endogenous gastrin expression strengthens AJs and
TJs
Comparison of ASG and VO DLD-1 cells showed that expression of gastrin gene
products reduced the expression of ZO-1, claudin-1, claudin-2 and FAK, but not
of ß-catenin, E-cadherin and actin
(Fig. 3A). Expression of
occludin was not significantly decreased in all clones, as clone DLD-1/VO2
showed a similar level of expression as ASG clones. The reason for this
higher-than-expected expression is unknown, as this clone behaved similarly to
the other VO DLD-1 clones in all other respects. Constitutive expression of
progastrin-derived peptides by DLD-1/VO cells greatly reduced or abolished the
membrane localisation of ZO-1, occludin, claudin-1 and E-cadherin
(Fig. 3B), as well as
ß-catenin (F.H., unpublished), and induced partial dissociation of the
E-cadherin/ß-catenin (Fig.
3C) and occludin/ZO-1 complexes (F.H., unpublished).
Interestingly, the significant increase in membrane localisation of AJ and TJ
proteins seen in ASG clones was largely reversed by treatment with exogenous
progastrin6-80 (Fig.
3B, bottom panels). Similarly, progastrin6-80 also
induced a time-dependent decrease of E-cadherin/ß-catenin association in
DLD-1/ASG clones (Fig. 3C).
|
Functional consequences of the progastrin-induced disruption of
cell-cell adhesion
The effect of progastrin on paracellular permeability was assessed by
measuring the flux of [3H]mannitol through confluent monolayers of
DLD-1 or IMGE-5 cells. The permeability was reduced by almost 30% over a 24
hour period in DLD-1/ASG cells compared with DLD-1/VO cells
(Fig. 4A). Conversely, the
mannitol flux was significantly increased from 1 to 4 hours after treatment of
IMGE-5 cells (Fig. 4B) or
DLD-1/ASG cells (Fig. 4A) with
5 nM progastrin6-80.
|
We also assessed whether the decrease in cell-cell adhesion induced by progastrin in gastrointestinal epithelial cells was coupled to an effect on their motility. When a confluent cell monolayer was wounded using a pipette tip, the spontaneous motility displayed by DLD-1/VO clones (Fig. 4C, columns 1-2) was greatly reduced in clones expressing the ASG construct (Fig. 4C, columns 3-4). Interestingly, motility was partly restored when the latter clones were treated with exogenous progastrin6-80 (Fig. 4C, column 5). Similarly, progastrin6-80 was found to stimulate the migration of IMGE-5 cells, with a maximal effective dose of 5 nM (Fig. 4D).
Involvement of Src kinase in the progastrin-induced delocalisation of
TJ proteins
In DLD-1 colorectal carcinoma cells, basal Src-kinase activity was higher
in VO than in ASG clones (Fig.
5A). Furthermore, we found that progastrin6-80 induced
a three-to-fourfold stimulation of Src kinase activity in ASG clones, whereas
it barely affected the same activity in VO clones. The specificity of this
effect was illustrated by the failure of progastrin6-80 to
stimulate cells transfected with a dominant-negative mutant of Src
(Fig. 5A). Similar results were
obtained on IMGE-5 cells (Fig.
5B). In both cell lines, Src kinase activation was already
detectable within 1 minute after treatment, and maximal stimulation was
obtained using 5 nM progastrin6-80 (F.H., unpublished). Using
IMGE-5 cells expressing a dominant-negative Src (IMGE-5/Src-/-), we
then showed that activation of Src was essential for the effect of
progastrin6-80 on the delocalisation of the TJ proteins ZO-1 and
symplekin (Fig. 5C). By
contrast, Src was not essential for the progastrin6-80-induced
delocalisation of the AJ protein ß-catenin. Concomitantly, the partial
dissociation of the occludin/ZO-1 complex induced by progastrin6-80
was also completely abolished in the IMGE-5/Src-/- clones
(Fig. 5D). A similar result was
obtained on the weaker association detected between occludin and ZO-1 in DLD-1
cells (F.H., unpublished). However, the expression of Src dominant-negative
mutants had no effect on the progastrin-induced dissociation of the
E-cadherin/ß-catenin complex in IMGE-5 cells
(Fig. 5E). Finally, as expected
in view of the well-documented proliferative role of Src
(Porter and Vaillancourt,
1998), expression of this dominant-negative mutant also slowed
down growth in both cell lines (F.H., unpublished).
|
PI3-kinase is involved in the progastrin-induced cytoplasmic shift of
ß-catenin
Basal and progastrin6-80-stimulated Src kinase activity was
assessed in IMGE-5 cells transfected with a dominant-negative mutant of the
p85 regulatory subunit of PI3-kinase (IMGE-5/PI3-k-/-)
(Fig. 6A). Expression of this
mutant was found to decrease IMGE-5 cell proliferation (F.H., unpublished),
confirming the previously described role of PI3-kinase in cell growth
(Porter and Vaillancourt,
1998). Both unstimulated and stimulated Src activity were similar
in IMGE-5/PI3-k-/- cells and in cells transfected with vector only
(IMGE-5/VO), indicating that the activation of Src is not dependent on
PI3-kinase activity in these cells. Furthermore, contrary to the results
obtained in IMGE-5/Src-/- cells, expression of the PI3-kinase
dominant-negative mutant was found to prevent the progastrin-induced
cytoplasmic shift of ß-catenin, without affecting the delocalisation of
the TJ proteins ZO-1 and symplekin (Fig.
6B). However, the latter effect was blocked when cells from these
same clones were preincubated with the Src kinase inhibitor PP2 before
progastrin6-80 treatment. Conversely, the necessity for PI3-kinase
activity for mediation of the progastrin6-80 effect on AJs was
confirmed by the fact that the PI3-kinase inhibitor LY294002 was able to block
the cytoplasmic shift of ß-catenin in wild-type (F.H., unpublished) or
Src-/-IMGE-5 cells (Fig.
6B). Finally, the specificity of PI3-kinase action in mediating
the disruptive effect of progastrin on AJs but not TJs was further shown by
the failure of the PI3-k-/- mutant to prevent the partial
dissociation of the ZO-1/occludin complex induced by progastrin6-80
in IMGE-5 cells, although the mutant completely blocked the dissociation of
the ß-catenin/E-cadherin complex (Fig.
6C). Although the short-term effects of progastrin6-80
were assessed by stimulation of IMGE-5 cells for up to 4 hours, the long-term
effects of progastrin could be assessed in DLD-1 Src-/- and
PI3-k-/- mutants, which constitutively secrete progastrin.
Interestingly, we noted a significant increase in membrane staining for ZO-1
in DLD-1/Src-/- mutants compared with VO cells
(Fig. 6B and
Fig. 3B, respectively). By
contrast, there was no significant difference in E-cadherin
(Fig. 6B) and ß-catenin
localisation (F.H., unpublished) in DLD-1/ PI3-k-/- cells compared
with DLD-1/VO cells (Fig. 3B and F.H., unpublished, respectively).
|
Involvement of PKC in the progastrin-induced delocalisation of
junction proteins
Within 30 minutes of stimulation, progastrin6-80 induced a
delocalisation of GFP-PKC to a perinuclear area, as well as to the
plasma membrane (Fig. 7Aa,b,c).
Interestingly, only the delocalisation of GFP-PKC
to the membrane was
abolished when cells were preincubated with the PI3-kinase inhibitor LY294002
(Fig. 7Af), whereas the Src
inhibitor PP2 had no effect (Fig.
7Ae).
|
Furthermore, in wild-type IMGE-5 cells, progastrin6-80 was found
to induce a rapid but transient association of PKC with a complex
co-immunoprecipitating with ß-catenin
(Fig. 7B). The association was
detected 5 minutes after stimulation with 5 nM progastrin6-80, was
maximal after about 15 minutes and was not detectable 60 minutes after
stimulation.
Although the PKC inhibitor calphostin was found to reverse the progastrin-induced cytoplasmic shift of ß-catenin in wild-type (F.H., unpublished) and IMGE-5/Src-/- cells (Fig. 7C), it only had a partial effect on the delocalisation of TJ proteins induced by progastrin in IMGE-5/PI3-k-/- cells (Fig. 7C). Similarly, the role of PKC in the progastrin-induced regulation of paracellular permeability was found to be ambiguous. Calphostin C only partially inhibited the stimulation of [3H]mannitol leakage by progastrin6-80. By contrast, progastrin6-80-stimulated mannitol leakage was completely blocked by the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and unaffected by the PI3-kinase inhibitor LY294002 (Fig. 7D), thereby confirming the participation of Src in the regulation of TJs by progastrin.
![]() |
Discussion |
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Our work shows that physiological levels of Src are essential for the
disruption of TJs in confluent IMGE-5 and DLD-1 cells. The Src family of
tyrosine kinases has previously been implicated in the regulation of cell-cell
contacts for example, during TJ reassembly after an oxidative stress
in MDCK cells (Meyer et al.,
2001), or after mitogen-activated protein kinase kinase 1 (MEK-1)
inhibition (Chen et al.,
2000a
). Conversely, results on confluent MDCK cells already
expressing TJs have shown that tyrosine phosphorylation was essential for TJ
dissociation (Collares-Buzato et al.,
1998
). Interestingly, in post-confluent gastrointestinal tumour
(Caco-2) cells, overexpression of an oncogenic form of Src was found to
disrupt AJs without affecting the structure and function of TJs
(Gomez et al., 1999
). In view
of our current results and data showing Src involvement in Gam-induced
disruption of AJs in MDCK cells (Bierkamp
et al., 2002
), it is probable that differences in the time-course
of activation, in the local microenvironment and in downstream targets, are
all responsible for the diversity of Src-mediated effects on cell adhesion and
motility in various cell types.
In addition, we also showed that the stimulation of PI3-kinase activity is
essential to the disruption of AJs by progastrin, but has no bearing on the
regulation of TJs by this peptide. Evidence for the involvement of PI3-kinase
in TJ regulation has been limited, although recent results have suggested that
it could be involved in the glucocorticoid-induced stimulation of
trans-epithelial resistance, without structurally remodelling TJs
(Woo et al., 1999). We
recently showed in gastric epithelial cells that PI3-kinase was also crucial
for the Ggly-induced disruption of AJs
(Hollande et al., 2001a
),
suggesting that progastrin and Ggly share common signalling pathways. These
results also corroborate the crucial role played more generally by PI3-kinase
in AJ modulation, as shown previously by the direct association between
ß-catenin and PI3-kinase (Espada et
al., 1999
). Interestingly, the effect of PI3-kinase activation can
clearly differ depending on the conditions in which epithelial cells are kept,
and, in particular, on the degree of confluence. Thus, in subconfluent mammary
epithelial cells PI3-kinase activation is required for the formation of AJs
(Somasiri et al., 2000
), but
in confluent cells PI3-kinase was found to mediate epithelial cell
dissociation induced by hepatocyte growth factor
(Royal et al., 1997
) and Gam
(Bierkamp et al., 2002
). The
multiple effects of the activation of this enzyme are further shown by the
fact that PI3-kinase activation can also be a consequence of the formation of
E-cadherin-mediated contacts in MDCK cells
(Pece et al., 1999
). It
remains to be shown whether this flexibility is also connected to a
tissue-specificity of the effect of PI3-kinase towards epithelial or
nonepithelial AJs, or both.
In view of published data showing that Gam induces translocation of
PKC from cytoskeleton to membrane in colonic cells
(Yassin and Little, 1995
), and
implicating PKC
in the regulation of cell-cell contacts
(Chen et al., 2000b
;
Chen et al., 2002
;
Vallentin et al., 2001
), we
investigated the involvement of PKC
in the progastrin-induced
dissociation of TJs and AJs. Our results showed that PKC
behaved as a
downstream effector of PI3-kinase in the regulation of AJs by progastrin.
Activation of PKC, along with inhibition of glycogen-synthase kinase-3ß,
seems to be involved in the cytoplasmic accumulation of ß-catenin in
response to growth factors like Wnt (wingless)
(Chen et al., 2000b
).
Furthermore, a clear colocalisation between ß-catenin and PKC
at
cell-cell contacts was recently shown in GH3B6 pituitary epithelial cells
after phorbol 12-myristate 13-acetate treatment, although no physical
association between the two proteins was evident
(Vallentin et al., 2001
). To
our knowledge, the current study shows, for the first time, a physical
association between PKC
and ß-catenin in epithelial cells, as well
as of cooperation between PI3-kinase and PKC
in the regulation of
ß-catenin localisation at the membrane, although the exact nature of the
action of PKC
on AJs needs to be elucidated.
The specific involvement of Src and PI3-kinase in the disruption by
progastrin of TJs and AJs, respectively, supports the concept of an
independent regulation of both types of junction during the early stages of
cell dissociation induced by an extracellular stimulus. Although progastrin
induced a disruption of both AJs and TJs in the current study, data from
IMGE-5 cells stably expressing Src-/- or PI3-k-/-
dominant-negative mutants seem to show that the early stages of TJ and AJ
disruption could be independent from one another. Nevertheless, recent results
argue in favour of a cross-talk between AJs and TJs, as overexpression of the
PDZ domains of ZO-1 (Reichert et al.,
2000) or treatment with occludin peptides
(Vietor et al., 2001
) was
found to upregulate the cytoplasmic availability and the transcriptional
activity of ß-catenin. However, the duration of treatment with progastrin
in our study was significantly shorter than the time allowed for measurement
of an effect of occludin fragments on the activation of the
ß-catenin/T-cell factor (TCF)/Lef transcriptional pathway in mammary
epithelial cells (Vietor et al.,
2001
). The occurrence and the importance of cross-talk between TJs
and AJs in DLD-1 cells is supported by the greatly decreased membrane
localisation of AJ proteins in DLD-1/PI3-k-/- cells. If PI3-kinase
specifically mediates the effect of progastrin on AJs, as shown in IMGE-5
cells, any effect of endogenous progastrin on AJs in DLD-1/PI3-k-/-
cells is likely to occur indirectly via the presence of higher concentrations
of free TJ proteins, such as ZO-1, in the cytosol. Alternatively, long-term
constitutive progastrin stimulation in these DLD-1/PI3-k-/- cells
could bypass the PI3-kinase pathway, thereby allowing a resumption of AJ
regulation in these cells. By contrast, DLD-1/Src-/- cells did not
show a significant disruption of their TJ protein localisation, indicating
that the interaction between TJs and AJs during the cell-cell dissociation
period in this model could be monodirectional, from the TJ towards the AJ.
The results presented in this study show that tumour cells expressing
progastrin and Ggly displayed major perturbations in cell-cell adhesion, as
well as spontaneous motility. This shows that such hormone precursors, which
are known to be overexpressed during foetal development
(Luttichau et al., 1993), as
well as in colon carcinoma (Ciccotosto et
al., 1995
), could act through an autocrine loop to chronically
disrupt adhesion and motility of colorectal carcinoma cells. The existence of
such a loop could be crucially important in vivo, where progastrin
overexpressed in transgenic mice was previously shown to act as a
cocarcinogen, following treatment with the colonic carcinogen azoxymethane
(Singh et al., 2000b
;
Singh et al., 2000a
).
Antibodies raised against a gastrin immunogen were shown to inhibit the
spontaneous metastasis of a human colorectal tumour cell line producing
progastrin and Ggly when injected into immunodeficient mice
(Watson et al., 1999
). In
humans, a strong correlation was shown between a higher incidence of liver
metastasis from colorectal carcinoma and elevated serum gastrin concentrations
(>150 pg/ml) in a panel of 140 patients
(Kameyama et al., 1993
).
Furthermore, the role of progastrin-derived peptides on cell motility clearly
has other implications than in cancer development, as shown in vivo by a
gastrin-induced parietal cell migration in the mouse gastric mucosa
(Kirton et al., 2002
).
Interestingly, several progastrin-derived peptides have been shown to
regulate the morphology, adhesion and motility of gastrointestinal epithelial
cells. The fully processed form Gam has recently been shown to induce
branching morphogenesis in gastric cancer cells
(Pagliocca et al., 2002), loss
of adhesion and scattering of G/CCK-B receptor-transfected MDCK cells
(Bierkamp et al., 2002
), as
well as increased matrix metalloproteinase-9 expression in gastric cells in
vitro and in the stomach of multiple endocrine neoplasia (MEN-1) patients with
elevated concentrations of plasma gastrin
(Wroblewski et al., 2002
).
Ggly has previously been shown to increase the invasiveness of the human colon
carcinoma cell line LoVo (Kermorgant and
Lehy, 2001
) and to induce the dissociation of AJ complexes, as
well as the migration of gastric epithelial cells
(Hollande et al., 2001a
). The
current work presents the first description of a regulatory role for the early
precursor progastrin in the dissociation and migration of nontumoral and
tumoral gastrointestinal cells.
Several observations indicate that the dissociating effect of progastrin is
probably mediated by a membrane receptor: (1) no effect of progastrin is
detected on cell types where regulation by gastrointestinal peptides is not
expected to occur, such as MDCK kidney cells (F.H., unpublished); (2)
progastrin6-80 is a large peptide (MW 8500) and is therefore
unlikely to penetrate membranes quickly, if at all, in order to act on an
intracellular target. However, in the current study, the onset of Src kinase
activation, as well as dissociation of junctional complexes after
progastrin6-80 treatment, was very rapid; (3) the
progastrin-induced dissociation of AJs and TJs, as well as the stimulation of
paracellular permeability and migration, were found to be dose dependent and
saturable at the low nanomolar concentrations typical of membrane receptors.
The nature of the receptor mediating this effect of progastrin on adhesion and
motility is yet to be determined, as we have so far been unable to iodinate
progastrin in a reproducible manner on its single tyrosine residue
(Baldwin et al., 2001). The
receptor could be similar to the only identified progastrin receptor, recently
found to mediate proliferation on rat intestinal epithelial cells (IEC)
(Singh et al., 2002
), and
further investigation should determine whether it is related to the receptor
involved in the effects of Ggly on cell dissociation and motility (Kermorgant
et al., 2001; Hollande et al.,
2001a
). The affinity of the receptor identified by Singh et al.
for progastrin-derived peptides lacking the N-terminal sequence was found to
be lower than for the full-length progastrin peptide
(Singh et al., 2002
). This
observation is in agreement with the data presented in this manuscript showing
that the maximal effective concentration of progastrin6-80, which
lacks the six N-terminal amino acids, is slightly higher than the
concentration of progastrin1-80 inducing maximal proliferation of
IEC-6 cells (Singh et al.,
2002
).
To date, most studies of the effects of progastrin-derived peptides on gastrointestinal cell adhesion and motility have been performed on cell lines in vitro, thereby enabling a more specific description of the molecular and cellular events involved. However, more studies in vivo are clearly necessary to clarify the individual roles and evaluate the impact of progastrin-derived peptides on the regulation of epithelial cell morphology and migration during development, and in the potentiation of the invasive properties of colorectal tumours.
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