From the Département d'Anatomie et Biologie
Cellulaire, Faculté de Médecine, Université de
Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and the
Department of Cell Biology, Institut Cochin,
75014 Paris, France
Received for publication, January 14, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein-tyrosine phosphatase SHP-1 is expressed
at high levels in hematopoietic cells and at moderate levels in many
other cell types including epithelial cells. Although SHP-1 has been shown to be a negative regulator of multiple signaling pathways in
hematopoietic cells, very little is known about the biological role of
SHP-1 in epithelial cells. In order to elucidate the mechanism(s) responsible for the loss of proliferative potential once committed intestinal epithelial cells begin to differentiate, the role and regulation of SHP-1 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 SHP-1 was
expressed in the nuclei of all intestinal epithelial cell models as
well as in epithelial cells of intact human fetal jejunum and colon. Expression and phosphatase activity levels of SHP-1 were much more
elevated in confluent growth-arrested intestinal epithelial cells and
in differentiated enterocytes as well. Overexpression of SHP-1 in
intestinal epithelial crypt cells significantly inhibited dhfr, c-myc, and cyclin D1 gene expression but
did not interfere with c-fos gene expression. In contrast,
a mutated inactive form of SHP-1 had no effect on these genes. SHP-1
expression significantly decreased
Protein phosphorylation and dephosphorylation of tyrosyl residues
are important events involved in the regulation of cell growth and
differentiation. Protein-tyrosine phosphatases
(PTPs)1 act both as positive
and negative regulators of signal transduction (1). Numerous PTPs have
been identified to date, including the extensively studied SHP-1
(initially designated as SHPTP-1, SHP, HCP, and PTP1C) and SHP-2
(initially designated SHPTP-2, Syp, PTP2C, and PTP1D). Whereas SHP-1 is
highly expressed in hematopoietic cells and moderately in many other
cell types such as epithelial cells, SHP-2 has a more widespread
distribution (2-4). Both proteins have similar structures, comprising
two tandem SH2 domains at the N terminus, a single central catalytic
domain and a C-terminal domain. The SH2 domains recruit SHP-1 and SHP-2
to tyrosine-phosphorylated molecules, enabling dephosphorylation to be
performed by the catalytic domain. Understanding the biological roles
of SHP-1 has been vastly invigorated by the discovery that
loss-of-function mutations in the gene encoding SHP-1 are responsible
for the profound immunological dysfunction observed in mice homozygous
for the motheaten (me/me) or allelic viable
motheaten (mev/mev) mutations (5,
6). These mice express either no SHP-1 or a catalytically defective
SHP-1 protein consequent to splice site mutations in the
SHP-1 gene (6). These defects, in turn, engender severe hematopoietic disruption as exemplified by an enormous expansion
and tissue accumulation of myeloid/monocytic cells that lead to patchy
dermatitis, extramedullary hematopoiesis, splenomegaly, and hemorrhagic
pneumonitis resulting in death at about 2-3
(me/me) or 9-12
(mev/mev) weeks. Lymphocyte
ontogeny and function are also dramatically altered. Cells isolated
from me and mev mice have enabled the
identification of SHP-1 target proteins in hematopoietic cells (7, 8).
SHP-1 has been shown to negatively regulate downstream signaling of the
erythropoietin receptor and to dephosphorylate several target molecules
such as c-Kit, the granulocyte/macrophage colony-stimulating factor
receptor, the B and T cell antigen receptor, the adapter protein
SLP-76, the cytosolic tyrosine kinase ZAP-70, and the lymphoid-specific
Src family kinase Lck (7, 9-13). Very little is known on the other hand about the biological roles of SHP-1 in epithelial cells, although
the existence of an epithelium-specific isoform of SHP-1 (the
epithelial and hematopoietic variants differ in the sequence of 4 amino
acids at the N terminus) is suggestive of specific function(s) in these
cells (14). Recently, Keilhack et al. (15) have shown that
SHP-1 is an important downstream regulator of ROS receptor
signaling in epididymal epithelium (15). Furthermore, recent evidence
indicates that SHP-1 associates with and dephosphorylates p120 catenin
in EGF-stimulated A431 cells (16), suggesting a role for this PTP in
the regulation of catenin function and cadherin-mediated epithelial
cell-cell adhesion.
The intestinal epithelium remains a model of choice to study regulation
of signal transduction pathways during cell proliferation and
differentiation largely due to its constant differentiating system with
a rapid and orderly turnover of cells (17). Cell differentiation begins
with a sudden loss of proliferative ability, a process characterized by
marked changes in cell ultrastructure and by the expression of several
newly acquired end products, including the expression of the gut
disaccharidase sucrase-isomaltase (17, 18). Committed intestinal
epithelial cells withdraw from the cell cycle to differentiate during
the G1 phase. Hence, molecules that stimulate or inhibit
G1 phase progression are thereby likely candidates for
controlling cell cycle and differentiation in developing tissue. The
Wnt/ In the present study, Animals and Human Specimens--
Fifteen SHP-1 mutant
mev/mev mice (C57aBL/6J-Hcph
mev (homozygous males) and 12 control animals
(C57BL/6J) were purchased from The Jackson Laboratory (Bar Harbor, ME).
Mice were fed Purina chow ad libitum and kept in a
controlled temperature and light cycle environment (20 °C; 12 h
light, 12 h darkness). All studies were conducted in agreement
with the principles and procedures outlined in the Canadian Guidelines
for Care and Use of Experimental Animals. Tissues from human fetuses
varying in age from 18 to 20 weeks of gestation (post-fertilization
fetal ages were estimated according to Streeter (32)) were obtained
from normal elective pregnancy terminations. No tissue was collected
from cases associated with a known fetal abnormality or fetal death.
Studies were approved by the Institutional Human Subject Review Board.
Indirect Immunofluorescence in Human Intestine--
Segments of
human 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. 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--
Human intestinal epithelial cells (HIEC) were
cultured as described previously (36) in Dulbecco's modified Eagle's
medium (DMEM; Invitrogen) supplemented with 4 mM glutamine,
20 mM HEPES, 50 units/ml penicillin, 50 µg/ml
streptomycin, 5 ng/ml recombinant human EGF (all obtained from
Invitrogen), 0.2 IU/ml insulin (Connaught Novo Laboratories,
Willowdale, Ontario, Canada), and 5% fetal bovine serum (FBS). The rat
intestinal epithelial crypt cell line IEC-6 (37) and the Caco-2/15 cell
line 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 (34, 35, 38-39) and was originally selected as expressing
the highest levels of sucrase-isomaltase among 16 clones obtained by
random cloning. Both cell lines were cultured in DMEM containing 10%
FBS, as described previously (34). Primary cultures of human
differentiated enterocytes (PCDE) prepared from fetal small intestines
ranging from 18 to 20 weeks of age were cultured as described above for
HIEC. When tested after 5-7 days, these primary cultures remained well
preserved, and both goblet and absorptive cells exhibited all the main
characteristics of intact villus intestinal cells (40). Human embryonic
kidney 293 cells (American Type Culture Collection) were cultured in DMEM containing 10% FBS.
Immunofluorescence Microscopy on Cultured Cells--
HIEC,
Caco-2/15, and IEC-6 cells grown on sterile glass coverslips were
washed twice with ice-cold PBS, fixed in methanol/acetone (30-70%)
for 15 min at Expression of Proteins in Intestinal Tissue--
After mice were
sacrificed by cervical dislocation, the jejunum and colon were rapidly
removed and the mucosae scraped and homogenized (1 mg of tissue/50
µl) in Triton buffer (150 mM NaCl, 1 mM EDTA,
40 mM Tris-HCl, pH 7.6, 1% Triton X-100, 0.2 mM orthovanadate) supplemented with protease inhibitors
(0.2 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml
leupeptin, 2 µg/ml pepstatin, 20 µg/ml aprotinin). Homogenates were
cleared by centrifugation (13,000 × g, 10 min).
One-half of the homogenates was mixed in Laemmli's buffer, boiled for
5 min, and frozen until preparation of Western blots; the other half
was stored at 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% Immune Complex Phosphatase Assay--
Cells were lysed on ice
for 10 min with 1 ml/dish of lysis buffer (150 mM NaCl, 1 mM EDTA, 40 mM Tris-HCl, 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).
Lysates (800 µg) cleared by centrifugation (10,000 × g, 10 min) were incubated for 1 h at 4 °C with
protein A-Sepharose (Amersham Biosciences) that had been initially
preincubated for 2 h with anti-SHP-1. Immunocomplexes were washed
four times with ice-cold lysis buffer and three times with ice-cold
phosphatase buffer (25 mM HEPES, pH 7.2, 50 mM
NaCl, 2.5 mM EDTA, 5 mM dithiothreitol) prior
to initiating the phosphatase assay according to Upstate
Biotechnologies, Inc. (Lake Placid, NY), and as described previously
(42). Levels of immunoprecipitated SHP-1 were analyzed by Western
blotting. The phosphatase reaction was initiated by incubating the
immunocomplexes at 30 °C in the presence of 750 µM of
the phosphopeptide RRLIEDAEpYAARG (where pY is phosphotyrosine).
After 45 min, the reaction was stopped by addition of malachite green
solution. Sample absorbance was measured at 655 nm after allowing for
color development. The increase in phosphatase activity (absorbance) in
PCDE was calculated relative to the level observed in HIEC, which was
set at 1, whereas the increase in phosphatase activity (absorbance) in
differentiating Caco-2/15 cells was calculated relative to the level
measured in subconfluent Caco-2/15, which was also set at 1. Specific
phosphatase activity was calculated relative to the amount of
immunoprecipitated SHP-1. Band intensities in Western blots were
quantified by laser densitometry using an Alpha Imager 1200 documentation and analysis system (Alpha Innotech, San Leandro, CA).
Coimmunoprecipitation Experiments--
Cells were washed twice
with ice-cold PBS, lysed in chilled lysis buffer (150 mM
NaCl, 1 mM EDTA, 40 mM Tris-HCl, pH 7.6, 1%
Triton X-100, 0.1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml
pepstatin, 10 µg/ml aprotinin, 0.1 mM orthovanadate, and
40 mM Expression Vectors and Reporter Constructs--
Plasmid
DHFR-luciferase, which contains a high affinity E2F-binding site in the
dihydrofolate reductase promoter (43), was a kind gift of Dr. P. Farnham (University of Wisconsin). The dhfr gene, which is
required for DNA synthesis, is transcribed at the G1/S
transition. The cyclin D1 reporter construct, which contains the cyclin
D1 gene promoter from nucleotides Transient Transfections and Luciferase Assays--
Subconfluent
HIEC were seeded in 12-well plates and cotransfected by lipofection
(LipofectAMINE 2000, Invitrogen) with 0.2 µg of DHFR-luciferase,
c-Myc-luciferase, cyclin D1-luciferase, c-Fos-luciferase or TOPFLASH
reporters and 0.2 µg of the relevant expression vector (pcDNAneo)
containing SHP-1 or SHP-1C453/S. The pRL-SV40 Renilla
luciferase vector (Promega, Nepean, Ontario, Canada) was used as a
control for transfection efficiency. Two days after transfection,
luciferase activity was measured as described previously (34, 35),
according to the Promega protocol.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from HEK293 cells overexpressing SHP-1 or SHP-1C/S or
Caco-2/15 cells at different times of confluency, according to Stein
et al. (46). Electrophoretic mobility shift assays were
performed as described previously (35, 47). Samples were
electrophoresed in a 4% polyacrylamide gel containing 0.5% Tris
borate buffer and 2% glycerol. The high affinity TCF/LEF-1 DNA-binding
site (5'-GCACCCTTTGATCTTACC-3') previously employed in Caco-2 cells
(48) was used for electrophoretic mobility shift assays.
Proliferation Assay in Mice--
Control and
motheatenv mice were injected with 10 mM
BrdUrd (1 ml/100 g body weight) 2 h prior to sacrifice. Segments
of jejunum and colon were rinsed with 0.15 M NaCl, cut into
small fragments, embedded in optimum cutting temperature compound, and
quickly frozen in liquid nitrogen. Frozen sections 2-3 µm thick were
spread on silane-coated glass slides and air-dried 1 h at room
temperature before storage at Data Presentation and Statistical Analysis--
Luciferase
assays were performed in either duplicate or triplicate, and results
were analyzed by the Student's t test. Differences were
considered significantly different at p < 0.05. Typical Western blots shown are representative of at least three
independent experiments.
Expression of SHP-1 in the Human Fetal Intestinal Epithelium, in
the Caco-2/15 Intestinal Cell Line, and in Normal Human and
Rat Intestinal Epithelial Crypt Cells--
Expression of SHP-1 was
first investigated in intact fetal intestinal epithelium (20 weeks of
gestation). The use of a specific antibody against SHP-1 revealed that
this PTP was expressed in the nuclei of all jejunal and colonic
epithelial cells (Fig. 1, A
and B), with strongest nuclear staining observed in cells
occupying the lower third of the villus in the jejunum (Fig.
1A, see arrows) and the mid-region of colonic
crypts (Fig. 1B, see arrowheads). In the colonic
cancer cell line Caco-2/15 (Fig. 1C) and in normal undifferentiated crypt-like HIEC (Fig. 1D) and IEC-6 (Fig.
1E) cells, indirect immunofluorescence revealed that SHP-1
was well expressed and primarily nuclear, while exhibiting very weak,
albeit detectable, cytoplasmic staining.
Increased Expression and Activity of SHP-1 during Cell Cycle Arrest
and Differentiation of Intestinal Epithelial Cells--
Caco-2/15
cells, which spontaneously differentiate into 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
(50), it was deemed important to validate the above results in normal
human intestinally derived cells. Expression and activity of SHP-1 were
therefore analyzed in lysates of the following cell models: crypt-like
HIEC cells which are proliferative and undifferentiated (36), and PCDE
which are primary cultures of differentiated, nonproliferative villus
enterocytes (40). As shown in Fig. 2B, Rb protein was
exclusively detected in its hyperphosphorylated state in HIEC compared
with that found in both PCDE and intestinal mucosae extract, indicating
that these latter cells were arrested in G0/G1
phase. Moreover, expression (Fig. 2B) and specific
phosphatase activity (Fig. 2C) of SHP-1 were significantly
lower in proliferative HIEC cells compared with that found in PCDE
cells and intestinal mucosae extract. Overall these data indicate that
increased SHP-1 expression and phosphatase activity do correlate with
cell cycle arrest and induction of differentiation in intestinal
epithelial cells.
SHP-1 Negatively Controls Cell Cycle Progression of Intestinal
Epithelial Cells--
An important early event in terminal
differentiation of cells, especially in tissues exhibiting a rapid
turnover such as the intestinal epithelium, is their withdrawal from
the cell cycle (2, 49). To evaluate the role of SHP-1 in
intestinal cell cycle progression, we generated a
catalytically inactive SHP-1 by mutating the catalytic cysteine 453 to
serine (SHP-1/C453S). This mutation completely abolished phosphatase
activity of SHP-1 (45, 51). This construct was transiently transfected
in HEK293 cells, and its expression was verified by Western blotting
(data not shown). To evaluate the role of SHP-1 in intestinal cell
cycle progression, SHP-1 wild-type and SHP-1/C453S constructs were
tested on dihydrofolate reductase (dhfr) expression in subconfluent
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 its promoter. In
addition, microinjection of E2F into quiescent fibroblasts provokes S
phase re-entry, underscoring the importance of E2F in cell growth
control (52). 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 (43, 53). The results shown in Fig.
3A demonstrate that ectopic
expression of wild-type SHP-1, but not SHP-1/C453S mutant,
significantly inhibited dhfr gene expression by roughly
60%, suggesting that SHP-1 negatively regulates intestinal epithelial
cell cycle progression. To clarify further the role of SHP-1 in cell
proliferation, the effects of either enforced SHP-1 or SHP-1/C453S
expression were compared on the transcriptional activity of a range
of promoters such as the cyclin D1 and c-myc gene promoters.
The activation of these promoters represents one of the earliest cell
cycle-regulated events occurring during the
G0/G1 to S phase transition (53, 54). As shown
in Fig. 3, B and C, transcriptional activities of
c-myc and cyclin D1 promoters were both significantly
attenuated (49 and 55%, respectively) by ectopic expression of the
wild-type SHP-1 mutant but not by the SHP-1/C453S mutant.
Because overexpression of the wild-type SHP-1 inhibited the expression
of the two key cell cycle regulatory genes c-myc and cyclin
D1, possible SHP-1 control of SHP-1 Overexpression Does Not Interfere with the DNA Binding
Capacity of SHP-1 Associates with Association of SHP-1 and Dephosphorylation of
A Subset of Motheatenv Mice Exhibits a Moderate Increase in
Tyrosine Phosphorylation and Expression Levels of
In vivo pulse labeling of cells in S phase with BrdUrd was
performed to verify whether the rate of intestinal epithelial cell proliferation is perturbed in the subset of
mev/mev mice in which the
expression levels of cyclin D1 and c-Myc were increased. Mice were
sacrificed shortly (2 h) after post-injection at a time when only
actively cycling cells (S phase) were labeled. As shown in Fig.
7C, no significant difference in the rate of epithelial cell
proliferation in either jejunum or colon (latter not shown) was found
between mev/mev mice and their controls.
Whereas multiple targets for SHP-1 have been identified in
hematopoietic cells (3, 7, 8), very little is known about the function
of SHP-1 in epithelial cells. In this report, we suggest for the first
time the involvement of SHP-1 in the negative control of intestinal
epithelial cell proliferation. Indeed, increased SHP-1 expression and
phosphatase activity coincide with cell cycle arrest and induction of
differentiation in intestinal epithelial cells. Results show that the
expression and phosphatase activity of SHP-1 are significantly
increased in differentiated intestinal epithelial cells in comparison
to undifferentiated cells. The nuclear localizations of SHP-1 in the
lower third of the small intestinal villi and in the mid-region of
colonic crypts are in essence a reflection of the distribution of cells
that have ceased proliferation. Ectopic expression of SHP-1 in human
intestinal crypt cells inhibits E2F-dependent
transcriptional activity and decreases the expression of
c-myc and cyclin D1 genes, the activation of which
represents one of the earliest cell cycle-regulated events occurring
during the transition from G0/G1 to S phase. In
addition, we propose that SHP-1 may regulate the nuclear
transcriptional function of Generally, SHP-1 has been shown to act as a negative regulator of
signal transduction in hematopoietic cells, terminating signals from a
diverse range of signaling molecules including the EGF receptor,
interleukin 3 receptor, c-Kit, colony stimulating factor-1 receptor, B
and T cell antigen receptors, and receptor-associated JAK kinases (for
review see Ref. 59). However, it appears that the role of SHP-1 is
dependent on cell type (60, 61). Indeed, overexpression of the
catalytically inactive mutant of SHP-1 in HEK293 cells strongly
suppresses mitogen-activated pathways and results in decreased cell
growth, DNA synthesis, and the transcription of early response genes
(60). Furthermore, transfection of HeLa cells with inactive SHP-1
reduces STAT DNA binding induced by interferon It is widely assumed that SHP-1 is a cytoplasmic protein (2-4, 59).
However, a novel nuclear localization for SHP-1 has been demonstrated
in nonhematopoietic cells (62). Therefore, SHP-1 localization differs
between nonhematopoietic and hematopoietic cells, with SHP-1 protein
being virtually exclusively cytoplasmic in hematopoietic cell lines.
These results have implications regarding the nuclear function of SHP-1
in nonhematopoietic cells. Few nuclear tyrosine-phosphorylated proteins
have been identified. One potential target is the STAT proteins, which
are activated by tyrosine phosphorylation and translocate to the
nucleus (61, 63). In the present study, we were able to identify
Tyrosine phosphorylation of SHP-1 could be targeted to nuclear The observation that most of the
mev/mev mice did not exhibit any
alterations in intestinal epithelium might suggest that the residual
activity of SHP-1 in these
mev/mev mice (5, 6) is probably
sufficient to prevent intestinal disorders. However, it is of note that
a moderate increased expression levels of In conclusion, our data indicate that increased SHP-1 expression and
activity coincide with cell cycle arrest and induction of
differentiation of intestinal epithelial cells. The observed association of SHP-1 with -catenin/TCF-dependent transcription in intestinal epithelial crypt cells. Immunoprecipitation experiments revealed that
-catenin is one of the main binding partners and a substrate for
SHP-1. Taken together, our results indicate that SHP-1 may be involved
in the regulation of
-catenin transcriptional function and in the
negative control of intestinal epithelial cell proliferation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin signaling pathway plays a central role in the
regulation of gastrointestinal proliferation, and mutations in this
pathway have been detected in over 80% of colorectal cancers (19, 20).
Indeed,
-catenin plays an essential role in intestinal epithelial
cells, not only as a cadherin-associated complex but also as a
signaling molecule in the nucleus. Specifically,
-catenin has been
reported to transactivate gene expression through binding with its
counterpart in intestinal cells, TCF-4 (21, 22). Downstream targets of
the
-catenin-TCF complex include cyclin D1, c-Myc, peroxisome
proliferator-activated receptor
, and E-cadherin (23-27). In order
to mobilize into the nucleus and function as a signaling molecule, it
is reasonable to assume that
-catenin must first dissociate from the
cadherin complexes and be stabilized in the cytoplasm. Increased
cytoplasmic pools of
-catenin can be prompted by several mechanisms
such as down-regulation of E-cadherin (28), point mutation of
-catenin itself (29), or by inhibition of the degradation pathway
mediated by adenomatous polyposis coli, axin, glycogen synthase
kinase-3
, and
-TrCP (30, 31).
-catenin is revealed as one of the key binding
partners for SHP-1 in human intestinal crypt cells and a substrate for
SHP-1 in these cells. Furthermore, our results indicate that SHP-1
negatively regulates
-catenin-dependent transcriptional activity resulting in the inhibition of cyclin D1 and c-myc
gene expression, the activation of which represents one of the earliest cell cycle-regulated events occurring during the transition from G0/G1 to S phase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C (33-35). For
indirect immunofluorescence, sections were fixed with 2% formaldehyde
in PBS (pH 7.4; 45 min, 4 °C) prior to detection of
4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and
SHP-1 (a kind gift from Dr. A. Veillette, University of Montreal,
Quebec, Canada) (1:250, 2 h, room temperature). Secondary antibodies consisted of goat anti-rabbit IgG-fluorescein isothiocyanate from Roche Diagnostics. Negative controls (no primary antibody) were
included in all experiments.
20 °C, permeabilized with 0.1% Triton X-100 in PBS
for 10 min, and blocked with PBS/bovine serum albumin 2% (20 min at
room temperature). Cells were then immunostained for 1 h with
primary antibody for the detection of SHP-1 or
-catenin and 30 min
with the secondary conjugated antibody. Negative controls (no primary
antibody) were included in all experiments.
80 °C for
-catenin immunoprecipitation. Proteins
were determined by the modified Lowry procedure described by Peterson
(41).
-mercaptoethanol, 0.005% bromphenol blue, 1 mM PMSF). Proteins (40 µg) from whole cell lysates were separated by SDS-PAGE in 7.5 or 10% gels and detected immunologically following electrotransfer onto nitrocellulose membranes (Amersham Biosciences). Protein and molecular weight markers (Bio-Rad) were localized with Ponceau Red. After blocking for 1 h at 25 °C in PBS, 0.05% Tween containing 5% powdered milk, membranes were first incubated for 2-4 h at 25 °C with antibodies against either SHP-1,
-catenin, E-cadherin and TCF-4 (BD Biosciences), actin, cyclin D1,
and antiphosphotyrosine (PY99, Santa Cruz Biotechnologies, Santa Cruz,
CA), pRb (Pharmingen), c-Myc (Roche Diagnostics), or sucrase-isomaltase
(HSI-14 from A. Quaroni, Cornell University) in blocking solution,
followed by a second incubation with horseradish peroxidase-conjugated
goat anti-mouse or anti-rabbit (1:1000) IgG (both from Sigma) in
blocking solution for 1 h. The blots were visualized by the
Amersham ECL system (Amersham Biosciences). Protein concentrations were
measured using a modified Lowry procedure with bovine serum albumin as
standard (41).
-glycerophosphate), and lysates cleared of
cellular debris by centrifugation. Primary antibodies were added to 800 µg of each cell lysate and incubated for 2 h at 4 °C under
agitation. Forty µg of protein A-Sepharose (Amersham Biosciences) was
subsequently added for 1 h (4 °C under agitation).
Immunocomplexes were harvested by centrifugation and washed four times
with ice-cold lysis buffer. Proteins were solubilized in Laemmli's
buffer and separated by SDS-PAGE.
944 to +139 cloned upstream of the
luciferase gene of the pXP2 reporter construct, has been described
previously (44) and was kindly provided by R. Muller (Institute of
Molecular Biology and Tumor Research, Philipps-University Marburg,
Germany). The c-fos-luciferase reporter vector was provided
by Dr. C. Czernilofsky (Bender and Co., Vienna, Austria). The pRL-SV40
Renilla luciferase and the c-Myc-luciferase reporter vectors
were from Promega (Nepean, Ontario, Canada). The T cell factor
(TCF) reporter construct TOPFLASH was purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). The constitutive active mutant
of mouse c-Src p60Y529F was a kind gift from Dr.
Josée Lavoie (Université Laval, Quebec, Canada). The
full-length mouse SHP-1 cDNA (M. Thomas, Howard Hughes Medical
Institute, St. Louis, MO) was subcloned into pcDNAneo vector
and described previously (45). Mutation of the critical cysteine 453 of
the catalytic site of the molecule for serine (SHP-1C453S) was
performed by site-directed mutagenesis of double-stranded DNA according
to the Clontech protocol.
80 °C (33-35). For indirect
immunofluorescence, sections were fixed according to the In Situ Cell
Proliferation Kit from Roche Diagnostics.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (103K):
[in a new window]
Fig. 1.
Expression of SHP-1 in the human fetal
intestinal epithelium, in the Caco-2/15 intestinal cell line, and in
normal human and rat intestinal epithelial crypt cells. Frozen
sections of fetal jejunum (A) and colon (B)
between 18 and 20 weeks of gestation were stained with antibodies to
SHP-1, and nuclei were stained with 4,6-diamidino-2-phenylindole.
A, the crypt-villus axis is oriented perpendicular to
the figure with the crypt at the bottom. SHP-1 was mostly
localized in the nuclei of villus cells (see arrows).
Scale bar, 20 µm. B, in the colon, SHP-1
was mostly detected in the nuclei of cells in the mid-region of colonic
crypts (see arrowheads). Dapi,
4,6-diamidino-2-phenylindole. Scale bar, 20 µm.
Subconfluent Caco-2/15 (C), HIEC (D), and IEC-6
(E) cells were fixed with methanol/acetone and permeabilized
with a solution of 0.1% Triton X-100 for immunofluorescence and
staining for SHP-1 protein. In situ indirect
immunofluorescence shown here is representative of three independent
experiments. Scale bars, 20 µm.
2) and 100%
confluence (day 0) and at 6, 9, and 12 days post-confluence and
analyzed by Western blotting to confirm timing of cell cycle arrest in
G1 phase and induction of sucrase-isomaltase protein
expression. Consistent with previous observations (34, 35, 49),
decreased phosphorylation of p105Rb protein became apparent and
significant at day 6 post-confluence concomitant with the onset and
accumulation of sucrase-isomaltase expression (Fig.
2A). Expression and
phosphatase activity levels of SHP-1 were also analyzed by Western
blotting and immunoprecipitation, respectively. Although specific
activity of SHP-1 reached a peak at 6 days post-confluence (Fig.
2C), SHP-1 expression was induced as soon as cells reached
confluence (day 0) and progressively increased during Caco-2/15
differentiation (Fig. 2A).
View larger version (37K):
[in a new window]
Fig. 2.
Increased expression and activity of SHP-1
during cell cycle arrest and differentiation of intestinal epithelial
cells. A, Caco-2/15 cells were harvested at 70 (SC) and 100% confluence (day 0) and at 6, 9, and 12 days post-confluence. Cell extracts (40 µg) were separated by
10% SDS-PAGE and proteins analyzed by Western blotting for expression
of SHP-1, pRb, and sucrase-isomaltase. B, subconfluent
proliferating HIEC, primary cultures of human differentiated
enterocytes (PCDE), and intestinal mucosae were lysed and proteins
separated by SDS-PAGE. SHP-1 and pRb protein expression were analyzed
by Western blotting. Results are representative of three independent
experiments. C, subconfluent growing HIEC and PCDE as
well as Caco-2/15 cells at 70 ( 2) and 100% confluence
(day 0) and at 3, 6, and 9 days post-confluence were
harvested. Cell extracts (800 µg) were immunoprecipitated with a
specific antibody to SHP-1. Levels of immunoprecipitated SHP-1 were
analyzed by Western blotting. Phosphatase activity of SHP-1 was assayed
by using the phosphopeptide RRLIEDAEpYAARG as substrate, according to
the Upstate Biotechnologies Inc. protocol. The increase in phosphatase
activity (absorbance) in PCDE was calculated relative to the level
observed in HIEC that was set at 1, whereas the increase in phosphatase
activity (absorbance) in differentiating Caco-2/15 cells was
calculated relative to the level measured in subconfluent Caco-2/15
that was also set at 1. Specific phosphatase activity was calculated
relative to the amount of immunoprecipitated SHP-1. Data shown
are representative of that obtained in three independent
experiments.
View larger version (28K):
[in a new window]
Fig. 3.
SHP-1 inhibits dhfr,
c-myc, cyclin D1 gene expression and negatively
controls -catenin/TCF-dependent activity
(TOPFLASH) in intestinal epithelial cells. Subconfluent HIEC cells
were cotransfected with 0.2 µg of pcDNAneo I containing or
lacking the wild-type SHP-1 or the dominant negative mutant C453S of
SHP-1 (SHP-1/CS) with 0.2 µg of DHFR- (A), c-Myc-
(B), cyclin D1- (C), TOPFLASH- (D), or
c-Fos-luciferase (E) reporters. Two days after transfection,
cells were lysed, and luciferase activity was measured. The increase in
luciferase activity was calculated relative to the pcDNAneo I level
that 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).
-catenin/TCF transcriptional activity
was further investigated. Indeed, functional
-catenin/TCF-binding sites have been identified in the promoters of c-myc (24)
and cyclin D1 (25, 26) genes. HIEC cells were thereby transfected with
TOPFLASH reporter, which directly assays
-catenin/TCF activity (55).
As shown in Fig. 3D,
-catenin/TCF transcriptional
activity was significantly inhibited (58%) by the expression of
wild-type SHP-1 but not SHP-1/C453S mutant. The effect of wild-type
SHP-1 was specific because overexpression of wild-type SHP-1 or
SHP-1/C453S mutant did not influence c-fos gene expression,
which represents a sensitive reporter of growth factor-induced
transcriptional activity (56). This early gene promoter contains the
well characterized serum-responsive element, whose activity is induced
upon serum activation of the serum-responsive factor (57).
-Catenin-TCF Complex--
Consistent with a previous
study (48), gel shift analysis in Caco-2/15 cells demonstrated a
decrease in binding of
-catenin-TCF complex to
TCF/LEF-1-binding site with time of confluency and differentiation, correlating with the increased expression of SHP-1
(Fig. 4A). Thus, increased
expression of SHP-1 could inhibit
-catenin/TCF transcriptional
activity by interfering with its DNA binding capacity. Electrophoretic
mobility shift experiments were therefore performed in HEK293 cells to
determine whether the DNA binding capacity of
-catenin/TCF was
affected by SHP-1 and/or SHP-1/C453S overexpression. As shown in Fig.
4B, binding of nuclear proteins to the TCF/LEF-1 DNA-binding
site was not affected by using extracts prepared from either SHP-1 or
SHP-1/C453S-overexpressing HEK293 cells.
View larger version (48K):
[in a new window]
Fig. 4.
SHP-1 overexpression does not interfere with
the DNA binding capacity of -catenin-TCF
complex. Binding of nuclear proteins to a high affinity TCF/LEF-1
DNA-binding site was assessed in Caco-2/15 cells and HEK293
overexpressing SHP-1 and SHP-1/C453S. Caco-2/15 cells were harvested at
confluence (day 0) and at 3, 6, and 9 days post-confluence.
HEK293 cells were harvested 2 days after transfection. Total cell
extracts were separated by 10% SDS-PAGE and proteins analyzed by
Western blotting for expression of SHP-1. 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. Results are representative of
three independent experiments.
-Catenin in Intestinal
Epithelial Cells--
To investigate further the regulation of
-catenin function by SHP-1, interaction of SHP-1 with
-catenin
was tested in coimmunoprecipitation assays. Immunoprecipitations
demonstrated the formation of a complex between SHP-1 and
-catenin
in intestinal crypt cells. Indeed, Fig.
5A shows that
SHP-1-
-catenin association in IEC-6 and HIEC cells was significantly
diminished once cells reached confluence (day 0) and decreased even
further at day 5 of post-confluence. Localization of SHP-1 and
-catenin proteins was further analyzed in asynchronously growing
subconfluent IEC-6 cells and in quiescent confluent IEC-6 cells. As
illustrated in Fig. 5B, bright nuclear SHP-1 staining was
evident in proliferative subconfluent (panel 1) and in
quiescent confluent (panel 2) IEC-6 cells, although some
SHP-1 staining was also observed in the cytoplasm of 5-day post-confluent cells (panel 3). In subconfluent growing
IEC-6 cells,
-catenin protein staining was partially localized in
the nucleus but was also clearly visible at the sites of cell-cell contacts (Fig. 5B, panel 4). Nuclear localization
of
-catenin was completely altered upon confluency, as shown by the
accumulation of
-catenin staining at sites of cell-cell contact in
confluent (Fig. 5B, panel 5) and post-confluent (Fig.
5B, panel 6) IEC-6 cells. These results suggest
that SHP-1 binds
-catenin in intestinal epithelial cells and that
this association decreases in confluent cells possibly because of
recruitment of
-catenin to cell junctions.
View larger version (51K):
[in a new window]
Fig. 5.
SHP-1 associates with
-catenin in intestinal epithelial cells.
A, SHP-1 and
-catenin were immunoprecipitated from
800 µg of lysates of pre-confluent, confluent (day 0), and
5-day post-confluent IEC-6 and HIEC cells. Proteins from
immunoprecipitates were solubilized in Laemmli buffer and separated by
SDS-PAGE. Proteins were analyzed by Western blotting to determine the
amount of SHP-1 and
-catenin in immunoprecipitates. Blots shown are
representative of three independent experiments. B,
subconfluent IEC-6 cells were fixed with methanol/acetone and
permeabilized with a solution of 0.1% Triton X-100 for
immunofluorescence and staining for SHP-1 or
-catenin proteins.
In situ indirect immunofluorescence shown is representative
of three independent experiments. Scale bar, 20 µm.
-Catenin by SHP-1 in HEK293 Cells--
To assess whether
SHP-1/
-catenin interaction results in SHP-1-mediated
-catenin
dephosphorylation, a constitutively active mutant of c-Src
(p60Y529F) was coexpressed with SHP-1 or with the
catalytically inactive SHP-1/C453S mutant in HEK293.
-Catenin is
heavily tyrosine-phosphorylated in Src-transformed cells and is
considered as a good substrate for c-Src (58). Overexpression of
p60Y529F in HEK293 cells led to massive tyrosine
phosphorylation of endogenous
-catenin (Fig.
6, top panel, lane 2). The
C453S mutant of SHP-1 had virtually no effect on
p60Y529F-induced tyrosine phosphorylation of
-catenin
(Fig. 6, top panel, lane 4), whereas SHP-1
expression resulted in a strong decrease in tyrosine-phosphorylated
-catenin (Fig. 6, top panel, lane 3). This
suggests that
-catenin may indeed be a specific target for SHP-1. In
addition, the catalytically inactive mutant of SHP-1 (C453S), which has
been described as a substrate-trapping mutant (4), exhibited strongly
elevated binding (Fig. 6, bottom panel, lanes 3 and 4), suggesting that
-catenin is a very efficient SHP-1 substrate.
View larger version (62K):
[in a new window]
Fig. 6.
Association of SHP-1 and dephosphorylation
of -catenin by SHP-1 in HEK293 cells.
HEK293 cells were transfected with the empty vectors pCMV and
pCDNAneo I (EV), activated c-Src alone
(c-src/CA + pCDNAneo), or together with SHP-1
(c-src/CA + SHP-1) or SHP-1/C453S
(c-src/CA + SHP-1/CS). Twenty four
hours after transfection,
-catenin was immunoprecipitated from 800 µg of lysates. Proteins from immunoprecipitates were solubilized in
Laemmli buffer and separated by SDS-PAGE. Tyrosine phosphorylation
of immunoprecipitated proteins was analyzed by immunoblotting
using PY-99 antiphosphotyrosine. Proteins were analyzed by
Western blotting (WB) to determine the amount of
SHP-1 and
-catenin in immunoprecipitates. Blots shown are
representative of three independent experiments.
-Catenin in
Intestinal Epithelium--
We next tested whether impairment of SHP-1
activity in mev/mev mice could
affect
-catenin signaling by analyzing tyrosine phosphorylation as
well as levels of immunoprecipitated
-catenin in
mev/mev intestinal epithelium. As
shown in Fig. 7A, jejunal
samples from control and mev/mev
mice revealed three bands, whereas colon samples maintained the presence of only one major 94-kDa band in both wild-type and
mev/mev mice. Moreover,
expression levels of
-catenin were much more elevated in the colon
than in the jejunum of all mice (Fig. 7A). Of note, the
colon of a substantial subset of
mev/mev mice (about one-third)
exhibited increased
-catenin tyrosine phosphorylation compared with
that in control (wild-type) animals, although colonic mucosae did
generate a greater quantity of immunoprecipitated
-catenin protein.
In this regard, in this subset of
mev/mev mice, the jejunum and
colon exhibited a moderate, but consistent, increase in
-catenin
protein levels compared with that in control (wild-type) animals (Fig.
7A). There was no difference, however, in E-cadherin and
TCF-4 expression levels as well as in tyrosine phosphorylation of TCF-4
between these mev/mev and control
mice (data not shown). Expression levels of cyclin D1 and c-Myc
proteins, two targets of
-catenin-TCF complex, were next analyzed in
this subset of mev/mev mice. As
shown in Fig. 7B, there was a dramatic increase in
expression of c-Myc in the jejunal and colonic epithelium of these
mev/mev mice. Cyclin D1
expression was also increased in these
mev/mev mice but at a much lesser
scale than c-Myc. Equal protein loading of each lane was confirmed by
anti-actin antibody labeling.
View larger version (37K):
[in a new window]
Fig. 7.
A subset of
motheatenv mice exhibits enhanced tyrosine
phosphorylation and -catenin expression levels
in intestinal epithelium. Wild-type and
mev/mev mice were sacrificed, the
jejunum and colon rapidly removed, and their respective mucosae scraped
and homogenized as described under "Experimental Procedures."
A,
-catenin was immunoprecipitated from 800 µg of
cleared mucosal lysates from jejunum and colon. Proteins from
immunoprecipitates were solubilized in Laemmli buffer and separated by
SDS-PAGE. Proteins were analyzed by Western blotting to determine the
amount of
-catenin in immunoprecipitates. Tyrosine phosphorylation
of immunoprecipitated proteins was also analyzed by immunoblotting
using PY-99 antiphosphotyrosine. B, cleared extracts
were separated by 10% SDS-PAGE and proteins analyzed by Western
blotting for expression of c-Myc, cyclin D1, and actin. Blots shown are
representative of three independent experiments. C,
wild-type and mev/mev mice were
injected with BrdUrd 2 h prior to sacrifice in order to label only
cells in S phase. Immunofluorescence was performed on sections of
jejunum and colon (not shown) of both wild-type (left panel)
and mev/mev mice (right
panel). Note the similarity in the number of labeled nuclei
between wild-type and mev/mev
mice. Scale bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin by its dephosphorylation.
Finally, in agreement with a functional interaction of
-catenin and
SHP-1, there is increased expression of cyclin D1 and c-Myc proteins in
the jejunum and colon of mev/mev
mice, whose SHP-1 activity is strongly compromised.
and EGF (61). In the
present study, we propose a negative role of SHP-1 in the control of
the intestinal epithelial cell cycle. As yet, the molecular basis for
the apparent opposite effects of SHP-1 in different cell systems has to
be defined, although the phosphatase domain appears to be critical.
-catenin as a binding partner and substrate for SHP-1 in epithelial cells.
-catenin has been shown to correlate
with tumorigenesis, cell migration, and developmental processes (64).
Furthermore, various growth factors (e.g. EGF, hepatocyte growth factor, and vascular epidermal growth factor) and cytoplasmic Src kinases are known to phosphorylate the tyrosine residue(s) on
-catenin (58, 65, 66). Experimental data suggest that tyrosine
phosphorylation of
-catenin is implicated as a means for release
from the E-cadherin complex (58, 67), although there is some
controversy over its direct effect on cell adhesiveness. A recent study
(68) reported that treatment of NIH 3T3 fibroblasts or HCT116 cells
with the tyrosine phosphatase inhibitor pervanadate increased tyrosine
phosphorylation of
-catenin and led to relocation from cell-cell
interfaces to the cytoplasm but did not change its binding activity to
LEF-1, nor did it enhance cyclin D1 transactivation. This is, however,
at odds with another recent study that clearly points out that
phosphorylation of Tyr654, a residue placed in the last
armadillo repeat of
-catenin, decreases its binding to E-cadherin
but stimulates the association of
-catenin to the basal
transcription factor TATA-binding protein (TBP) (69). Interestingly,
this greater association between TBP and
-catenin correlates with a
higher stimulation of
-catenin/TCF transcriptional activity. In
addition, other nuclear factors have been shown to interact with the N-
and C-terminal transactivation domains of
-catenin, including Pontin
(70), Teashirt (71), Sox 17 and 13 (72), histone deacetylase (73),
Brg-1 (74), and SMAD4 (75). Hence, we propose a hypothetical model of
SHP-1-induced inhibition of
-catenin/TCF transcriptional activity in
intestinal epithelial cells whereby SHP-1 dephosphorylates
-catenin
leading to a decreased interaction of the
-catenin-TCF-4 complex
with TBP or other nuclear coactivators or to an increased interaction with corepressors.
-catenin by recognizing the
consensus sequence for ligands of the N-terminal SH2 domain of SHP-1,
i.e. hXY(P)XXh (where h = hydrophobic and X = any amino acid). Indeed, three
tyrosine residues in the sequence of human
-catenin partially
resemble the consensus sequence (16). Tyrosine 333 (Tyr-Thr-Tyr333-Glu-Lys-Leu), 604 (Leu-Leu-Tyr604-Ser-Pro-Ile), and 654 (Ala-Thr-Tyr654-Ala-Ala-Ala) in their phosphorylated forms
are therefore candidates for SHP-1 interaction sites in
-catenin.
Interestingly, one of the phosphorylated
-catenin tyrosine residues
previously mapped was Tyr654 (58). One could therefore
speculate that binding of SHP-1 to Tyr654 of
-catenin
results in the dephosphorylation of
-catenin on Tyr654
(or other phosphotyrosines) eventually leading to dissociation of TBP
or other coactivators from the
-catenin-TCF complex. However, further studies are required at this point to identify the
phosphorylated tyrosine on
-catenin and its roles in
-catenin
function in intestinal epithelial cells and to clarify the mechanism by
which SHP-1 specifically inhibits
-catenin/TCF transcriptional activity.
-catenin in the jejunal
and colonic mucosa of a subset of
mev/mev mice was reproducibly
observed in comparison to that observed in control mice. In contrast to
the relatively well known degrading effects of serine phosphorylation
on
-catenin, the effects of its tyrosine phosphorylation are not
well characterized beyond an effect on cell adhesiveness. An attractive
possibility is that tyrosine phosphorylation may increase the entry of
-catenin into the nucleus by increasing its stabilized pools in the
cytoplasm. However, preliminary experiments demonstrate that SHP-1
overexpression in HEK293 cells did not result in dysregulation of
-catenin stability.2
Although we did not observe an altered localization of
-catenin in
mev/mev mice (data not shown),
one cannot rule out the possibility that an increased tyrosine
phosphorylation may induce subtle changes in stability and subcellular
localization, which are sufficient to affect its transactivating
function. In fact, our data did demonstrate an increased expression of
both cyclin D1 protein and even more predominantly of c-Myc in a subset
of mev/mev mice. However, any
significant increase in proliferation of gut epithelial cells was
observed in mev/mev mice
suggesting that the increased expression of cyclin D1 and c-Myc was not
sufficient for S phase entry in these cells.
-catenin in intestinal epithelial cells and its dephosphorylation by SHP-1 provide evidence that SHP-1 may be
involved in the regulation of the
-catenin/TCF signaling pathway and
therefore modulate proliferation of these cells. Interestingly, down-regulation of the
-catenin/TCF pathway is associated with the
promotion of a more differentiated phenotype in colonic epithelial cells (48). Although further studies are needed to pinpoint the role of
tyrosine phosphorylation in
-catenin nuclear function and signaling,
our study provides novel fundamental insights into the function of
SHP-1 in the control of intestinal epithelial cell proliferation and in
the early events of intestinal epithelial cell differentiation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Vézina for technical assistance and P. Pothier for the critical reading of the manuscript. We also thank Drs. C. Poulin and F. Jacot, obstetricians from the Département de la Santé Communautaire du Centre Universitaire de Santé de l'Estrie, for invaluable collaboration in providing the tissue specimens used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada.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.
§ Both authors contributed equally to this work.
¶ Student scholar from the Fonds pour la Recherche en Santé du Québec.
** Recipient of a Canadian Research Chair in Signaling and Digestive Physiopathology. To whom correspondence should be addressed: Dépt. of d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-564-5271; Fax: 819-564-5320; E-mail: Nathalie.Rivard@USherbrooke.ca.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M300425200
2 C. Duchesne and N. Rivard, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTP, protein-tyrosine phosphatase; APC, adenomatous polyposis coli; BrdUrd, bromodeoxyuridine; dhfr, dihydrofolate reductase; EGF, epidermal growth factor; HEK, human embryonic kidney; HIEC, human intestinal epithelial cells; IEC, intestinal epithelial cells; PCDE, primary cultures of differentiated enterocytes; pRb, retinoblastoma protein; TBP, TATA-binding protein; TCF, T cell factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tonks, N. K., and Nell, B. G. (2001) Curr. Opin. Cell Biol. 13, 182-195[CrossRef][Medline] [Order article via Infotrieve] |
2. | Adachi, M., Fisher, E. H., Ihle, J., Imai, K., Jirik, F., Neel, B. G., Pawson, T., Shen, S.-H., Thomas, M., Ullrich, A., and Zhao, Z. (1996) Cell 85, 15[Medline] [Order article via Infotrieve] |
3. | Feng, G. S. (1999) Exp. Cell Res. 253, 47-54[CrossRef][Medline] [Order article via Infotrieve] |
4. | Qu, C. K. (2000) Cell Res. 10, 279-288[Medline] [Order article via Infotrieve] |
5. | Tsui, H. W., Siminovitch, K. A., Souza, L., and Tsui, F. W. L. (1993) Nat. Genet. 4, 124-129[Medline] [Order article via Infotrieve] |
6. | Shultz, L. D., Schweitzer, P. A., Rajan, T. V., Yi, T., Ihle, J. N., Matthews, R. J., Thomas, M. L., and Beier, D. R. (1993) Cell 73, 1445-1454[Medline] [Order article via Infotrieve] |
7. | Klingmüller, U., Lorenz, U., Cantley, L. C., Neel, B. G., and Lodish, H. F. (1995) Cell 80, 729-738[Medline] [Order article via Infotrieve] |
8. | Chen, H. E., Chang, S., Trub, T., and Neel, B. G. (1996) Mol. Cell. Biol. 16, 3685-3697[Abstract] |
9. | Neel, B. G. (1997) Curr. Opin. Immunol. 9, 405-420[CrossRef][Medline] [Order article via Infotrieve] |
10. | Jiao, H., Berrada, K., Yang, W., Tabrizi, M., Platanias, L. C., and Yi, T. (1996) Mol. Cell. Biol. 16, 6985-6992[Abstract] |
11. |
Binstadt, B. A.,
Billadeau, D. D.,
Jevremovic, D.,
Williams, B. L.,
Fang, N.,
Yi, T.,
Koretzky, G. A.,
Abraham, R. T.,
and Leibson, P. J.
(1998)
J. Biol. Chem.
273,
27518-27523 |
12. | Plas, D. R., Johnson, R., Pingel, J. T., Matthews, R. J., Dalton, M., Roy, G., Chan, A. C., and Thomas, M. L. (1996) Science 272, 1173-1176[Abstract] |
13. |
Chiang, G. G.,
and Sefton, B. M.
(2001)
J. Biol. Chem.
276,
23173-23178 |
14. | Banville, D., Stocco, R., and Shen, S. H. (1995) Genomics 27, 165-173[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Keilhack, H.,
Müller, M.,
Böhmer, S.-A.,
Frank, C.,
Weidner, K. M.,
Birchmeier, W.,
Ligensa, T.,
Berndt, A.,
Kosmehl, H.,
Günther, B.,
Müller, T.,
Birchmeier,
and Böhmer, F. D.
(2001)
J. Cell Biol.
152,
325-334 |
16. |
Keilhack, H.,
Hellman, U.,
van Hengel, J.,
van Roy, F.,
Godovac-Zimmermann, J.,
and Böhmer, F.-D.
(2000)
J. Biol. Chem.
275,
26376-26384 |
17. | Babyatsky, M. W., and Podolsky, D. K. (1999) in Growth and Development of the Gastrointestinal Tract (Yamada, T., ed), 3rd Ed. , pp. 547-584, J. B. Lippincott, Philadelphia |
18. | Gordon, J. I., and Hermiston, M. L. (1994) Curr. Opin. Cell Biol. 6, 795-803[Medline] [Order article via Infotrieve] |
19. | Smith, K., Bui, T. D., Poulsom, R., Kaklamanis, L., Williams, G., and Harris, A. L. (1999) Br. J. Cancer 81, 496-502[CrossRef][Medline] [Order article via Infotrieve] |
20. | Morin, P. J. (1999) Bioessays 21, 1021-1030[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Cadigan, K. M.,
and Nusse, R.
(1997)
Genes Dev.
11,
3286-3305 |
22. | Miller, J. R., Hocking, A. M., Brown, J. D., and Moon, R. T. (1999) Oncogene 18, 7860-7872[CrossRef][Medline] [Order article via Infotrieve] |
23. | He, T. C., Chan, T. A., Vogelstein, B., and Kinzler, K. W. (1999) Cell 99, 335-345[Medline] [Order article via Infotrieve] |
24. |
He, T. C.,
Sparks, A. B.,
Rago, C.,
Hermeking, H.,
Zawel, L.,
da Costa, L. T.,
Morin, P. J.,
Vogelstein, B.,
and Kinzler, K. W.
(1998)
Science
281,
1509-1512 |
25. | Tetsu, O., and McCormick, F. (1999) Nature 398, 422-426[CrossRef][Medline] [Order article via Infotrieve] |
26. | Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., D'Amico, M., Pestell, R., and Ben-Ze'ev, A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 55522-555527 |
27. |
Roose, J.,
Huls, G.,
van Beest, M.,
Moerer, P.,
van der Horn, K.,
Goldschmeding, R.,
Logtenberg, T.,
and Clevers, H.
(1999)
Science
285,
1923-1926 |
28. | Umbas, R., Schalken, J. A., Aalders, T. W., Carter, B. S., Karthaus, H. F., Schaafsma, H. E., Debruyne, F. M., and Isaacs, W. B. (1992) Cancer Res. 52, 5104-5109[Abstract] |
29. |
Morin, P. J.,
Sparks, A. B.,
Korinek, V.,
Barker, N.,
Clevers, H.,
Vogelstein, B.,
and Kinzler, K. W.
(1997)
Science
275,
1787-1790 |
30. | Rubinfeld, B., Albert, I., Porfori, E., Fiol, C., Munemitsu, S., and Polakis, P. (1996) Science 272, 1023-1026[Abstract] |
31. |
Liu, C.,
Kato, Y.,
Zhang, Z.,
Do, V. M.,
Yankner, B. A.,
and He, X.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6273-6278 |
32. | Streeter, G. L. (1920) Contrib. Embryol. 11, 143-179 |
33. | Vachon, P. H., and Beaulieu, J. F. (1992) Gastroenterology 103, 414-423[Medline] [Order article via Infotrieve] |
34. | Aliaga, J. C., Deschênes, C., Beaulieu, J. F., Calvo, E. L., and Rivard, N. (1999) Am. J. Physiol. 277, G631-G641[Medline] [Order article via Infotrieve] |
35. |
Houde, M.,
Laprise, P.,
Jean, D.,
Blais, M.,
Asselin, C.,
and Rivard, N.
(2001)
J. Biol. Chem.
276,
21885-21894 |
36. | Perreault, N., and Beaulieu, J. F. (1996) Exp. Cell Res. 224, 354-364[CrossRef][Medline] [Order article via Infotrieve] |
37. | Quaroni, A., Wands, J., Trelstad, R. L., and Isselbacher, K. J. (1979) J. Cell Biol. 80, 248-265[Abstract] |
38. | Beaulieu, J. F., and Quaroni, A. (1991) Biochem. J. 280, 599-608[Medline] [Order article via Infotrieve] |
39. | Vachon, P. H., and Beaulieu, J. F. (1995) Am. J. Physiol. 268, G857-G867[Medline] [Order article via Infotrieve] |
40. | Perreault, N., and Beaulieu, J. F. (1998) Exp. Cell Res. 254, 34-42[CrossRef] |
41. | Peterson, G. L. (1977) Anal. Biochem. 83, 346-356[Medline] [Order article via Infotrieve] |
42. |
Yoshida, K.,
and Kufe, D.
(2001)
Mol. Pharmacol.
60,
1431-1438 |
43. | Slansky, J. E., Li, Y., Kaelin, W. G., and Farnham, P. G. (1993) Mol. Cell. Biol. 13, 1610-1618[Abstract] |
44. |
Lavoie, J. N.,
L'Allemain, G.,
Brunet, A.,
Müller, R.,
and Pouysségur, J.
(1996)
J. Biol. Chem.
271,
20608-20616 |
45. |
Rivard, N.,
McKenzie, F. R.,
Brondello, J.-M.,
and Pouysségur, J.
(1995)
J. Biol. Chem.
270,
11017-11024 |
46. | Stein, B., Rahmsdorf, H. J., Steffen, A., Litfin, M., and Herrlich, P. (1989) Mol. Cell. Biol. 9, 5169-5181[Medline] [Order article via Infotrieve] |
47. | Pelletier, N., Boudreau, F., Yu, S.-J., Zannoni, S., Boulanger, V., and Asselin, C. (1998) FEBS Lett. 439, 275-280[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Mariadason, J. M.,
Bordonaro, M.,
Aslam, F.,
Shi, L.,
Kurugichi, M.,
Velcich, A.,
and Augenlicht, L. H.
(2001)
Cancer Res.
61,
3465-3471 |
49. | Deschênes, C., Vézina, A., Beaulieu, J. F., and Rivard, N. (2001) Gastroenterology 120, 423-438[Medline] [Order article via Infotrieve] |
50. | Engle, M. J., Goetz, G. S., and Alpers, D. H. (1998) J. Cell. Physiol. 174, 362-369[CrossRef][Medline] [Order article via Infotrieve] |
51. | Pei, D., Lorenz, U., Klingmüller, U., Neel, B. G., and Walsh, C. T. (1994) Biochemistry 33, 15483-15493[Medline] [Order article via Infotrieve] |
52. | La Thangue, N. B. (1994) Curr. Opin. Cell Biol. 6, 443-450[Medline] [Order article via Infotrieve] |
53. | Prober, D. A., and Edgar, B. A. (2001) Curr. Opin. Genet. & Dev. 11, 19-26[CrossRef][Medline] [Order article via Infotrieve] |
54. | Shackney, S. E., and Shankey, T. V. (1999) Cytometry 35, 97-116[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Korinek, V.,
Barker, N.,
Morin, P. J.,
van Wichen, D.,
de Weger, R.,
Kinzler, K. W.,
Vogelstein, B.,
and Clevers, H.
(1997)
Science
275,
1784-1787 |
56. | Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157[CrossRef][Medline] [Order article via Infotrieve] |
57. | Treisman, R. (1990) Semin. Cancer Biol. 1, 47-58[Medline] [Order article via Infotrieve] |
58. |
Roura, S.,
Miravet, S.,
Piedra, J.,
Garcia de Herreros, A.,
and Dunach, M.
(1999)
J. Biol. Chem.
274,
36734-36740 |
59. | Zhang, J., Somani, A.-K., and Siminovitch, K. A. (2000) Semin. Immunol. 12, 361-378[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Su, L.,
Zhao, Z.,
Bouchard, P.,
Banville, D.,
Fisher, E. H.,
Krebs, E. G.,
and Shen, S.-H.
(1996)
J. Biol. Chem.
271,
10385-10390 |
61. |
You, M.,
and Zhao, Z.
(1997)
J. Biol. Chem.
272,
23376-23381 |
62. |
Craggs, G.,
and Kellie, S.
(2001)
J. Biol. Chem.
276,
23719-23725 |
63. |
Darnell, J. E.
(1997)
Science
277,
1630-1635 |
64. | Daniel, J. M., and Reynolds, A. B. (1997) Bioessays 19, 883-891[Medline] [Order article via Infotrieve] |
65. |
Danilkovitch-Miagkova, A.,
Miagkov, A.,
Skeel, A.,
Nakaigawa, N.,
Zbar, B.,
and Leonard, E. J.
(2001)
Mol. Cell. Biol.
21,
5857-5868 |
66. | Ilan, N., Mahooti, S., Rimm, D. L., and Madri, J. A. (1999) J. Cell Sci. 18, 3005-3014 |
67. | Hiscox, S., and Jiang, W. G. (1999) Anticancer Res. 19, 509-517[Medline] [Order article via Infotrieve] |
68. | Kim, K., and Lee, K.-Y. (2001) Cell Biol. Int. 25, 421-427[CrossRef][Medline] [Order article via Infotrieve] |
69. |
Piedra, J.,
Martinez, D.,
Castano, J.,
Miravet, S.,
Dunach, M.,
and Garcia de Herreros, A.
(2001)
J. Biol. Chem.
276,
20436-204443 |
70. |
Bauer, A.,
Huber, O.,
and Kemler, R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14787-14792 |
71. |
Gallet, A.,
Angelats, C.,
Erkner, A.,
Charroux, B.,
Fasano, L.,
and Kerridge, S.
(1999)
EMBO J.
18,
2208-2217 |
72. | Zorn, A. M., Barish, G. D., Williams, B. O., Lavender, P., Klymkowsky, M. W., and Varmus, H. E. (1999) Mol. Cell 4, 487-498[Medline] [Order article via Infotrieve] |
73. |
Billin, A. N.,
Thirlwell, H.,
and Ayer, D. A.
(2000)
Mol. Cell. Biol.
20,
6882-6890 |
74. |
Barker, N.,
Hurlstone, A.,
Musisi, H.,
Bienz, M.,
and Clevers, H.
(2001)
EMBO J.
20,
4935-4943 |
75. | Nishita, M., Hashimoto, M., Ogata, S., Laurent, M., Ueno, N., Shibaya, H., and Cho, K. (2000) Nature 6771, 781-785 |