1 Groupe du Conseil de
Recherches Médicales sur le Développement Fonctionnel et la
Physiopathologie du Tube Digestif, The intracellular signaling pathways responsible
for cell cycle arrest and establishment of differentiated cells along
the gut axis remain largely unknown. In the present study, we analyzed the regulation of p42/p44 mitogen-activated protein kinase (MAPK) in
the process of proliferation and differentiation of human intestinal cells. In vitro studies were done in Caco-2/15 cells, a human colon
cancer cell line that spontaneously differentiates into an enterocyte
phenotype. In vivo studies were performed on cryostat sections of human
fetal intestinal epithelium by indirect immunofluorescence. We found
that inhibition of the p42/p44 MAPK signaling by the PD-98059 compound
or by ectopic expression of the MAPK phosphatase-1 strongly attenuated
E2F-dependent transcriptional activity in Caco-2/15 cells. p42/p44 MAPK
activities dramatically decreased as soon as Caco-2/15 cells reached
confluence. However, significant levels of activated p42 MAPK were
detected in differentiated Caco-2/15 cells. Addition of PD-98059 during
differentiation interfered with sustained activation of p42 MAPK and
sucrase-isomaltase expression. Although p42/p44 MAPKs were expressed in
both the villus tip and crypt cells, their phosphorylated and active
forms were detected in the undifferentiated crypt cells. Our results
indicate that elevated p42/p44 MAPK activities stimulate cell
proliferation of intestinal cells, whereas low sustained levels of MAPK
activities correlated with G1
arrest and increased expression of sucrase-isomaltase.
epithelium; proliferation; mitogen-activated protein kinase
phosphatases; Ras signaling; sucrase-isomaltase
THE CONTROL OF CELL division and differentiation is
mediated by interactions of signaling molecules at the cell surface,
which ultimately lead to long-term changes in gene expression. In most cell types, the mitogen-activated protein kinase (MAPK) cascade, a
relay of cytoplasmic protein kinases expressed in organisms as diverse
as yeast and mammalian cells, transmits the mitogenic (8, 37) or the
differentiating signals (37, 42). p42 and p44 MAPKs, also termed ERK2
and ERK1 for extracellular signal regulated kinases, the most widely
studied members, are ubiquitously expressed (1, 37, 58). An interesting
feature of this family of kinases is that they require dual
phosphorylation on specific threonine and tyrosine residues for their
activation (1). MAPK activation is mediated by a dual specificity
kinase termed MAP kinase kinase (MEK), which in turn is activated by
Raf oncoproteins (1, 8). Raf proteins appear to be regulated by both
the Ras family of oncoproteins (35, 66) and the recently described 14-3-3 proteins (24). Cellular activation of Ras is mediated by the
guanine nucleotide exchange factor Sos, which, when associated in a
complex with the adaptor protein Grb2, binds to activated receptor
tyrosine kinases (21). Hence, the cascade leading from receptors with
intrinsic tyrosine kinase activity to MAPK activation is relatively complete.
On stimulation, p42/p44 MAPKs translocate to the nucleus where they may
phosphorylate nuclear transcription factors and thus regulate gene
expression (17, 33). Whereas the mechanisms of MAPKs regulation are
relatively well understood, the precise physiological roles of these
enzymes remain to be established. However, activated MAPKs can
phosphorylate and regulate many downstream targets, including
additional kinases, receptors, and transcription factors such as Elk-1,
ATF-2, c-Jun, and CHOP (25, 36, 42, 52). Strong evidence exists for the
critical involvement of MAPKs in the regulation of cell proliferation;
indeed, a close correlation was established between MAPK activation and
DNA synthesis (11, 20, 39), and inhibition of cellular MAPK activity
was shown to block cell cycle progression (10, 46). Recently, it has
been demonstrated that
L-glutamine, tumor necrosis
factor- The intestinal epithelium remains a model of choice to study regulation
of signal transduction pathways during differentiation because it is a
constant differentiating system with a rapid and orderly turnover of
cells (62). Differentiation of cells starts with a sudden loss of their
proliferative ability when they reach the upper third of the crypts;
this process is characterized by marked changes in cell ultrastructure
and by the expression of several new cell products that include the
expression of the gut disaccharidase sucrase-isomaltase (51, 62, 65).
Evers et al. (22) have recently suggested that Caco-2 cell
differentiation may be dependent on the induction of the cell cycle
inhibitor p21Cip. However, the
intracellular signaling pathways responsible for cell-cycle arrest and
establishment of differentiated cells occupying specific positions
along the gut axis remain largely unknown.
In the present study, we analyzed the regulation of p42/p44 MAPK in the
process of proliferation and differentiation of the human fetal
intestinal epithelium (18-20 wk) and of the gut-derived Caco-2
cell line. Caco-2, a human colon cancer cell line, provides a unique
and well-characterized model system for the evaluation of gut
differentiation because these cells undergo differentiation to a small
bowel-like phenotype in culture with microvilli, dome formation, and
expression of sucrase-isomaltase occurring several days after the cells
have reached confluence (5-7, 22, 49, 50). Moreover, our interest
in intestinal development led us to also examine IEC-6 intestinal
epithelial cells, a nontransformed rat jejunal cell line that shares
characteristics of undifferentiated small intestinal crypt cells (4,
26, 53). In this study, we established that p42/p44 MAPK activities are
necessary for both cell cycle progression and differentiation of the
intestinal cells. Indeed, elevated p42/p44 MAPK activities correlated
with E2F-dependent transcriptional activity and DNA synthesis. In
contrast, low sustained levels of MAPK activities correlated with
G1 arrest and increased expression
of sucrase-isomaltase.
Materials.
[ Specimens and indirect immunofluorescence.
Tissues from 10 fetuses varying in age from 18 to 20 wk of gestation,
postfertilization fetal ages were estimated according to Streeter (61),
were obtained from normal elective pregnancy terminations. No tissue
was collected from cases associated with known fetal abnormality of
fetal death. Specimens of adult intestinal jejunum were also analyzed.
Studies were approved by the Institutional Human Subject Review Board.
Segments of fetal small intestine were rinsed with 0.15 M NaCl, cut
into small fragments, embedded in optimum cutting temperature compound,
and quickly frozen in liquid nitrogen (64, 65). Frozen sections 2- to
3-µm thick were spread on silane-coated glass slides and then air
dried 1 h at room temperature before storage at Cell culture.
The rat intestinal epithelial crypt cell line IEC-6 (53) and the cell
line Caco-2/15 were 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 extensively
characterized elsewhere (5, 7, 64). Both cell lines were cultured in
plastic dishes in DMEM (GIBCO) containing 10% FCS, as described
previously (64). Caco-2/15 cells were used between
passages 53 and
78. Studies were performed on culture
at subconfluence (50-70% confluence), confluence, and between 2 and 20 days postconfluence. Primary culture of human differentiated
enterocytes prepared with specimens of small intestines from fetuses
ranging from 18 to 20 wk of age were cultured as described (47) in DMEM
supplemented with 4 mM glutamine, 20 mM HEPES, 50 U/ml penicillin, 50 mg/ml streptomycin, 5 ng/ml recombinant human EGF (all obtained from
GIBCO BRL), 0.2 IU/ml insulin, and 5% FCS. When tested after 5-7
days, these primary cultures of differentiated enterocytes remained
well preserved, and both goblet and absorptive cells exhibit all the
main characteristics of intact villus intestinal cells (47).
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%
MAPK in gel assays.
MAPK activity present in IEC-6 cells was determined in renatured SDS
polyacrylamide gels according to the method of Kameshita and Fujisawa
(30). Briefly, cell extracts (20 µg proteins) were resolved on a 10%
SDS-PAGE gel copolymerized with 0.25 mg/ml myelin basic protein. After
electrophoresis, gels were washed with four changes of 50 mM Tris, pH
8.0, containing 20% propanol. The gels were then denatured with two
changes of 60 min each of 120 ml denaturing buffer containing 6 M
guanidine hydrochloride, 50 mM Tris, pH 8.0, and 5 mM mercaptoethanol.
The enzymes on gel were then renatured with four changes (2 times at 60 min, 1 time overnight, and 1 time at 60 min) of 250 ml renaturing
buffer containing 50 mM Tris, pH 8.0, 0.4% Tween 20, and 5 mM
mercaptoethanol at 4°C. The renatured gels were then incubated in
an assay buffer containing 40 mM HEPES, pH 8.0, 10 mM
MgCl2, 2 mM dithiothreitol, and
0.1 mM EGTA at room temperature for 30 min. The MAPK activities were determined by incubating gels into 20 ml of the assay buffer containing 20 µM ATP and 100 µCi
[32P]ATP at room
temperature for 2 h. The reaction was then stopped by adding 250 ml of
a solution containing 5% TCA and 10 mM sodium pyrophosphate, followed
by washing with the same solution nine times over a period of 1.5 h to
eliminate nonspecific radioactivity in the gels. Gels were exposed to
Kodak X-OMAT film overnight at DNA synthesis reinitiation.
Subconfluent and 1-wk confluent IEC-6 cells were serum-starved for 24 h
in DMEM. Cells were then stimulated for 20 h with 50 ng/ml of EGF in
fresh DMEM medium (without serum) containing 0, 20, or 40 µM of the
MEK-1/2 inhibitor PD-98059. At the end of the incubation, 0.25 µCi/ml
and 3 µM
[methyl-3H]thymidine
were added for an additional 1 h. Cells were thereafter fixed and
washed three times with ice-cold TCA (5%), harvested with 0.1 N NaOH,
and the radioactivity incorporated was counted as previously described
(55).
p21Ras Activation assays.
The assay to measure the activity status of
p21Ras was as described (67).
Briefly, Caco-2/15 cells were harvested at 70% confluence, 100%
confluence (day 0), and 4, 8, and 14 days postconfluence in lysis buffer A
(50 mM Tris · HCl, pH 7.5, 15 mM NaCl, 20 mM MgCl2, 5 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µM
pepstatin A, 1% Triton X-100, 1%
N-octyl glucoside) for 15 min at
4°C. Insoluble material was removed by centrifugation at 12,000 g for 2 min at 4°C. Proteins from
lysates (900 µg) were incubated with 30 µg of GST-RBD fusion
protein, where RBD is amino acids 81-131 of Raf-1 and is the
minimal domain required for binding of Ras-GTP (29), preadsorbed to
glutathione-sepharose beads for 2 h at 4°C. Precipitates were
washed three times with buffer A. The
presence of p21Ras was detected by
resuspending the final pellet in 25 µl of Laemmli buffer, followed by
protein separation on 12.5% polyacrylamide gels, and Western blotting
with antisera OP40 recognizing
p21Ras.
Expression vectors and reporter constructs.
The sucrase-isomaltase reporter construct (SI-luciferase) used for
luciferase assays contains the human sucrase-isomaltase promoter from
residues Northern blots analysis.
Total RNA from subconfluent and postconfluent Caco-2/15 cells were
extracted after homogenization in 4 M guanidine thiocyanate prepared as
suggested by Chirgwin et al. (18) and subjected to electrophoresis on
1% agarose-2.2 M formaldehyde for quality control. For Northern blots,
20 µg of total RNA, quantified by measuring absorbency at 260 nm,
were size fractionated on a 1% agarose gel containing 2.2 M
formaldehyde and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Keene NH). For RNA probes, the membranes were
prehybridized at 65°C for 2-4 h in solution containing
5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM HEPES (pH
6.8), 1% SDS, 5× Denhardt's, 5 mM EDTA, 50% formamide, and
sonicated salmon sperm DNA (100 µg/ml). Hybridization
was performed at 65°C for 16-20 h in the above solution
containing [32]UTP-labeled cRNA
probes (1 × 106 counts per
min/ml); an 18S ribosomal
[32P]dCTP-labeled cDNA
probe was used as a control probe to evaluate RNA loading and transfer.
The sizes of the RNA transcripts were estimated according to the
position of the 18S and 28S rRNAs. Autoradiograms were quantified using
a laser densitometer (Bio-Rad imaging densitometer; Bio-Rad,
Mississauga, ON). The relative densities of the bands were expressed as
arbitrary absorbency units; to correct for differences in loading of
total RNA on Northern blots, a ratio of the relative density of each
band to the relative density of the 18S ribosomal band was calculated
before comparisons were made. The templates (for human MKP-3 cRNA
probe) were linearized with Not I and
labeled with [32P]UTP
using T3 RNA polymerase (Promega transcription riboprobe system;
Promega, Madison, WI).
Transient transfections and luciferase assays.
Subconfluent and 3 days postconfluent Caco-2/15 cells were seeded in a
24-well plate and cotransfected by the lipofectine technique with 0.1 µg of E2F-SV40- or SI-luciferase reporters and 0.1 µg of the
relevant expression vector (pECE or pcDNAneo) containing epitope-tagged
hyperactivated MEK-1 (S218D/S222D) or wild-type MEK-1 (11) or
dominant-negative mutant of p44 (p44MAPK-T192A) (46) or MKP-1 (10). Two
days after transfection, luciferase activity was measured according to
the Promega protocol.
Determination of brush-border lactase-phlorizin hydrolase activity.
Five-day postconfluent Caco-2/15 cells were scraped in water and
sonicated. The homogenates were used for enzymatic determinations of
lactase, assayed according to Dahlqvist as modified by Ménard and
Arsenault (40). Protein content of the homogenates was determined using
a modified Lowry procedure with BSA as standard (48). Data were
expressed in international units (µmol of substrate hydrolyzed per
minute) per gram of protein.
Data presentation and statistical analysis.
Assays were performed in either duplicate or triplicate. The data
presented are from representative experiments performed at least twice.
Results were analyzed by Student's
t-test. Results were considered
significantly different at P < 0.05.
Requirement of p42/p44 MAPK activities for DNA synthesis in IEC-6
and Caco-2/15 cells.
To investigate the role of the p42/p44 MAPK pathway in intestinal cell
proliferation, we first examined the enzymatic activation of p42/p44
isoforms by growth factors in IEC-6 cells. Cell lysates were prepared
from subconfluent and 1-wk postconfluent cells, serum starved for 24 h,
and stimulated for the indicated time periods with serum. As previously
reported in other cell types (9, 20, 39, 46), serum stimulation of
p42/p44 MAPK in subconfluent IEC-6 cells was rapid and maximal within 5 min. p42/p44 MAPK activities then declined slowly to reach their
minimal activities at 3 h (Fig.
1A).
Interestingly, activity of the p44 isoform detected was always superior
to that of p42 at any given time point. The same kinetic of MAPK
activation was observed in the postconfluent IEC-6 cells. However, the
level of stimulation by serum was significantly less than observed in
subconfluent cells, suggesting that cell density might regulate the
enzymatic activity of p42/p44 MAPK.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and epidermal growth factor (EGF) stimulate proliferation
of intestinal crypt cells (IEC-6) by activating the MAPK pathway (26,
45, 54). Furthermore, studies with constitutively active and
dominant-negative mutants of MEK-1 (20) together with pharmacological
blockade experiments (2) also demonstrated the absolute requirement for
the MAPK pathway in neuronal differentiation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP,
[methyl-3H]thymidine,
and the enhanced chemiluminescence (ECL) immunodetection system were
obtained from Amersham (Montréal, PQ). Antiserum E1B, which
specifically recognizes p42 and p44 MAPK on Western blots (38), and
antiserum Alb1, which specifically recognized MAPK phosphatase-1
(MKP-1) and MKP-2 (9), were kind gifts from Drs. Fergus McKenzie and
Jacques Pousségur (Université de Nice, Nice, France).
Rabbit polyclonal antibodies against phosphorylated and active forms of
p42/p44 MAPK were from Promega (Madison, WI). Antiserum OP40
specifically recognizing p21Ras
was from Oncogene Sciences (Calbiochem, San Diego, CA). Monoclonal antibody HSI-14 (6) against sucrase-isomaltase was kindly provided by
Dr. Andrea Quaroni (Cornell University, Ithaca, NY). Goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC, and sheep anti-rabbit IgG-rhodamine were from Boehringer Mannheim (Laval, PQ). The specific inhibitor of
MEK-1/2, PD-98059, was purchased from New England Biolabs (Mississauga, ON). EGF was from Collaborative Biomedicals (Bedford, MA). Insulin was
from Connaught Novo Laboratories (Willowdale, ON). All other materials
were obtained from Sigma unless otherwise stated.
80°C. For
indirect immunofluorescence, sections were fixed with methanol (10 min,
20°C) in PBS (pH 7.4, 45 min, 4°C) before
immunostaining. Negative controls (no primary antibody) were included
in all experiments.
-mercaptoethanol, 0.005% bromphenol blue, 1 mM phenylmethylsulfonyl
fluoride); proteins (40 µg) from whole cell lysates were separated by
SDS-PAGE in 10% gels. Proteins were detected immunologically following
electrotransfer onto nitrocellulose membranes. The blots were then
incubated with different antibodies in blocking solution for 2-4 h
at 25°C and then incubated with horseradish peroxidase-conjugated
goat anti-mouse or anti-rabbit (1:1,000) IgG in blocking
solution for 1 h. The blots were visualized by the Amersham ECL system.
Protein concentrations were measured using a modified Lowry procedure
with BSA as standard (48).
70°C before development.
201 to +139 cloned upstream of the luciferase gene of
the pGL2 reporter construct as described previously (Dr. P. G. Traber,
Univ. of Pennsylvania, Philadelphia, PA) (63). Plasmid E2F
SV40-luciferase, which contains a high- affinity E2F binding site from
the dihydrofolate reductase (DHFR) promoter coupled to a luciferase
gene (60), was a kind gift of Dr. P. Farnham (Univ. of Winscosin). The
DHFR gene, which is required for DNA synthesis, is transcribed at the
G1/S transition and contains E2F-dependent sites in the promoter. Microinjection of E2F into quiescent fibroblasts provokes S phase reentry, underscoring the importance of E2F to cell growth control (32). The pCH110 reporter construct contains the SV40 promoter upstream of a
-galactosidase gene (Pharmacia Biotech, Baie d'Urfé, PQ). The expression
vectors for MEK-1, constitutively active MEK-1 mutant
(MEK-1-S218D/S222D, in which the Raf-1-dependent regulatory
phosphorylation sites were substituted by aspartic residues) (11) and
p44 MAPK, dominant-negative mutant p44 MAPK-T192A were previously
cloned into the pECE and pcDNAneo (Invitrogen) vectors, respectively
(kindly provided by Dr. J. Pouysségur, Université de Nice,
Nice, France) (46). Human MKP-1 construct (kindly provided
by Dr N. Tonks, Cold Spring Harbor, NY) was described previously (10).
Human MKP-3 (GenBank accession number X93920) construct was a gift from
Dr. S. Meloche (Université de Montréal, Montréal, PQ)
(27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 1.
Regulation and role of mitogen-activated protein kinase (MAPK) cascade
in the control of DNA synthesis in IEC-6 cells.
A: 50 µg of IEC-6 cell extracts were
harvested at different time periods (0-3 h after serum
addition) and MAP kinase activity present in the extracts was
determined in renatured SDS polyacrylamide as described in
MATERIALS AND METHODS.
B: subconfluent and 1-wk confluent
IEC-6 cells were arrested for 24 h in serum-free DMEM medium.
Reinitiation of DNA synthesis in response to 50 ng/ml epidermal growth
factor (EGF) was measured as described in MATERIALS
AND METHODS. Results are means ± SE of at least 3 separate experiments. * Significantly different from control at
P < 0.05 (Student's
t-test). PD, PD-98059.
|
Repression of p42/p44 MAPK activities in postconfluent Caco-2/15 cells. Caco-2/15 cells, which differentiate spontaneously to small bowel phenotype when they reach a postconfluent state (7, 50), were harvested at 70% (subconfluent), 100% confluence (day 0), and 3, 6, 10, 12, and 15 days postconfluence and analyzed by Western blot to confirm the timing of induction of the sucrase-isomaltase protein expression. Consistent with previous observations (7, 64), sucrase-isomaltase protein levels significantly increased 6 days postconfluence (Fig. 2B). Within this time course, we next analyzed the protein expression of p42/p44 MAPK. Neither p42 nor p44 abundance changed with differentiation as indicated with the p42/p44 MAPK antibody (E1B), recognizing the phosphorylated and unphosphorylated forms of the enzymes (see MATERIALS AND METHODS; Fig. 2C). However, differential regulation of their kinase activities was observed during the differentiation of Caco-2/15 cells. Western blot analysis with an antibody recognizing the biphosphorylated and active MAPK isoforms revealed that p42/p44 MAPK activities dramatically decreased as soon as Caco-2/15 cells reached confluence to become almost inactive by day 6 postconfluence (Fig. 2D).
Caco-2/15 cell differentiation is associated with progressive
decrease in GTPase activity of Ras.
Although alternative pathways have been suggested, the principal
mechanism of p42/p44 MAPK activation requires active
p21Ras (8, 21, 29). Attempts to
measure p21Ras activation using
the standard immunoprecipitation technique following metabolic cell
labeling with 32Pi
were unsuccessful (not shown). Hence, we used a novel assay to detect
p21Ras activity based on the
Ras-binding domain of Raf as a specific "trap" to selectively
precipitate p21Ras only in its
GTP-bound state and hence activated form (see
MATERIALS AND METHODS). Figure
3A shows
that p21Ras activity of Caco-2/15
cells was significantly reduced when the cells reached confluence
(day 0), activation which
progressively decreased during their differentiation. However, as
previously observed (14), Western blot performed on cell extracts using antibodies to p21Ras revealed that
expression of p21Ras was
significantly increased during differentiation of these cells (Fig.
3B). Our data suggest that an
impairment to p21Ras function
might be required for the repression of p42/p44 MAPK activities in
confluent Caco-2/15 cells.
|
Role of MKPs in regulation of p42/p44 MAPK activities during
Caco-2/15 cell differentiation.
The observation that p21Ras
remained significantly activated during the early days of
postconfluence led us to investigate whether the activity of p42/p44
MAPK can be negatively regulated by some protein phosphatases in these
cells. To address the potential role of protein serine-threonine
phosphatases in the control of MAPK activities, 2-day confluent
Caco-2/15 cells were treated with okadaic acid, a potent inhibitor of
PP2A and PP1 (19). As shown in Fig.
4A,
treatment of confluent Caco-2/15 cells with okadaic acid decreased
p42/p44 MAPK activities, indicating that PP2A or PP1 is unlikely to
play a major role in the downregulation of p42/p44 MAPK activities in
these cells. We next examined the hypothesis that p42/p44 MAPK might be
negatively regulated by a protein tyrosine phosphatase by treating
confluent cells with the potent inhibitor vanadate (28). Figure
4A also demonstrates that vanadate
treatment alone resulted in activation of p42/p44 MAPK in confluent
Caco-2/15 cells. Moreover, treatment of these cells with a combination
of okadaic acid and vanadate had no additive effect on p42/p44 MAPK
activities compared with vanadate alone (data not shown). Thus these
experiments support the existence of a vanadate-sensitive mechanism
that negatively regulates MAPK isoforms activities in confluent
Caco-2/15 cells.
|
Sucrase-isomaltase gene and protein expression are regulated by MAPK
cascade.
To determine whether MAPK activation plays a significant role in
differentiation, we examined the consequences of blocking or activating
specific components of the MEK/MAPK signaling pathway on
sucrase-isomaltase expression in both subconfluent and confluent Caco-2/15 cells. The luciferase gene driven by the human
sucrase-isomaltase promoter represents a sensitive reporter of
intestinal cell differentiation (63). In transiently tranfected
subconfluent Caco-2/15 cells, sucrase-isomaltase gene expression was
significantly stimulated by more than twofold above control with the
MEK-1/2 inhibitor, PD-98059 (Fig.
5A,
left). In contrast, expression of
the hyperactivated MEK-1 (MEK-1 SS/DD), but not wild-type MEK-1,
significantly reduced below control values sucrase-isomaltase gene
expression by 75%. Interestingly, this inhibitory effect of active
MEK-1 can be overcome by treatment with PD-98059 (data not shown).
These results suggest that the high p42/p44 MAPK activities observed in
subconfluent Caco-2/15 cells negatively regulate sucrase-isomaltase
gene expression. However, activated p42 MAPK was detectable in
differentiated Caco-2/15 cells, suggesting that sustained MAPK
activation may be involved in enterocyte differentiation. When the
MAPK cascade was blocked in 5-day postconfluent cells by the
PD-98059 compound (20 µM), by expression of the
dominant-negative p44 MAPK (p44DN = p44 MAPK-T192A), or by expression
of MKP-1, the sucrase-isomaltase gene expression was significantly
inhibited by 49, 44, and 81%, respectively (Fig. 5A,
right). Conversely, addition of
PD-98059 at day 7-12
postconfluence dose dependently reduced both p42/p44 MAPK activities
and sucrase-isomaltase expression (Fig.
5B) observed at day
12 postconfluence. However, treatment with 20 µM
PD-98059 was shown to significantly reduce the enzymatic activity of
lactase-phlorizin hydrolase [control cells (8.02 ± 1.01 IU)
vs. PD-98059-treated cells (6.48 ± 0.09 IU),
P < 0.05 on day
6 postconfluency]. Moreover, higher
concentrations of PD-98059 were able to completely block p42 MAPK
activity, sucrase-isomaltase expression, and lactase-phlorizin
hydrolase activity but protein synthesis was also reduced (unpublished
observations). However, this inhibitor when used at optimal
concentrations (20-40 µM) allows us to conclude that p42/p44
MAPK cascade activation is required for maximal induction of
sucrase-isomaltase in postconfluent Caco-2/15 cells.
|
Regulation of p42/p44 MAPK activities in human fetal intestinal
epithelium.
MAPKs were further investigated in the intact intestinal epithelium. We
first localized p42 and p44 MAPK isoforms in the human fetal small
intestine by immunofluorescence staining. p44 MAPK was seen mostly
localized in the nuclei of the cells present in the crypt and lower
third of the villus and in cytoplasm and nucleus of the villus cells
(Fig.
6A).
Interestingly, p42 MAPK was found predominantly at the luminal surface
of all enterocytes, according to an increasing crypt-villus gradient of
expression (Fig. 6B). The use of a
specific antibody to the p42 and p44 isoforms phosphorylated on their
TEY motif revealed that the active forms of MAPKs were detected
primarily in the nuclei of epithelial cells present in the crypts and
lower third of the villi (Fig. 6C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mucosal lining of the gut is a constantly renewing system with multiple and diverse functions. As the enterocytes progress from crypt to villus, they loose their ability to proliferate and acquire differentiated characteristics that include among others the expression of the gut dissacharidase sucrase-isomaltase (62). Specific gene expression changes and proteins that mediate growth arrest and induction of differentiation in the gut remain to be fully elucidated. Factors that determine whether cells continue to proliferate or cease dividing and begin to differentiate appear to operate during the first gap phase (G1) of the cell cycle. Activation of p42/p44 MAPKs is necessary for growth factor-dependent proliferation of fibroblasts (10, 46). The two enzymes are coordinately activated during the G0 to G1 transition, and their activity remained elevated up to S phase entry, implicating this family of protein kinases in the control of G1 progression (11, 20, 39). However, in another cell model, the PC-12 cells, it is differentiation that is promoted by a sustained activation of MAPKs in response to nerve growth factor (20, 37). These observations raised the important question of whether activation of the MAPK cascade is involved in the proliferation and differentiation processes of intestinal epithelial cells.
To answer this question, we employed a variety of separate but complementary experimental approaches, using the specific inhibitor of MEK activity PD-98059 (2) and transfection of specific MEK or MAPK constructs. Based on our observations, we can conclude that MAPKs are clear positive regulators of the intracellular pathways leading to intestinal epithelial cell proliferation and differentiation. As previously suggested in other cell systems (20, 37, 57, 58), the intensity and the duration of p42/p44 MAPK activities seem critical for cell-signaling decisions. We provided several evidences that there is a direct relationship between elevated p42/p44 MAPK activities, particularly p44 MAPK activity, and intestinal cell proliferation: 1) p44 MAPK activity is markedly stimulated by serum in IEC-6 cells; 2) inhibition of p42/p44 MAPK activities by treatment of IEC-6 cells with PD-98059 lead to G1 arrest; 3) in subconfluent Caco-2/15 cells, when the MAPK cascade was blocked by PD-98059, by expression of dominant-negative p44 MAPK (p44 MAPK-T192A) or by expression of MKP-1, the E2F-dependent transcriptional activity was significatively repressed; 4) p42/p44 MAPK activities, particularly p44 MAPK, were markedly reduced in postconfluent Caco-2/15 cells, suggesting that MAPK are selectively inactivated during enterocyte differentiation; 5) the p44 isoform is primarily localized in the nucleus of intestinal crypt cells; 6) the phosphorylated and active forms of MAPKs were detected only in the nuleus of undifferentiated crypt cells; 7) the MEK inhibitor PD-98059 significantly increased transcriptional activity of sucrase-isomaltase gene promoter. Furthermore, the forced expression of the constitutive active mutant of MEK-1 in Caco-2/15 cells significantly inhibited transcriptional activity of sucrase-isomaltase gene promoter.
Previous studies reported that transfection of Caco-2 cells with an activated human Val-12 Ha-ras gene repressed the expression of sucrase-isomaltase and villin, suggesting that this oncoprotein and its downstream effectors, which include MAPK (8, 21, 42, 66), may exert a general antagonizing effect on the enterocyte-like differentiation of Caco-2 cells (14). These observations are in agreement with those of Mamajiwalla and Burgess (34), who recently demonstrated that although p42 MAPK was expressed in both crypt and villus cells of adult chicken epithelium it was phosphorylated on tyrosine and active only in the crypt cells (34).
As various Caco-2 cell lines (7, 49, 50), Caco-2/15 cell line has been extensively characterized for their ability to differentiate gradually between day 0 and day 20 (5-7, 64). For instance, the sucrase-isomaltase transcript begins to increase as soon as Caco-2/15 cells reached confluency (day 0) (5). As show herein, significant levels of activated MAPK were detected in differentiated Caco-2/15 cells, predominantly p42 MAPK. This suggests that sustained p42 MAPK activation could be involved in enterocyte differentiation. Using the PD-98059 inhibitor and an antibody recognizing the active forms of p42/p44 MAPKs, we demonstrated that a certain level of MAPK activity is required for maximal increases of sucrase-isomaltase protein levels. Indeed, 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 are involved in the regulation of sucrase-isomaltase expression in Caco-2/15 cells. Moreover, when the MAPK cascade was blocked in 5-day postconfluent cells with the PD-98059 compound, by ectopic expression of the dominant-negative p44MAPK, or by ectopic expression of MKP-1, the sucrase-isomaltase gene expression was significantly inhibited. Most importantly, treatment of primary culture of human differentiated enterocytes with the PD-98059 compound strongly reduced expression of sucrase isomaltase. All of these data support the notion that the p42/p44 MAPK cascade plays a positive role during differentiation of intestinal cells. These results are in apparent disagreement with those of Font de Mora et al. (23) who recently demonstrated that PD-98059 accelerates adipocytic differentiation of 3T3-L1 cells. This antagonistic relationship between MAPK activation and cell differentiation contrasts, however, with observations in PC-12 cells where MAPK is required for neuronal proliferation and differentiation (20, 37). Moreover, activated p42 MAPK was detectable in differentiated C2C12 cells, suggesting that sustained MAPK activation may also be involved in myogenic differentiation (57). Furthermore, insulin-induced differentiation of 3T3-L1 is blocked by MAPK antisense oligonucleotides (56). Taken together, these data indicate that downstream cellular responses initiated by p42/p44 MAPKs may vary and trigger mutually exclusive events (37, 58).
The enzymatic activity of MAPK isoforms is positively regulated by the upstream cascade Ras > Raf > MEK-1/2 and negatively regulated by ill-defined protein phosphatases. In the colon, Ras is probably the most well-studied upstream activator of MAPK. Unfortunately, virtually nothing is known on how upstream activators of MAPKs are regulated during intestinal differentiation. Although the Ki-ras gene is frequently mutated in human colon cancer (12, 44, 59), the Ha-ras protooncogene is highly expressed in most differentiated cells of the intestinal mucosa (14). However, our data indicate that an impairment to p21Ras functions might be required for the maintenance of a differentiated intestinal phenotype since a progressive decrease in p21Ras activity was observed during differentiation of the Caco-2/15 cells.
In the present report, we demonstrated that a significant Ras activity remained detectable at the time Caco-2/15 cells reached confluence. This observation implies that downregulation of the MAPK pathway in newly confluent Caco-2/15 cells does not result from a major failure to activate the upstream protein kinases. The role of protein phosphatases in the regulation of MAPK activities was addressed by a combination of approaches. Treatment of confluent cells with okadaic acid had no stimulatory effect on the enzymatic activity of MAPK isoforms. However, treatment with vanadate restored MAPK activities to a level comparable to what was observed in subconfluent cells. These observations indicate that MAPK is predominantly regulated by a vanadate-sensitive mechanism in confluent Caco-2/15 cells, which most probably involves a protein tyrosine phosphatase. Our results with the inhibitor drugs also suggest that PP2A and PP1 are not major regulators of MAPK activities in Caco-2/15 cells. This is apparently different from the situation prevailing in PC-12 cells where PP2A was identified as the major MKP in cellular extracts (3).
Recently, an increasing number of "dual specificity" phosphatases have been cloned. Members of this family of dual-specificity phosphatases are capable of inactivating p42/p44 MAPK isoforms in vitro and are sensitive to vanadate (31). It was therefore of interest to determine whether one of these phosphatases was upregulated in differentiating Caco-2/15 cells. Interestingly, it was recently reported that the erp transcript (MKP-1 mRNA) was detected in the differentiated villus cells but not in the proliferating cells within the intestinal crypts of the adult intestinal epithelium (43). Consistent with these observations, an increase of the expression of MKP-1 was detected from day 6 postconfluence in Caco-2/15 cells. However, the expression of MKP-3 mRNA was greatly enhanced in cells reaching confluence. It is also of note that the expression of MKP-3 mRNA parallels reduction in p42/p44 MAPK activities during the first days of postconfluence. Interestingly, MKP-3 was localized exclusively in cytosolic compartments of a number of cell types (41). In this respect, MKP-3 appears distinct from other dual specificity phosphatases that are clearly nuclear (10). Experiments using purified proteins as well as expression in COS cells showed that MKP-3 displays clear selectivity for inactivation of ERK MAPK family members (41). This finding, together with our data, could indicate an important role for MKP-3 in inactivating selectively cytosolic p42/p44 MAPKs during induction of Caco-2/15 differentiation. Such compartmentalized regulation of MAPKs following confluence may be of fundamental importance in molecular processes underlying Caco-2/15 cell differentiation. Thus our results are consistent with the hypothesis that induction of MKP-1 and MKP-3 might be responsible for the impairment of p42/p44 MAPK activities in the cytoplasm and nucleus of postconfluent Caco-2/15 cells. The involvement of MKP-3 during the nerve growth factor-induced differentiation of PC-12 cells was also recently demonstrated (13). However, whereas MAPK activities remained low through differentiation of confluent Caco-2/15 cells and whereas it is suggested that MKP-1 and MKP-3 may be regulating the activity of MAPK, their expressions decreased significantly after day 6. This suggests that the impairment in the p21Ras function observed in differentiating Caco-2/15 cells is required for the long-term repression of p42/p44 MAPK activities.
We demonstrated here for the first time that p42 and p44 MAPK isoforms are localized in different compartments of the intestinal epithelial cells. The p44 isoform is primarily localized in the nucleus of the crypt cells and in the cytoplasm and nucleus of the villus cells. However, the p42 isoform is primarily detected at the apical surface along the crypt-villus axis. These observations are in agreement with those of Mamajiwalla and Burgess (34) who also recently reported that p42 MAPK was localized to the apex and nuclei of cells throughout the crypt-villus axis of the adult chicken intestinal epithelium.
In summary, our results indicate that p42/p44 MAPK and their regulators are tightly controlled during enterocyte differentiation and implicate the MAPK pathway as a key signaling pathway in the normal development of the intestinal epithelium.
![]() |
NOTE ADDED IN PROOF |
---|
While this work was being reviewed, Taupin and Podolsky (Gastroenterology 116: 1072-1080, 1999) also reported a loss of activation of the MAP kinases p42/p44 in HT29-N2 cells on change to glucose-free growth medium, preceding the change in differentiated phenotype. However, in contrast to our data showing an inhibition of sucrase-isomaltase gene expression with the MEK inhibitor, Taupin and Podolsky demonstrated that the PD-98059 inhibitor enhanced expression of the differentiation markers sucrase-isomaltase, intestinal trefoil factor, and the mucin gene MUC2 in HT29-N2 cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the technical assistance of P. Pothier and P. Chailler. Special thanks go to Dr. J. Morisset for constant encouragement, judicious comments, and fruitful discussion in the course of this work. We wish to thank Drs. C. Poulin and F. Jacot, obstetricians from the Département de la Santé Communautaire du Centre Universitaire de Santé de l'Estrie, for excellent collaboration in providing tissue specimens used in this study.
![]() |
FOOTNOTES |
---|
This research was supported by a group grant from the Medical Research Council of Canada (GR-15186). Nathalie Rivard is a "chercheur-boursier" from the Fonds de la Recherche en Santé du Québec.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. Rivard, Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4 (E-mail: nrivard{at}courrier.usherb.ca).
Received 30 March 1999; accepted in final form 8 June 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahn, N. G.,
J. E. Weier,
C. P. Chan,
and
E. G. Krebs.
Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells.
J. Biol. Chem.
265:
11487-11494,
1990
2.
Alessi, D. R.,
A. Cuenda,
P. Cohen,
D. T. Dudley,
and
A. R. Saltiel.
PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J. Biol. Chem.
270:
27489-27494,
1995
3.
Alessi, D. R.,
N. Gomez,
G. Moorhead,
T. Lewis,
S. M. Keyse,
and
P. Cohen.
Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines.
Curr. Biol.
5:
283-295,
1995[Medline].
4.
Barnard, J. A.,
W. H. Polk,
H. L. Moses,
and
R. J. Coffey.
Production of tranforming growth factor- by normal rat small intestine.
Am. J. Physiol.
261 (Cell Physiol. 30):
C994-C1000,
1991
5.
Basora, N.,
P. H. Vachon,
F. E. Herring-Gillam,
N. Perreault,
and
J. F. Beaulieu.
Relation between integrin 7B
1 expression in human intestinal cells and enterocyte differentiation.
Gastroenterology
113:
1510-1521,
1997[Medline].
6.
Beaulieu, J. F.,
B. Nichols,
and
A. Quaroni.
Post-translational regulation of sucrase-isomaltase in intestinal villus cells.
J. Biol. Chem.
264:
20000-20011,
1989
7.
Beaulieu, J. F.,
and
A. Quaroni.
Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells.
Biochem. J.
280:
599-608,
1991[Medline].
8.
Blenis, J.
Signal transduction via the MAP kinases: proceed at your own RSK.
Proc. Natl. Acad. Sci. USA
90:
5889-5892,
1993[Abstract].
9.
Brondello, J. M.,
A. Brunet,
J. Pouyssegur,
and
F. R. McKenzie.
The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44 MAPK cascade.
J. Biol. Chem.
272:
1368-1376,
1997
10.
Brondello, J. M.,
F. R. McKenzie,
H. Sun,
N. K. Tonks,
and
J. Pouysségur.
Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibroblasts.
Oncogene
10:
1895-1904,
1995[Medline].
11.
Brunet, A.,
G. Pagès,
and
J. Pouysségur.
Constitutively active mutants of MAP kinase kinase (MEK1) induce growth factor-relaxation and oncogenicity when expressed in fibroblasts.
Oncogene
9:
3379-3387,
1994[Medline].
12.
Burmer, G. C.,
and
L. Loeb.
Mutations in the KRAS2 oncogene during progressive stages of human colon carcinoma.
Proc. Natl. Acad. Sci. USA
86:
2403-2407,
1989[Abstract].
13.
Camps, M.,
C. Chabert,
M. Muda,
U. Boschert,
C. Gillieron,
and
S. Arkinstall.
Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.
FEBS Lett.
425:
271-276,
1998[Medline].
14.
Celano, P.,
C. M. Berchtold,
M. Mabry,
M. Carroll,
D. Sidransky,
R. A. Casero,
and
R. Lupu.
Induction of markers of normal differentiation in human colon carcinoma cells by the v-rasH oncogene.
Cell Growth Differ.
4:
341-347,
1993[Abstract].
15.
Charles, C. H.,
H. Sun,
L. F. Lau,
and
N. K. Tonks.
The growth factor-inducible immediate-early gene 3CH134 encodes a protein-tyrosine phosphatase.
Proc. Natl. Acad. Sci. USA
90:
5292-5296,
1993[Abstract].
16.
Chastre, E. S.,
E. Empereur,
Y. di Gioia,
N. El Mahdani,
M. Mareel,
K. Vleminckx,
F. van Roy,
V. Bex,
S. Emami,
D. A. Spandidos,
and
C. Gespach.
Neoplastic progression of human and rat intestinal cell lines after transfer of the ras and polyoma middle T oncogenes.
Gastroenterology
105:
1776-1789,
1993[Medline].
17.
Chen, R. H.,
C. Sarnecki,
and
J. Blenis.
Nuclear localization and regulation of erk- and rsk-encoded protein kinases.
Mol. Cell. Biol.
12:
915-927,
1992[Abstract].
18.
Chirgwin, J. M.,
A. E. Przybyla,
R. J. MacDonald,
and
W. J. Rutter.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[Medline].
19.
Cohen, P.,
C. F. B. Holmes,
and
Y. Tsukitani.
Okadaic acid: a new probe for the study of cellular regulation.
Trends Biochem. Sci.
15:
98-102,
1990[Medline].
20.
Cowley, S.,
H. Paterson,
P. Kemp,
and
C. J. Marshall.
Activation of MAP kinase kinase is both necessary and sufficient for PC12 differentiation and for tranformation of NIH 3T3 cells.
Cell
77:
841-852,
1994[Medline].
21.
Egan, S. E.,
B. W. Giddings,
M. W. Brooks,
L. Buday,
A. M. Sizeland,
and
R. A. Weinberg.
Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation.
Nature
363:
35-51,
1993.
22.
Evers, B. M.,
T. C. Ko,
J. Li,
and
E. A. Thompson.
Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G722-G727,
1996
23.
Font de Mora, J.,
A. Porras,
N. Ahn,
and
E. Santos.
Mitogen-activated protein kinase activation is not necessary for but antagonizes 3T3-L1 adipocytic differentiation.
Mol. Cell. Biol.
17:
6068-6075,
1997[Abstract].
24.
Freed, E.,
M. Symons,
S. G. Macdonald,
F. McCormick,
and
R. Ruggieri.
Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation.
Science
265:
1713-1716,
1994[Medline].
25.
Gille, H.,
A. D. Sharrocks,
and
P. Shaw.
Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter.
Nature
358:
414-418,
1992[Medline].
26.
Goke, M.,
M. Kanai,
K. Lynch-Devaney,
and
D. K. Podolsky.
Rapid mitogen-activated protein kinase activation by transforming growth factor alpha in wounded rat intestinal epithelial cells.
Gastroenterology
114:
697-705,
1998[Medline].
27.
Gopalbhai, K.,
and
S. Meloche.
Repression of mitogen-activated protein kinases ERK1/ERK2 activity by a protein tyrosine phosphatase in rat fibroblasts transformed by upstream oncoproteins.
J. Cell. Physiol.
174:
35-47,
1998[Medline].
28.
Gordon, J. A.
Use of vanadate as protein-phosphotyrosine phosphatase inhibitor.
Methods Enzymol.
201:
477-482,
1991[Medline].
29.
Herrmann, C.,
G. A. Martin,
and
A. Wittinghoffer.
Quantitative analysis of the complex between p21Ras and the Ras-binding domain of the human Raf-1 protein kinase.
J. Biol. Chem.
269:
2901-2905,
1995.
30.
Kameshita, I.,
and
H. Fujisawa.
A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate polyacrylamide gel.
Anal. Biochem.
183:
139-143,
1989[Medline].
31.
Keyse, S. M.
An emerging family of dual specificity MAP kinase phosphatases.
Biochim. Biophys. Acta
1265:
152-160,
1995[Medline].
32.
La Thangue, N. B.
DP and E2F proteins: components of a heterotrimeric transcription factor implicated in cell cycle control.
Curr. Opin. Cell Biol.
6:
443-450,
1994[Medline].
33.
Lenormand, P.,
C. Sardet,
G. Pagès,
G. L'Allemain,
A. Brunet,
and
J. Pouysségur.
Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44 mapk) but not of their activator MAP kinase kinase.
J. Cell Biol.
122:
1079-1088,
1993[Abstract].
34.
Mamajiwalla, S. N.,
and
D. R. Burgess.
Differential regulation of the activity of the p42 kD mitogen-activated protein kinase (p42mapk) during enterocyte differentiation in vivo.
Oncogene
11:
377-386,
1995[Medline].
35.
Marais, R.,
Y. Light,
H. F. Paterson,
C. S. Mason,
and
C. J. Marshall.
Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras amd tyrosine kinases.
J. Biol. Chem.
272:
4378-4383,
1997
36.
Marais, R.,
J. Wynne,
and
R. Treisman.
The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain.
Cell
73:
381-393,
1993[Medline].
37.
Marshall, C. J.
Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.
Cell
80:
179-185,
1995[Medline].
38.
McKenzie, F. R.,
and
J. Pouysségur.
cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition.
J. Biol. Chem.
271:
13476-13483,
1996
39.
Meloche, S.,
K. Seuwen,
G. Pages,
and
J. Pouysségur.
Biphasic and synergistic activation of p44MAPK (ERK1) by growth factors: correlation between late phase activation and mitogenicity.
Mol. Endocrinol.
638:
845-854,
1992.
40.
Ménard, D.,
and
P. Arsenault.
Explant culture of human small intestine.
Gastroenterology
88:
691-700,
1985[Medline].
41.
Muda, M.,
U. Boschert,
R. Dickinson,
J. C. Martinou,
I. Martinou,
M. Camps,
W. Schlegel,
and
S. Arkinstall.
MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.
J. Biol. Chem.
271:
4319-4326,
1996
42.
Nishida, E.,
and
Y. Gotoh.
The MAP kinase cascade is essential for diverse signal transduction pathways.
Trends Biochem. Sci.
18:
128-131,
1993[Medline].
43.
Noguchi, T.,
R. Metz,
L. Chen,
M. G. Mattéi,
D. Carrasco,
and
R. Bravo.
Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a non transmembrane protein tyrosine phosphatase, and effect of ERP on cell growth.
Mol. Cell. Biol.
13:
5195-5205,
1993[Abstract].
44.
Oldham, S. M.,
G. Clark,
L. M. Gangarosa,
R. J. Coffey,
and
C. J. Der.
Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.
Proc. Natl. Acad. Sci. USA
93:
6924-6928,
1996
45.
Oliver, B. L.,
R. I. Sha'afi,
and
J. J. Hajjar.
Tranforming growth factor-alpha and epidermal growth factor activate mitogen-activated protein kinase and its substrates in intestinal epithelial cells.
Proc. Soc. Exp. Biol. Med.
210:
162-170,
1995[Abstract].
46.
Pagès, G.,
P. Lenormand,
G. L'Allemain,
J. C. Chambard,
S. Meloche,
and
J. Pouysségur.
Mitogen-activated protein kinases p42MAPK and p44MAPK are required for fibroblast proliferation.
Proc. Natl. Acad. Sci. USA
90:
8319-8323,
1993
47.
Perreault, N.,
and
J. F. Beaulieu.
Primary cultures of fully differentiated and pure human intestinal epithelial cells.
Exp. Cell Res.
245:
34-42,
1998[Medline].
48.
Peterson, G. L.
A simplification of the protein assay method of Lowry et al. which is more generally applicable.
Anal. Biochem.
83:
346-356,
1977[Medline].
49.
Peterson, M. D.,
W. M. Bement,
and
M. S. Mooseker.
As in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2Bbe cells.
J. Cell Sci.
105:
461-472,
1993
50.
Pinto, M.,
S. Robine-Leon,
M. D. Appay,
M. Kedinger,
N. Triadou,
N. Bussaulx,
B. Lacroix,
P. Simon-Assmann,
K. Haffen,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and proliferation of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell
47:
323-330,
1983.
51.
Podolsky, D. K.
Regulation of intestinal epithelial proliferation: a few answers, many questions.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G179-G186,
1993
52.
Pulverer, B. J.,
J. M. Kyriakis,
J. Avruch,
E. Nikolalaki,
and
J. R. Woodgety.
Phosphorylation of c-jun mediated by MAP kinases.
Nature
353:
670-674,
1991[Medline].
53.
Quaroni, A.,
J. Wands,
R. L. Trelstad,
and
K. J. Isselbacher.
Epitheloid cell cultures from rat small intestine: characterization by morphologic and immunologic criteria.
J. Cell Biol.
80:
248-265,
1979[Abstract].
54.
Rhoads, J. M.,
R. A. Argenzio,
W. Chen,
R. A. Rippe,
J. K. Westwick,
A. D. Cox,
H. M. Berschneider,
and
D. A. Brenner.
L-glutamine stimulates intestinal cell proliferation and activates mitogen-activated protein kinases.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G943-G953,
1997
55.
Rivard, N.,
G. L'Allemain,
J. Bartek,
and
J. Pouysségur.
Abrogation of p27Kip1 by cDNA antisense suppresses quiescence (G0) state in fibroblasts.
J. Biol. Chem.
271:
18337-18341,
1996
56.
Sale, E. M.,
P. G. P. Atkinson,
and
G. L. Sale.
Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis.
EMBO J.
14:
674-684,
1995[Abstract].
57.
Sarbassov, D. D.,
L. G. Jones,
and
C. A. Peterson.
Extracellular signal-regulated kinase-1 and -2 respond differently to mitogenic and differentive signaling pathways in myoblasts.
Mol. Endocrinol.
11:
2038-2047,
1997
58.
Seger, R.,
and
E. G. Krebs.
The MAPK signaling cascade.
FASEB J.
9:
726-735,
1995
59.
Shirasawa, S.,
M. Furuse,
N. Yokoyama,
and
T. Sasazuki.
Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.
Science
260:
85-88,
1993[Medline].
60.
Slansky, J. E.,
Y. Li,
W. G. Kaelin,
and
P. G. Farnham.
A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.
Mol. Cell. Biol.
13:
1610-1618,
1993[Abstract].
61.
Streeter, G. L.
Weight, sitting head, head size, foot length, and menstrual age of the human embryo.
Contr. Embryol.
11:
143-179,
1920.
62.
Traber, P. G.
Differentiation of intestinal epithelial cells: lessons from the study of intestine-specific gene expression.
J. Lab. Clin. Med.
128:
467-477,
1994.
63.
Traber, P. G.,
G. D. Wu,
and
W. Wang.
Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.
Mol. Cell. Biol.
12:
3614-3627,
1992[Abstract].
64.
Vachon, P. H.,
and
J. F. Beaulieu.
Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line.
Gastroenterology
103:
414-423,
1992[Medline].
65.
Vachon, P. H.,
and
J. F. Beaulieu.
Extracellular heterotrimeric laminin promotes differentiation in human enterocytes.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G857-G867,
1995
66.
Vojtek, A. B.,
and
J. A. Cooper.
Rho family members: activators of MAP kinase cascades.
Cell
82:
527-529,
1995[Medline].
67.
Warne, P. H.,
P. R. Viciana,
and
J. Downward.
Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.
Nature
364:
1031-1034,
1993.