From the CIHR Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
Received for publication, January 10, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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The intracellular signaling pathways responsible
for cell cycle arrest and differentiation along the crypt-villus axis
of the human small intestine remain largely unknown. p38
mitogen-activated protein kinases (MAPKs) have recently emerged as key
modulators of various vertebrate cell differentiation processes. In
order to elucidate further the mechanism(s) responsible for the loss of
proliferative potential once committed intestinal cells begin to
differentiate, the role and regulation of p38 MAPK with regard to
differentiation were analyzed in both intact epithelium as well as in
well established intestinal cell models recapitulating the crypt-villus
axis in vitro. Results show that phosphorylated and active
forms of p38 were detected primarily in the nuclei of differentiated
villus cells. Inhibition of p38 MAPK signaling by 2-20
µM SB203580 did not affect E2F-dependent
transcriptional activity in subconfluent Caco-2/15 or HIEC cells. p38
MAPK activity dramatically increased as soon as Caco-2/15 cells reached
confluence, whereas addition of SB203580 during differentiation of
Caco-2/15 cells strongly attenuated sucrase-isomaltase gene and protein expression as well as protein expression of villin and alkaline phosphatase. The binding of CDX2 to the sucrase-isomaltase promoter and
its transcriptional activity were significantly reduced by SB203580.
Pull-down glutathione S-transferase and immunoprecipitation experiments demonstrated a direct interaction of CDX3 with p38. Finally, p38-dependent phosphorylation of CDX3 was observed
in differentiating Caco-2/15 cells. Taken together, our results
indicate that p38 MAPK may be involved in the regulation of CDX2/3
function and intestinal cell differentiation.
The epithelium of the small intestine is a highly dynamic system
continuously renewed by a process involving cell generation and
migration from the stem cell population located at the bottom of the
crypt to the extrusion of the terminally differentiated cells at the
tip of the villus (1, 2). The crypt-villus functional axis unit, which
develops relatively early during human ontogeny (being established by
mid-pregnancy), can be defined by typical morphological and functional
properties displayed by the mature villus enterocytes that distinguish
them from crypt cells (1-3). Indeed, the villi are mainly lined by
functional absorptive, goblet, and endocrine cells, whereas the crypts
contain stem cells, proliferative and poorly differentiated cells, as well as a subset of differentiated secretory cells, namely Paneth cells
(3). The differentiation of each cell type takes place as the cells
move either upward toward the villus (absorptive, mucus and endocrine
cells) or downward to concentrate at the bottom of the crypt (Paneth
cells) (2). The basic mechanisms responsible for induction of cell
differentiation are little understood. The decision to differentiate is
taken by the committed crypt cells abruptly, while in their most rapid
state of proliferation (4). The newly differentiated cells acquire
their distinctive ultrastructural features and cell surface markers
after leaving the proliferative cell cycle, at the top of the crypts or
the base of the villi (2, 5-7). It is noteworthy that in all species
studied, the crypt-villus axis junction represents a physical limit
from which enterocytes acquire their final functional characteristics
(1-7).
The process of cell differentiation in the intestinal epithelium has
been the subject of extensive studies, for which the morphological and
functional characteristics of the intestinal mucosa (8, 9), the growth
kinetics of the epithelial cells (10), and the chronological changes
that affect brush border enzyme activities during pre- and postnatal
development (11, 12) have all been well documented. However, the basic
mechanisms involved in the induction and the modulation of cell
differentiation in the upper portion of the crypts, and the cellular
interactions responsible for the orderly arrangement of the relative
numbers of proliferative, maturing, and functional epithelial cells are still largely unknown. Hormones, such as glucocorticoids, and growth
factors, such as epidermal growth factor
(EGF),1 have been implicated
in the regulation of intestinal growth and development (12, 13).
However, little is known about the molecular signals responsible for
the ontogenic changes in intestinal gene expression.
Several lines of evidence suggest that the intestinal specific,
caudal-related cdx1 and cdx2/3 homeobox genes
encode nuclear transcription factors that play critical roles in
intestinal cell proliferation and differentiation. CDX1 is mainly
expressed in the crypt compartment although not restricted to
proliferative cells (14), and its inhibition by antisense RNA reduces
cell proliferation in vitro (15). The CDX2/3 homeoproteins
(the protein designated CDX3 in the hamster and CDX2 in the mouse and
humans) are mainly expressed in differentiating enterocytes (16),
triggering growth retardation and cell differentiation by
overexpression in several intestinal lines in vitro (15, 17,
18). Furthermore, genes regulated by either CDX1 or CDX2/3 generally
define a functional differentiated phenotype (for example,
sucrase-isomaltase (18), glucagon (19), intestinal phospholipase
A/lysophospholipase (20), carbonic anhydrase (21), and lactase (22)).
However, little is known about the intracellular signaling pathways
that positively regulate the activities of CDX transcription factors, especially those involved in receiving and transducing extracellular cues.
In eukaryotic cells, the mitogen-activated protein kinase (MAPK) family
has been shown to play various important roles in regulating gene
expression via transcription factor phosphorylation (23-26). In
mammals, two distinct classes have been identified to date as follows:
p42-p44 (extracellular signal-regulated kinase) MAPKs inducible by
growth factors, and SAPKs (stress-activated protein kinases), which
include p38 MAPKs and p46-p54 JNKs, inducible by cytokines and cellular
stress (27). Unique structural features, specific activation pathways,
and varying substrate specificities support the contention that
different MAPKs are independently regulated and control different
cellular responses to extracellular stimuli (28, 29). We (30) and
others (31) recently analyzed the role and regulation of p42/p44 MAPKs
in the process of proliferation and differentiation of human intestinal
cells. Our results demonstrated that elevated p42/p44 MAPK activities
stimulated cell cycle progression of intestinal epithelial cells,
whereas low sustained levels were correlated with G1 arrest
and differentiation. However, the intracellular pathways responsible
for establishment of differentiated cells occupying specific positions
along the gut axis still remain largely unknown.
Several recent studies have demonstrated that p38 MAPK is involved in
various vertebrate cell differentiation processes, namely adipocytic
(32) and myogenic differentiation (33). The role of p38 MAPK in
intestinal cell differentiation is, however, not known. In the present
work, the role and regulation of p38 MAPK were analyzed in relation to
human intestinal cell proliferation and differentiation in the intact
epithelium as well as in well established intestinal cell models that
allow the recapitulation of the crypt-villus axis in vitro
as follows: Caco-2/15 cells, which have the ability to differentiate
into fully functional villus-like enterocytes (34-37); normal
crypt-like HIEC cells, which are proliferative and undifferentiated
(38); and finally PCDE cells, which are primary cultures of
differentiated and non-proliferative villus enterocytes (39). By using
a combination of different approaches, p38 MAPK was found to be
activated rapidly in intestinal cells induced to differentiate.
Specific inhibition of p38 significantly reduced the expression of
several differentiation markers including sucrase-isomaltase,
alkaline phosphatase, lactase, and villin. Finally, p38 exerted its
stimulatory effect on intestinal differentiation by directly
interacting with CDX2/3 and enhancing its transcriptional activity.
Materials--
[ Specimens and Indirect Immunofluorescence--
Tissues from five
fetuses of 20 weeks of gestation (post-fertilization fetal ages were
estimated according to Streeter (43)) were obtained from normal
elective pregnancy terminations. No tissue was collected from cases
associated with known fetal abnormalities or fetal death. All studies
were approved by the Institutional Human Subject Review Board. Segments
of fetal small intestine were rinsed with 0.15 M NaCl,
sectioned into small fragments, embedded in optimum cutting temperature
compound, and quickly frozen in liquid nitrogen (36). Frozen sections
2-3 µm thick were spread on silane-coated glass slides and air-dried
for 1 h at room temperature before storage at Cell Culture--
The Caco-2/15 cell line was obtained from A. Quaroni (Cornell University, Ithaca, NY). This clone of the parent
Caco-2 cell line (HTB 37; American Type Culture Collection, Manassas,
VA) has been characterized extensively elsewhere (30, 34, 36, 37) and
was selected originally as expressing the highest level of
sucrase-isomaltase among 16 clones obtained by random cloning. This
cell line was cultured in plastic dishes in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% fetal
bovine serum (FBS), as described previously. Caco-2/15 cells were used
between passages 53 and 78. Studies were performed on cultures at
subconfluence (50-70% confluence), confluence, and between 2 and 40 days post-confluence. Human intestinal epithelial cells (HIEC) were
cultured as described (38) in DMEM supplemented with 4 mM
glutamine, 20 mM HEPES, 50 units/ml penicillin, 50 µg/ml streptomycin, 5 ng/ml recombinant human epidermal growth factor, 0.2 IU/ml insulin, and 5% FBS. Primary cultures of human differentiated enterocytes (PCDE) prepared from specimens of small intestine from
fetuses ranging from 18 to 20 weeks of age, were cultured in
supplemented DMEM as described above for HIEC (39). When tested after
5-7 days, these primary cultures of differentiated enterocytes were
well preserved; both goblet and absorptive cells exhibited the main
characteristics of intact villus intestinal cells (39).
Protein Expression and Immunoblotting--
Cells were lysed in
SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.3% SDS,
10% glycerol, 5% p38 MAPK Assay--
The cells were lysed for 10 min on ice with
1 ml/dish of lysis buffer (150 mM NaCl, 1 mM
EDTA, 40 mM Tris, pH 7.6, 1% Triton X-100) supplemented
with protease inhibitors (0.1 mM PMSF, 10 µg/ml
leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin) and phosphatase inhibitors (0.1 mM orthovanadate, 20 mM
para-nitrophenyl phosphate, 40 mM
Expression Vectors and Reporter Constructs--
The
sucrase-isomaltase reporter construct used for luciferase assays
contained the human sucrase-isomaltase promoter from residues Northern Blot Analysis--
Total cellular RNAs were prepared
from Caco-2/15 cells at subconfluence, confluence, and 3 and 6 days
post-confluence by the guanidinium isothiocyanate/phenol method
(TRIZOL, Life Technologies, Inc.) as described before (49). RNAs were
subjected to agarose gel electrophoresis with formaldehyde and
transferred to nylon membranes (Nytran, Schleicher & Schuell). Equal
RNA loading was confirmed by hybridization to an Transient Transfections and Luciferase Assays--
First,
subconfluent Caco-2/15 cells were seeded in 24-well plates and
transfected by lipofection (Lipofectin, Life Technologies, Inc.) as
described before (30) with 0.1 µg of E2F-SV40-luciferase reporter per
well. One day after transfection, cells were exposed to 2-20
µM SB203580 or 20 µM PD98059 for 24 h,
and luciferase activity was measured. The increase in luciferase
activity was calculated relative to the basal level of
E2F-SV40-luciferase set at 1 and corrected for the empty vector
effects. Second, 1 day post-confluent Caco-2/15 cells were seeded in
24-well plates and co-transfected by lipofection (LipofectAMINE 2000, Life Technologies, Inc.) as described previously (30) with 0.1 µg of
SI-luciferase reporter and 0.1 µg of the relevant expression vector
(pECE) containing wild-type or dominant-negative mutant of p38 Gal4 Assays--
The 540-base pair sequence encoding the
180-amino acid transactivation domain of CDX3 (42) was PCR-amplified
and cloned in frame with the DNA-binding domain of GAL4 in the
mammalian expression vector pM2 (50). The expression vector was
transfected by lipofection (Lipofectin, Life Technologies, Inc.) in
Caco-2/15 cells with the pFR-luciferase reporter vector containing five tandem repeats of the GAL4-binding element upstream of a basic promoter
element (Stratagene, La Jolla, CA). Luciferase activity was assessed
after a 24-h treatment with Me2SO, 20 µM
SB203580, or 20 µM PD98059.
Determination of Brush Border Enzyme Activity--
Caco-2/15
cells, treated with or without 20 µM SB203580, were
harvested in water at confluence (day 0) and at 3, 6, and 9 days
post-confluence, and sonicated. The disaccharidases sucrase-isomaltase and lactase-phlorizin were assayed using the method of Dahlqvist as
modified by Ménard and Arsenault (51). Alkaline phosphatase was
assayed by the method of Eichholz (52). Dipeptidyl peptidase IV (DPPIV)
activity was assayed according to the method of Roncari and Zuber (53),
with glycyl-L-proline-p-nitroanilide as
substrate. Total homogenate protein content was determined using a
modified Lowry procedure with bovine serum albumin as standard (44). Data were expressed in international units (micromoles of substrate hydrolyzed per min) per g of protein.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from Caco-2/15 cells at subconfluence, confluence, 2, 6, 9, and 12 days post-confluence, according to Stein et al. (54).
Electrophoretic mobility shift assays were performed as described
previously (55). Samples were electrophoresed in a 4% polyacrylamide
gel containing 0.5% Tris borate buffer and 2% glycerol. The following
human sucrase-isomaltase promoter DNA-binding sites were used for
electrophoretic mobility shift assays: SIF1, the CDX2 DNA-binding site,
GST Fusion Protein Purification--
Hamster CDX3 was ligated
downstream of the glutathione S-transferase sequence in a
pGEX plasmid (Amersham Pharmacia Biotech). The recombinant plasmid was
introduced into Escherichia coli BL21 DE3, and the fusion
protein was produced by growing 50 ml of a bacterial culture to an
optical density between 0.9 and 1.1 and then treating the cultures with
0.5 mM isopropyl-1-thio- GST Pull-down Assays--
Caco-2/15 cells were grown in 60-mm
dishes to confluence in DMEM supplemented with 10% FBS. The cells were
lysed in 700 µl of lysis buffer (150 mM NaCl, 1 mM EDTA, 40 mM Tris, pH 7.6, 1% Triton X-100)
supplemented with protease inhibitors (0.1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin) and phosphatase inhibitors (0.1 mM orthovanadate, 20 mM para-nitrophenyl phosphate, 40 mM
Data Presentation and Statistical
Analysis--
Luciferase assays were performed in triplicate, and
results were analyzed by the Student's t test and were
considered significantly different at p < 0.05. Typical Western blots, representative of two or three independent
experiments, are shown. Densitometric analyses were carried out for
each Western blot.
Activity of p38 Activation of p38 MAPK during Differentiation of Intestinal
Cells--
Caco-2/15 cells that differentiate spontaneously to a small
bowel phenotype after confluence (30, 34-37) were harvested at 70 (day
Because Caco-2/15 cells are derived from a human colonic adenocarcinoma
(59), we wanted to support our results in normal human small
intestine-derived cells. We then analyzed the phosphorylation of p38
MAPK in normal human intestinal cell models as follows: the crypt-like
HIEC cells that are proliferative and undifferentiated (38), and the
PCDE cells that are primary cultures of differentiated and
non-proliferative villus enterocytes (39). Cell lysates were prepared
from subconfluent growing HIEC cells and from PCDE cells. As shown in
Fig. 2D, phosphorylation of p38 was significantly lower in
subconfluent growing HIEC cells compared with that found in PCDE cells.
Hence, p38 MAPK exhibits similar patterns of activity if we compare
pre- and post-confluent Caco-2/15 cells versus normal HIEC
and PCDE cells that together allow the in vitro reproduction of the normal crypt-villus axis (9).
p38 MAPK Is Not Involved in Cell Cycle Progression of Intestinal
Epithelial Cells--
An important early event in the terminal
differentiation of cells, especially in tissues exhibiting a rapid
turnover such as the intestinal epithelium, is their withdrawal from
the cell cycle (60, 61). To evaluate the role of p38 MAPKs in
intestinal cell cycle progression, SB203580 compound, a specific
inhibitor of p38
We recently demonstrated that Caco-2/15 cells slowed their cell cycle
at confluence to become almost completely arrested in the
G1 phase by day 6 post-confluence. Indeed, decreased
phosphorylation of retinoblastoma proteins and reduced Cdk2
activity correlated with the induction of differentiation markers,
namely sucrase-isomaltase and villin (61). To determine whether p38
MAPK activation plays a significant role in cell cycle arrest of
confluent Caco-2/15 cells, the consequences of blocking p38 MAPK with
SB203580 were examined on pRb phosphorylation by using specific
antibodies detecting the active hypophosphorylated form of p105Rb
protein (lower band) as well as the inactive
hyperphosphorylated forms of the protein (upper bands). As
shown in Fig. 3B, addition of SB203580 at days 0-6
post-confluence had no significant effect on the decrease in pRb
phosphorylation observed at days 3 and 6 post-confluence suggesting
that p38 MAPK is not involved in the loss of proliferative potential as
committed intestinal cells begin to differentiate.
p38 MAPKs appear to play important roles in the regulation of cell
survival in other cell types (63, 64). To verify the potential effect
of the inhibition of p38 MAPKs on Caco-2/15 cell survival, expression
of PARP, a well known substrate for caspase-3 (65), was measured in
cells treated with SB203580. As shown in Fig. 3B, treatment
of confluent Caco-2/15 cells with SB203580 had no effect on PARP
cleavage, suggesting that persistent inhibition of p38 MAPK did not
affect Caco-2/15 cell survival.
Inhibition of p38 MAPK Activity Prevents Enterocyte
Differentiation--
The dramatic induction of p38 MAPK activity led
us to verify whether this MAPK was associated with differentiation of
Caco-2/15 cells. Daily addition of SB203580 at confluence repressed
sucrase-isomaltase protein expression in confluent Caco-2/15 cells
(Fig. 4A). In addition,
induction of villin expression was also decreased by 2-3-fold at days
3, 7, and 12 post-confluence. Equal protein loading was confirmed by
using an anti-actin antibody (Fig. 4A). Enzymatic assays
were also performed in order to verify the induction pattern of other
differentiation markers, namely lactase, DPPIV, and alkaline phosphatase, in control and in SB203580-treated cells. As shown in
Table I, treatment of confluent Caco-2/15
cells with 20 µM SB203580 did not significantly affect
the induction of DPPIV at post-confluence, whereas induction of
lactase and alkaline phosphatase was significantly attenuated (Table
I).
The effect of p38 on sucrase-isomaltase gene expression was further
analyzed by transiently transfecting newly confluent Caco-2/15 cells
with the luciferase gene driven by the human sucrase-isomaltase promoter (45). As shown in Fig. 4B, sucrase-isomaltase gene expression was inhibited in a dose-dependent manner by the
p38 inhibitor, SB203580, with maximal effect observed at 20 µM (72% inhibition). Furthermore, in contrast to
wild-type p38
Of note, addition of SB203580 to differentiating Caco-2/15 cells
at days 6-12 post-confluence still reduced sucrase-isomaltase expression by 47%, as observed at day 12 post-confluence (Fig. 4C). In addition, treatment of primary cultures of normal
differentiated enterocytes (see "Experimental Procedures") with 20 µM SB203580 also significantly reduced the expression of
sucrase-isomaltase by 22 and 55% and villin by 38 and 57% after 2 and
4 days of treatment, respectively (Fig. 4D). Equal protein
loading was confirmed by using an anti-keratin-18 antibody. These data
suggest that p38 MAPK activity is required for maximal expression of
sucrase-isomaltase in differentiating Caco-2/15 cells and
differentiated normal enterocytes.
Transactivation Activity of CDX2/3 Is Down-regulated by Inhibition
of the p38 Pathway--
The dramatic effect of the p38 inhibitor on
the expression of sucrase-isomaltase prompted us to investigate whether
p38 also affected the activity of the transcription factor CDX2/3 (the protein designated CDX3 in the hamster and CDX2 in the mouse and humans
(66)), a key activator of sucrase-isomaltase transcription (45) and an
inducer of intestinal epithelial cell differentiation (18). We first
examined whether inhibition of p38 in confluent Caco-2/15 cells
affected the expression of CDX2. Northern blot analysis demonstrated
that addition of SB203580 at days 0-6 post-confluence had no
effect on CDX2 mRNA expression in confluent Caco-2/15 cells (Fig.
5A). Lysates from COS cells
were used as negative control.
The effect of p38 on CDX2 activity was then studied by transiently
transfecting Caco-2/15 cells with a CDX3 expression vector. As reported
previously (17) with CDX2, ectopic expression of CDX3 resulted in a
strong increase (13-fold induction) in sucrase-isomaltase promoter
activity. Addition of SB203580 reduced in a dose-dependent manner the inducing effect of CDX3 with maximal inhibition observed at
20 µM (70% inhibition) (Fig. 5B, upper
panel).
Previous studies revealed that p38 increases the transcriptional
activity of various transcription factors by phosphorylation of the
transactivation domain (67). To determine whether p38 had a similar
effect on CDX2/3, fusion proteins were used containing the
transactivation domain of CDX3 (amino acids 1-180) fused to Gal4(DBD)
(see "Experimental Procedures"). To assess the transcriptional activity of Gal4-CDX3 proteins, Caco-2/15 cells were co-transfected with a luciferase reporter gene containing five copies of a Gal4 DNA-binding site upstream of a minimal promoter and Gal4-CDX3 expression plasmids. As demonstrated above, inhibition of the p38
pathway by SB203580 significantly reduced CDX3-dependent
reporter gene expression (Fig. 5B, lower panel) suggesting
that p38 modulates the transcriptional activity of CDX3. In contrast,
inhibition of the p42/p44 MAPK pathway with the PD98059 inhibitor had
no effect on CDX3-dependent reporter gene expression (Fig.
5B, lower panel).
Effect of SB203580 on the DNA-binding Capacity of Transcription
Factors Involved in Sucrase-Isomaltase Expression--
There are three
positive regulatory elements for transcription within the
sucrase-isomaltase promoter region in intestinal epithelial cells known
as sucrase-isomaltase footprint (SIF)-1, SIF-2, and SIF-3. SIF-1 binds
the intestine-specific homeodomain transcription factor CDX2/3 (17),
whereas SIF-2 and SIF-3 bind the transcription factors HNF-1 CDX3 Specifically Interacts with p38 MAPK--
To determine
whether CDX2/3 can directly associate with p38 MAPK, pull-down assays
were performed using the GST-CDX3 fusion protein to absorb naive newly
confluent Caco-2/15 cell lysates. The absorbed material was analyzed by
Western blot with the p38 antibody. Immunoprecipitated p38 from newly
confluent Caco-2/15 cells was used as positive control. As shown in
Fig. 7A, a significant amount
of p38 MAPK bound to the GST-CDX3 fusion protein was detected. GST
protein alone did not pull down the p38 protein (data not shown).
Interestingly, the GST-CDX3 fusion did not pull down other MAPKs such
as p42 MAPK or JNK1 (data not shown). To determine whether CDX3-p38
association may have some functional relevance, the capacity of pulled
down p38 to phosphorylate the GST-CDX3 fusion protein was evaluated in
a kinase assay. As shown in Fig. 7B, pulled down p38
efficiently phosphorylated the GST-CDX3 protein. More importantly, this
phosphorylation was inhibited in a dose-dependent fashion
by the addition of low concentrations of the specific p38 inhibitor
SB203580 (50% inhibition with 0.5 µM of SB203580). In
this regard, an amino acid sequence analysis revealed that CDX2/3
contains putative phosphorylation sites for p38 MAPK (17, 67). An
in vitro kinase assay using bacterially expressed GST-CDX3 protein or MBP as substrates and p38 immunoprecipitated from newly confluent Caco-2/15 cells revealed that p38 MAPK was able to potently phosphorylate MBP and, more importantly, phosphorylate GST-CDX3 to a
significant level (Fig. 7C). This suggests that CDX2/3 may indeed be a specific target for p38 MAPK.
We further verified whether CDX3-p38 association could be detected
in vivo. CDX3 was co-transfected into 293T cells with a plasmid encoding wild-type HA-p38 EGF Represses p38 MAPK Activity and Sucrase-Isomaltase Expression
in Differentiating Caco-2/15 Cells--
Sucrase-isomaltase
expression has been reported to be down-regulated by growth factors
such as keratinocyte growth factor (69) and EGF (70). However,
the mechanism involved in the repression of sucrase-isomaltase
expression by growth factors is unknown. The effect of EGF was
therefore examined on both sucrase-isomaltase protein expression and
p38 activity. As shown in Fig. 8, chronic treatment (from days 0 to 15) of confluent Caco-2/15 cells with 100 ng/ml EGF repressed sucrase-isomaltase protein expression compared
with untreated cells (over 95% inhibition). Of interest, treatment
with EGF significantly and persistently down-regulated p38 MAPK
activity with maximal effect observed after 2 and 4 h of treatment
(Fig. 8). Finally, the inhibitory action of EGF on sucrase-isomaltase
expression was not attributable to the re-activation of the p42/p44
MAPK pathway since the specific MEK inhibitor, PD98059, blocked p42/p44
MAPK activation but did not interfere with the repressive effect of EGF
on sucrase-isomaltase expression.
The molecular mechanisms orchestrating cellular transitions and
changes in gene expression during intestinal epithelial differentiation are largely unknown. In this report, we suggest for the first time the
possible involvement of p38 MAPK in intestinal cell differentiation. We
show that the activity of p38 was induced as soon as Caco-2/15 cells
reached confluence and began differentiating into a small bowel-like
phenotype with microvilli formation and expression of disaccharidases.
Furthermore, the nuclear localization of p38 MAPK activity in the
villi, which is indicative of its functional role in transcription,
reflects the distribution of differentiated cells. Inhibition of p38
activity by the specific inhibitor SB203580 did not interfere with cell
cycle progression of committed cells but inhibited intestinal cell
differentiation and expression of various differentiation markers
(namely sucrase-isomaltase, alkaline phosphatase, villin, and lactase).
The effects of p38 MAPK on sucrase-isomaltase transcription revealed a
regulation of sucrase-isomaltase expression, an effect mediated by
CDX2/3. The latter was demonstrated previously to be an important
modulator of enterocyte differentiation (17-18, 68).
A key issue in intestinal development is what triggers the
differentiation process. Members of the CDX family have been shown to
be involved in enterocyte lineage specification (18, 71). Once
specified, intestinal cells continue to proliferate until they receive
a differentiation signal that has yet to be identified. However,
in vitro cell culture experiments have shown that cell-cell contact can trigger differentiation and therefore substitute the in vivo signal. In Caco-2 cells, the establishment of
cell-cell contact is a critical step initiating both cell cycle exit
and induction of the differentiation process (30, 31, 34-37, 59, 61,
72). Indeed, as with various clones of Caco-2 cell line (35, 59,
72), the Caco-2/15 clone has been extensively characterized for its
ability to differentiate gradually between days 0 and 20 of
post-confluence (34, 36, 37, 41). For instance, sucrase-isomaltase
transcription increases as soon as Caco-2/15 cells reach confluency
(73). In this regard, junctional cell interactions play an important
role in the control of cell differentiation during intestinal ontogeny
and the continuous cell renewal of the mature organ (74, 75). Our
results illustrate a pathway by which cell-cell contacts can modulate
enterocyte differentiation through activation of a distinct MAPK, the
p38 MAPK. With respect to this, it has been observed that the p38
pathway is more efficiently activated in confluent muscle cells than in
subconfluent myocytes cultured under the same conditions (33).
Furthermore, we have recently shown that in contrast to p38 MAPK,
p42/p44 MAPK (30) and p46/p54
JNK3 activities dramatically
decreased as soon as Caco-2/15 cells reached confluence and began to
differentiate. Hence, persistent activation of p38 in differentiating
Caco-2/15 cells, in the absence of a parallel JNK activation,
distinguishes this pathway from those activated in response to stress
or cytokines (24, 27-29, 76). Similar observations were recently
reported in differentiating muscle cells (33) and in PC12 (77). Recent
data have shown that assembly of E-cadherin-mediated adherens junctions
is sufficient to trigger the activation of the PI3-kinase/Akt (78) and
p42/p44 MAPK cascades in renal epithelial cells (79). However, the
exact mechanisms through which cell-cell contacts activate the p38 MAPK pathway in intestinal epithelial cells remain to be determined. Cell-cell contacts may stimulate the p38 pathway by silencing the
activity of a mitogen-dependent factor (e.g. a
phosphatase). All together, these observations underscore the
importance of cell density in the activation of p38 during cell differentiation.
Members of the CDX family act within a regulatory network that
establishes the differentiated phenotype of intestinal epithelial cells. Indeed, these homeobox proteins activate the expression of many
intestine-specific genes (68). p38 MAPK appears to be a potential
activator of CDX2/3 and could control intestinal differentiation. The
fact that CDX2/3 plays a role in mediating p38 function in the
activation of sucrase-isomaltase transcription and enterocyte differentiation is suggested by the following: 1) the inhibition of p38
activity with the SB203580 inhibitor repressed CDX3-induced sucrase-isomaltase gene transcription in a dose-dependent
manner; 2) the transcriptional activity of a Gal4-CDX3 fusion protein was reduced by SB203580; 3) similar to CDX2 (11), p38 MAPK activity was
primarily localized in the nucleus of villus cells; 4) CDX3 specifically associated with p38 MAPK both in vitro and
in vivo. p38 MAPK could be targeted to the transcription
factor CDX2/3 by a docking domain that is distinct from the
phosphoacceptor motifs. In fact, we have found a docking domain for p38
homologous to the general consensus sequence
(Arg/Lys)-Xaa-Xaa-Xaa-Xaa-(Leu/Ile)-Xaa-(Leu/Ile) (67) and localized
between amino acids at positions 19-26 of the transactivation domain
of CDX2 (17) and CDX3 proteins (48).
Four isoforms of p38 are known ( Recently, we reported that p42/p44 MAPK activity was repressed during
the differentiation of Caco-2/15 cells (30). However, significant
levels of activated MAPK were detected in differentiated Caco-2/15
cells, predominantly p42 MAPK. We demonstrated that inhibition of MEK
activation during differentiation interfered with sustained activation
of p42 MAPK and sucrase-isomaltase protein expression, consistent with
the conclusion that p42 MAPK is involved in the regulation of
sucrase-isomaltase expression in Caco-2/15 cells. However, our data
suggest that the p42/p44 and p38 MAPK pathways also exhibit distinct
activities. First, p42/p44 MAPK and p38 activities are differentially
modulated; p38 is induced whereas p42/p44 MAPK are strongly reduced in
differentiating intestinal cells. Second, inhibition of p42/p44 MAPK
with PD98059 only partially prevented sucrase-isomaltase protein
expression (30) and did not inhibit the expression of alkaline
phosphatase and villin, whereas the inhibition of p38 with SB203580
severely attenuated the expression of various intestinal specific
markers. Third, the transcriptional activity of Gal4-CDX3 fusion
protein was reduced by SB203580 but not by PD98059. These differences
suggest that these two pathways perform distinct functions and that
their combined activity may be required for the complete
differentiation process, as recently demonstrated in muscle cells
(33).
The marked difference observed in sucrase-isomaltase and alkaline
phosphatase activities but not in lactase and DPPIV activities in
SB203580-treated cells confirms that expression of these brush border
enzymes in Caco-2 cells is regulated in different ways (34, 70).
Furthermore, these results suggest that p38 MAPK targets specific
cellular functions rather than the overall program of cell
differentiation. Current experiments are in progress to identify other
signaling pathways activated early during intestinal differentiation.
EGF plays a major role in intestinal epithelial cell proliferation and
maturation (13). Caco-2/15 cells grown in the presence of high
concentrations of EGF exhibited increased DNA synthesis and
proliferation and formed poorly differentiated multilayers (70). We
have shown that EGF inhibits p38 activity as well as sucrase-isomaltase
expression. Hence, the p38 pathway may well be an important target for
inhibition of the intestinal differentiation program by growth factors
in proliferating cells.
An important role for p38
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and the enhanced
chemiluminescence (ECL) immunodetection system were obtained from
Amersham Pharmacia Biotech. Antiserum that specifically recognizes
p38
on Western blots (40) was a kind gift from Dr. J. Landry (Laval
University, Québec, Canada). Rabbit polyclonal antibodies against
phosphorylated and active forms of p38 MAPK were from New England
Biolabs (Mississauga, Ontario, Canada). Mouse monoclonal antibody
against pRb (14001A) was purchased from PharMingen (Mississauga,
Ontario, Canada). Monoclonal antibody HSI-14 (41) against
sucrase-isomaltase was kindly provided by Dr. A. Quaroni (Cornell
University, Ithaca, NY). Monoclonal antibody CII10 recognizing the
89-kDa apoptotic fragment and the 113-kDa non-cleaved fragment of
poly(ADP-ribose) polymerase (PARP) was a kind gift from Dr. G. G. Poirier (Laval University, Québec, Canada). Polyclonal antibodies
against CDX2/3 protein were provided by Dr. D. J. Drucker
(University of Toronto, Ontario, Canada) (42). The monoclonal HA
antibody raised against a peptide from influenza hemagglutinin HA1
protein was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).
Goat anti-rabbit IgG-fluorescein isothiocyanate (FITC) and goat
anti-mouse IgG-fluorescein isothiocyanate were from Roche Molecular
Biochemicals. The specific inhibitors of MEK1/2 (PD98059) and of
p38
/
(SB203580) were purchased from Calbiochem. EGF was obtained
from Collaborative Biomedicals (Bedford, MA), and insulin was from
Connaught Novo Laboratories (Willowdale, Ontario, Canada). All other
materials were obtained from Sigma-Aldrich unless stated otherwise.
80 °C. For
indirect immunofluorescence, sections were fixed with 2% formaldehyde
in phosphate-buffered saline (pH 7.4; 45 min, 4 °C), before
immunostaining as described previously (37). Negative controls (no
primary antibody) were included in all experiments. Nuclei were stained
with propidium iodide as per instructions of the manufacturer
(Molecular Probes, Eugene, OR).
-mercaptoethanol, 0.005% bromphenol blue, 1 mM phenylmethylsulfonyl fluoride (PMSF)). Proteins (40 µg) from whole cell lysates were separated by SDS-polyacrylamide gel
electrophoresis (PAGE) in 7.5 or 10% gels. Proteins were detected immunologically following electrotransfer onto nitrocellulose membranes
(Amersham Pharmacia Biotech). Protein and molecular weight markers
(Bio-Rad) were localized by staining with Ponceau Red. Membranes were
blocked for 3 h at 25 °C in phosphate-buffered saline
containing 10% powdered milk. Membranes were then incubated overnight
with primary antibodies in blocking solution and with horseradish
peroxidase-conjugated goat anti-mouse or anti-rabbit (1:1000) IgG for
1 h. The blots were visualized by the Amersham Pharmacia Biotech
ECL system. Protein concentrations were measured using a modified Lowry
procedure with bovine serum albumin as standard (44).
-glycerophosphate). Lysates (400 µg) cleared by centrifugation (10 000 × g, 10 min) were incubated for 2 h at
4 °C with protein A-Sepharose (Amersham Pharmacia Biotech) that had
been preincubated for 1 h with anti-p38
. Immunocomplexes were
then washed four times with ice-cold lysis buffer and three times with
ice-cold kinase buffer (20 mM para-nitrophenyl
phosphate, 10 mM MgCl2, 1 mM
dithiothreitol, 30 mM HEPES, pH 7.4) before performing the kinase assay. The kinase reaction was initiated by incubating the
immunocomplexes at 30 °C in the presence of the substrate myelin
basic protein (MBP) and [
-32P]ATP at 20-100
µM, 1-5 µCi/assay. After 30 min, the reaction was
stopped by addition of Laemmli's buffer. Radiolabeled substrates were
separated from immunocomplexes by SDS-PAGE and autoradiographed. Incorporation of 32P by MBP was linear over the
course of the kinase assay.
183 to
+54 cloned upstream of the luciferase gene of the pGL2 reporter
construct as described previously (Dr. P. G. Traber, University of
Pennsylvania, Philadelphia) (45). Plasmid E2F SV40-luc, which contains
a high affinity E2F-binding site from the dihydrofolate reductase
(DHFR) promoter coupled to a luciferase gene (46, 47), was a kind gift
of Dr. P. Farnham (University of Wisconsin). The expression vectors for
wild-type p38
and the dominant-negative mutant p38
(kindly
provided by Dr. J. Pouysségur, Université de Nice, Nice,
France) were previously cloned into pECE vector. The hamster CDX3
expression vector was a gift from Dr. W. J. Rutter (University of
California, San Francisco) (48).
-tubulin probe.
Hybridizations were performed with a random-primed
32P-labeled probe (Amersham Pharmacia Biotech) of a
PCR-amplified human CDX2 fragment from nucleotides 1102 to 1706.
per
well. In some experiments, 0.05 µg of wild-type CDX3 expression
vector was co-transfected. One day after transfection, cells were
treated with or without 2-20 µM of SB203580 for 24 h, and luciferase activity was measured. The pRL-SV40
Renilla luciferase vector (Promega, Madison, WI) was used as
a control for transfection efficiency. Two days after transfection,
luciferase activity was measured according to the Promega protocol.
58 to
33, 5'-AGGGTGCAATAAAACTTTATGAGTAG-3'; SIF3, the HNF1
DNA-binding site,
178 to
154, 5'-GTACAATTACTAATTAACTTAGATT-3'; SIF4, the E4BP4 DNA-binding site,
142 to
123,
5'-AACATTTATGTAAACTACTT-3' (45).
-D-galactopyranoside for 1.5-3 h. Cells were recovered and resuspended in 1.5 ml of SB
buffer (16 mM sodium phosphate, pH 7.4, 150 mM
NaCl, 15% glycerol, 0.02% Triton X-100, 1 mM
dithiothreitol, 15 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml
pepstatin A) and sonicated. Triton X-100 was added to the lysates to a
final concentration of 1%. The bacterial lysates were incubated on ice
for 15 min and centrifuged at 12,000 rpm for 15 min. The supernatants
were recovered and mixed with 55 µl of a 1:1 suspension of
glutathione-Sepharose 4B beads, and the mixture was rotated at 4 °C
for 1 h. The beads were then washed extensively in SB buffer
and used for in vitro binding assays, as described
(57).
-glycerophosphate). Lysates were centrifuged at 15,000 × g for 10 min. 5 µg of GST-CDX3 or GST proteins were coupled to 50 µl of glutathione-Sepharose (Amersham Pharmacia Biotech). The Caco-2/15 lysates were incubated for 2 h at 4 °C with the immobilized fusion proteins by end-over-end rotation. The
beads were washed four times with the lysis buffer. Laemmli buffer was
added to the beads, and the mixture was boiled for 5 min. Bound
proteins were visualized by SDS-PAGE (9% acrylamide gels) and
immunoblotting as described above. In other experiments, the beads were
washed four times with lysis buffer followed by three times with
ice-cold kinase buffer before performing the kinase assay in the
presence or absence of 0.1-20 µM SB203580.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MAPK in the Human Fetal Intestinal
Epithelium--
Phosphorylation and activity of p38 MAPKs were
investigated in intact fetal intestinal epithelium (20 weeks of
gestation). It is generally agreed that by 16-18 weeks of gestation,
the overall morphological appearance of the small intestine and the
expression of most of the functional markers including
sucrase-isomaltase are comparable to those in adult intestine (3, 58).
However, lactase represents an exception, with its activity increasing between 36 and 40 weeks of gestation (58). The use of a specific antibody against p38, phosphorylated on the TGY motif, revealed that
phosphorylated and active p38 MAPKs were mostly localized in the nuclei
of all villus cells (Fig. 1A,
see arrows), whereas the intensity of staining was
significantly decreased in the crypt.
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Fig. 1.
Expression of phosphorylated p38 MAPK in the
human fetal intestinal epithelium. Frozen sections of fetal
intestine at 20 weeks of gestation were stained with antibodies to
phospho-p38 MAPK (A). Nuclei were stained with propidium
iodide (B). The crypt-villus axis is oriented perpendicular
to figures in both panels, with the crypt at the bottom. Along the
crypt-villus axis, phosphorylated p38 staining was mostly detected in
the nuclei of all villus epithelial cells (see arrows).
Original magnification, × 97.
2) and 100% confluence (day 0), and 3, 6, 10, 16, 25, and 31 days
post-confluence, and analyzed by Western blot to confirm timing of
induction of sucrase-isomaltase protein expression. Consistent with
previous observations (30, 34, 37), sucrase-isomaltase protein levels
significantly increased at 3 days post-confluence (Fig.
2A). Expression and kinase
activity of p38
MAPK were also analyzed by immunoprecipitation. As
shown in Fig. 2B, p38
abundance did not change with
differentiation of Caco-2/15 cells. However, differential regulation of
p38
kinase activity was observed during the differentiation of
Caco-2/15 cells. As demonstrated in Fig. 2B,
immunoprecipitated p38
exhibited very low basal activity in
phosphorylating MBP in subconfluent growing Caco-2/15 cells, in
contrast to a dramatic induction of p38
activity when cells reached
confluence (day 0). This activation persisted during cell differentiation. Furthermore, Western blot analysis with an antibody recognizing the biphosphorylated and active p38 MAPK isoforms revealed
that p38
phosphorylation significantly increased as soon as
Caco-2/15 cells reached confluence (Fig. 2C). These results imply that p38
activation precedes the induction of
sucrase-isomaltase, a differentiation marker. Of note, p38
protein
was never detected in Caco-2/15 cells by Western blotting, and very low
levels of RNA were detected by reverse transcriptase-PCR
analysis.2 Thus, p38
MAPK
activation may be functionally linked to intestinal differentiation.
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Fig. 2.
Differentiation of intestinal epithelial
cells, correlation with activation of p38 MAPK phosphorylation and
activity. A and C, Caco-2/15 cells were
harvested at 70 ( 2) and 100% confluence (day 0) and 3, 6, 10, 16, 25, and 31 days post-confluence. Cell extracts (60 µg) were
separated by 10% SDS-PAGE, and proteins were analyzed by Western
blotting. A, expression of sucrase-isomaltase. C,
phosphorylation of p38 MAPK was analyzed with specific antibodies
against the active phosphorylated form of p38. B, Caco-2/15
cells were harvested at 70 (
2) and 100% confluence (day
0) and 3, 6, 10, 16, 25, and 31 days post-confluence. Cell
extracts (400 µg) were immunoprecipitated (Ip) with a
specific antibody to p38
. The levels of immunoprecipitated p38
were analyzed by Western blotting, and the kinase activity of p38 is
demonstrated by the phosphorylation of MBP. D, cell lysates
were prepared from subconfluent growing HIEC and from PCDE. Cell
extracts (60 µg) were separated by 10% SDS-PAGE, and proteins were
analyzed by Western blotting for sucrase-isomaltase expression and
phosphorylation of p38. Similar results were obtained in three
different experiments.
/
MAPKs (62), was tested on dihydrofolate
reductase (DHFR) expression in subconfluent Caco-2/15 and HIEC cells.
The DHFR gene, which is required for DNA synthesis and is transcribed at the G1/S transition, contains E2F-dependent
binding sites in the promoter. In addition, microinjection of E2F into
quiescent fibroblasts provokes S phase re-entry, underscoring the
importance of E2F in cell growth control (47). Therefore, the plasmid
construction containing the E2F-responsive DHFR promoter linked to a
luciferase reporter gene represents a sensitive reporter of cell cycle
progression and S phase entry (46). When the p38
/
MAPKs were
blocked with the SB203580 compound (2-20 µM),
E2F-dependent luciferase expression was not significantly
affected in either cell line (Fig.
3A). In contrast, the MEK1
inhibitor PD98059 significantly reduced E2F-regulated reporter gene
expression by 50% in Caco/15 cells and by 81% in HIEC compared with
control untreated cells. These results confirm our previous
observations (30) whereby the MEK>p42/p44 MAPK cascade is required for
intestinal cell cycle progression.
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Fig. 3.
Inhibition of p38 MAPK has no effect on cell
cycle progression and survival of Caco-2/15 cells. A,
subconfluent Caco-2/15 and HIEC cells (40-50% of confluency) were
transfected with 0.1 µg of DHFR-luciferase reporter. One day after
transfection, cells were exposed to 2-20 µM SB203580
(SB) or 20 µM PD98059 (PD) for
24 h, lysed (the cells were still not confluent), and luciferase
activity measured. The increase in luciferase activity was calculated
relative to the Me2SO (DMSO) level of
DHFR-luciferase, which was set at 1. Results are the mean ± S.E.
of at least three separate experiments. *, significantly different from
control at p < 0.05 (Student's t test).
B, confluent Caco-2/15 cells (day 0) were treated with or
without 20 µM SB203580 for 3 and 6 days and lysed, and
proteins were separated by 10% SDS-PAGE. Phosphorylation of pRb
proteins was analyzed with specific antibodies against the active
(lower band) as well as the inactive (upper
bands) forms of the proteins. PARP cleavage was analyzed by
Western blotting as described under "Experimental Procedures."
Similar results were obtained in two different experiments.
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Fig. 4.
Modulation of intestinal cell differentiation
by SB203580. A, confluent Caco-2/15 cells (day
0) were treated with Me2SO or 20 µM
SB203580 and were harvested at 3, 7, and 12 days post-confluence. 50 µg of cell extracts were separated by 10% SDS-PAGE, and proteins
were analyzed by Western blotting to determine the levels of expression
of sucrase-isomaltase, villin, and actin. B, 1 day
post-confluent Caco-2/15 cells were transfected with 0.1 µg of
sucrase-isomaltase-luciferase reporter vector and 0.1 µg of the
expression vector pECE alone or containing epitope-tagged wild-type
p38 (wt) or dominant-negative mutant of p38
(Thr180-Gly-Tyr182/Ala180-Gly-Phe182)
(DN). One day after transfection, cells were exposed to
5-50 µM SB203580 for 24 h, and luciferase activity
was measured. The increase in luciferase activity was calculated
relative to the Me2SO level (0) of
sucrase-isomaltase-luciferase, which was set at 1. Results are the
mean ± S.E. of at least three separate experiments. *,
significantly different from control at p < 0.05 (Student's t test). C, 6 days post-confluent
Caco-2/15 cells were exposed to 20 µM SB203580 for 6 days
and lysed at day 12, and proteins were separated by 10% SDS-PAGE.
Sucrase-isomaltase protein expression was analyzed by Western blotting.
D, primary cultures of human differentiated enterocytes
(PCDE) were isolated, and after 2 days cells were exposed to
20 µM SB203580 for 2 and 4 days and lysed, and proteins
were separated by SDS-PAGE. Sucrase-isomaltase and keratin-18 protein
expression were analyzed by Western blotting. Similar results were
obtained in two different experiments.
Modulation of cell differentiation by SB203580 in Caco-2/15 cells
, ectopic expression of the dominant-negative mutant of
p38
significantly reduced sucrase-isomaltase gene expression by
61%. Collectively, these results indicate that p38 activation is an
early and necessary event for activation of the intestinal
differentiation program, preceding the induction of various
differentiation markers.
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Fig. 5.
Transactivation activity of CDX2/3 is
down-regulated by inhibition of the p38 pathway. A,
expression of CDX2. Total RNA was extracted from COS cells (as negative
control) and from Caco-2/15 cells at different periods of confluence
( 2, 0, 3, and 6 days post-confluence), treated with or without 20 µM SB203580, and analyzed by Northern hybridization using
a 32P-labeled human CDX2 probe. A murine tubulin probe was
used to evaluate the relative amounts of mRNA transferred to the
membrane. B, modulation of the transcriptional activity of
CDX2 by SB203580. Upper panel, 1 day post-confluent
Caco-2/15 cells were transfected with 0.1 µg of the
sucrase-isomaltase-luciferase reporter vector and 0.05 µg of the CDX3
expression vector or the pBAT vector (EV). One day after
transfection, cells were exposed to 2.5-20 µM SB203580
for 24 h, and luciferase activity was measured. Lower
panel, 1 day post-confluent Caco-2/15 cells were transfected with
0.1 µg of the pFR-luciferase reporter vector and 0.05 µg of the pM2
expression vector encoding the transactivation domain of CDX3 (amino
acids 1-180) fused to Gal4(DBD). One day after transfection, cells
were exposed to 20 µM SB203580 or 20 µM
PD98059 for 24 h, and luciferase activity was measured. The
increase in luciferase activity was calculated relative to the empty
vector level of sucrase-isomaltase-luciferase, which was set at 1. Results are the mean ± S.E. of at least three separate
experiments. *, significantly different from control at
p < 0.05 (Student's t test).
and
HNF1-
(68). In addition, a negative cis-acting element SIF-4 may be
a binding site for the E4BP4 transcriptional repressor protein (11).
Electrophoretic mobility shift experiments were performed to determine
whether the DNA-binding capacity of these transcription factors was
affected by SB203580. As shown in Fig. 6,
binding of nuclear proteins to SIF-1 was not affected by using extracts
prepared from SB203580-treated cells except for increased binding
observed at day 6 post-confluence, which was reproducibly blocked by
SB203580. Furthermore, binding of nuclear proteins to the SIF-3 and
SIF-4 oligonucleotides was unchanged with extracts prepared from
SB203580-treated cultures.
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Fig. 6.
Electrophoretic mobility shift assay of SIF1,
SIF3, and SIF4 DNA-binding proteins in extracts prepared from control
and SB203580-treated Caco-2/15 cells. Binding of nuclear proteins
to the SIF1 (CDX2), SIF3 (HNF1), and SIF4
(E4BP4) elements was assessed in Caco-2/15 cells that were
incubated in either medium alone ( ) or medium containing 20 µM SB203580 since day 0. Cells were harvested at
subconfluence (
2), confluence (day 0), or 2, 6, 9 and 12 days post-confluence. Nuclear extracts were prepared and mixed
with 32P-labeled double-stranded oligonucleotides.
DNA-protein complexes were separated from the free probe on a native
polyacrylamide gel. The results are representative of two independent
experiments.
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Fig. 7.
Specific interaction of CDX2/3 proteins with
p38 MAPK. A, CDX3 associates with p38 kinase in
vitro. Lysates from newly confluent Caco-2/15 cells were prepared
and incubated with 4 µl of p38 antiserum bound to protein A-Sepharose
(Ip -p38) or 5 µg of GST alone (not shown)
or with purified GST-CDX3 bound to glutathione-Sepharose
(pull-down GST-CDX3). The beads were washed and resuspended
in SDS sample buffer, and the bound material was transferred onto
nitrocellulose after SDS-PAGE. Immunological detections were performed
using antibodies recognizing p38
MAPK. B, p38
phosphorylates CDX3 in a pull-down assay. Lysates from newly confluent
Caco-2/15 cells were prepared and incubated with 5 µg of GST alone
(not shown) or with purified GST-CDX3 bound to glutathione-Sepharose.
The beads were washed four times with lysis buffer followed by three
times with ice-cold kinase buffer before performing the kinase assay in
the presence or in the absence of 0.1-20 µM SB203580.
The kinase activity is demonstrated by the phosphorylation of GST-CDX3.
Similar results were obtained in three different experiments.
C, phosphorylation of CDX3 by immunoprecipitated p38 MAPK
in an in vitro kinase assay. Kinase assays were performed
for 30 min at 30 °C with 1 or 5 µg of GST-CDX3 or 2 µg of MBP,
as described under "Experimental Procedures." Similar results were
obtained in three different experiments. D,
co-immunoprecipitation of CDX3 with HA-p38. Co-transfection of CDX3 and
HA-p38
expression vectors was performed in 293T cells. 24 h
after transfection, cells were lysed. Immunoprecipitations
(Ip) of HA-p38
were performed using antibody against HA
and were analyzed by SDS-PAGE followed by electrotransfer onto
nitrocellulose. Immunological detections were performed with an
antibody against CDX3 or an antibody against HA. Immunoblots of total
lysates show the levels of HA-p38 and CDX3 expression. Similar results
were obtained in three different experiments.
, and co-immunoprecipitations were
performed on total cell lysates. As shown in Fig. 7D
(lane 2), CDX3-p38
association was easily detected upon
immunoprecipitation of HA-p38
in 293T cells. Parallel control
experiments using non-transfected cells did not precipitate the CDX3
protein under similar conditions (Fig. 7D, lane 1).
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Fig. 8.
EGF represses p38 MAPK activity and
sucrase-isomaltase expression in differentiating Caco-2/15 cells.
Confluent Caco-2/15 cells were exposed to 100 ng/ml EGF for 15 days, in
the presence (+) or absence ( ) of 20 µM PD98059, and
lysed. Sucrase-isomaltase protein expression and p42/p44 MAPK
phosphorylation were analyzed by Western blotting. p38 MAPK activity
was assessed as described under "Experimental Procedures." Similar
results were obtained in three different experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
, and
) (76). We
suggest that p38
is the major isoform involved in differentiation of
intestinal cells because 1) the inhibitor SB203580, specific to the p38
and
isoforms, inhibited the differentiation of Caco-2/15 cells;
2) specific immunoprecipitation of the p38
isoform from Caco-2/15
cell extracts significantly phosphorylated GST-CDX3; and 3) the p38
protein was not detected in differentiating Caco-2/15 cells and primary
cultures of differentiated enterocytes (data not shown). Interestingly,
the p38
isoform was mostly expressed in undifferentiated human
intestinal crypt cells (data not shown). Further studies are needed to
elucidate the role of the p38
isoform in intestinal cells.
MAPK in various mammal cell
differentiation processes also has been proposed recently. Adipocytic differentiation of 3T3-L1 fibroblasts induced by insulin was blocked by
expression of dominant-negative p38
or incubation of the cells with
SB203580 (32, 80). The transcription factors CCAAT/enhancer-binding protein
(C/EBP
) and peroxisome proliferator-activated receptor
(PPAR
) may be p38 targets during adipogenesis (80). In addition, differentiation of C2C12 and L8 myoblasts to myotubes was also mediated
by p38
activation (81, 82). The stimulation of muscle-specific gene
expression by p38 was apparently mediated by the myocyte enhancer
factor-2C (MEF2C) transcription factor, a p38 substrate known to be
essential in myogenesis (33, 56). Thus, the function of p38 in cell
differentiation correlates with the regulation of the activity (usually
associated with phosphorylation) of different transcription factors,
for example C/EBP, MEF2, and CDX2/3 in adipocyte, muscle, and
intestinal cell precursors. Although further studies are needed to
pinpoint the upstream pathways activating p38 in committed cells
induced to differentiate, our study provides novel fundamental insights
into the function of p38 in the early events of intestinal epithelial differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Anne Vézina and José Cristobal Aliaga for technical assistance. We also thank Pierre Pothier for the critical reading of the manuscript. We thank Drs. C. Poulin and F. Jacot, obstetricians from the Département de la Santé Communautaire du Center Universitaire de Santé de l'Estrie, for excellent collaboration in providing the tissue specimens used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported by Canadian Institutes of Health Research Grants MT-14405 and GR-15186.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.
Chercheur-boursier du Fonds de la Recherche en Santé du
Québec. To whom correspondence should be addressed: Dépt.
d'Anatomie et de Biologie Cellulaire, Faculté de Médecine,
Université de Sherbrooke, Sherbrooke, Québec J1H5N4,
Canada. Tel.: 819-564-5271; Fax: 819-564-5320; E-mail:
nrivard@courrier.usherb.ca.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M100236200
2 P. H. Vachon, and J. F. Beaulieu, personal communication.
3 M. Houde, P. Laprise, and N. Rivard, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: EGF, epidermal growth factor; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; DMEM, Dulbecco's modified Eagle's medium; HIEC, human intestinal epithelial cells; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein; DHFR, dihydrofolate reductase; PCR, polymerase chain reaction; DPPIV, dipeptidyl peptidase IV; PARP, poly(ADP-ribose) polymerase; HA, hemagglutinin; PCDE, primary cultures of differentiated enterocytes; pRb, retinoblastoma protein.
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REFERENCES |
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1. | Babyatsky, M. W., and Podolsky, D. K. (1999) Growth and Development of the Gastrointestinal Tract (Yamada, T., ed) , 3rd Ed. , pp. 547-584, J. B. Lippincott, Philadelphia |
2. | Karam, S. M. (1999) Front. Biosci. 4, D286-D298[Medline] [Order article via Infotrieve] |
3. | Ménard, D. (1989) in Growth-promoting Factors and the Development of the Human Gut (Lebenthal, E., ed) , pp. 123-150, Raven Press, Ltd., New York |
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