From the Department of Ophthalmology, Catholic
University of Korea, Seoul 137-040, Korea, the
§ Department of Genetics and Development, Columbia
University, New York, New York 10032, and the ¶ Department of Life
Science, The University of Seoul, Seoul 130-743, Korea
Received for publication, January 19, 2003
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ABSTRACT |
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Axin regulates Wnt signaling through
down-regulation of The Wnt signaling pathway has critical roles in embryonic
development, differentiation, and tumorigenesis (1-4). Currently, 19 Wnt genes have been identified in humans and most of them have homologs
in other organisms (5). The tightly controlled temporal and spatial
expression patterns of Wnt genes have implied that different Wnts have
specific roles in embryonic development, and ablation of specific Wnt
genes in mice have shown that this is true (6, 7). In particular,
targeted inactivation of Wnt-1, Wnt-3a, or Wnt-7a in mice suggest that
they have critical roles in neural development and neural cell fate
determination (8-10).
Wnt genes encode secreted glycoproteins that signal through the cell
surface receptor Frizzled, which has at least 10 orthologs in mammals,
and other coreceptors (for a review, see Ref. 11). Recent data suggest
that heterotrimeric G-proteins are involved in the signaling between
receptors and downstream pathways (12). Upon binding of Wnts to
Frizzled, Dvls are activated and antagonize the Axin was originally identified by an insertional mutation in transgenic
mice (AxinTg1), which caused developmental
defects similar to those in mice carrying spontaneous mutations at the
genetic locus called Fused (35, 36). Ectopic expression of Axin in
Xenopus embryos showed that Axin blocked embryonic axis
formation by inhibition of the canonical Wnt/ Among the diverse biological roles of Wnts, their
involvement in neural differentiation has been studied due to prominent expression of several Wnts in the developing central nervous system (6,
7). Indeed, absence of Wnt-1 and Wnt-3a leads to abnormal populations
of dorsal interneurons, implying that Wnt signaling has a role in the
determination of neuronal cell fate (46). An elegant finding by Hall
et al. (10), that secretion of Wnt-7a from postsynaptic
granule cell neurons remodels the axons and growth cones of developing
mossy fibers, is another good example for involvement of Wnt signaling
in neuronal differentiation. However, it has not been shown that Wnt
signaling plays any autocrine role in neurite extension.
The pluripotent P19 embryonal carcinoma (EC) cell line has been used as
a model system to study neuronal differentiation, because these cells
can be easily differentiated into neuronal cells that form neurite-like
structures upon simple retinoic acid treatment (47, 48). It has been
reported that the expression of diverse Wnts are regulated during
neuronal differentiation, and it has therefore been suggested that
different Wnts may have roles in this process (49). However, although
many Wnts display a dynamic expression pattern during the neuronal
differentiation of P19 cells, so far only Wnt1 has been tested for a
potential role in this process, and it remains unknown whether
endogenous canonical We show here that Axin is down-regulated and that Cell Culture and Differentiation--
P19 embryonal carcinoma
(EC) cells (obtained from ATCC) were cultured in Dulbecco's modified
Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine
serum (FBS, HyClone Laboratories Inc.), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine
(Invitrogen), in humidified 5% CO2. To induce neuronal differentiation by P19 EC cells, they were aggregated in
bacterial-grade Petri dishes with 1 µM
all-trans-retinoic acid (Sigma) at a density of 1 × 105/ml. After 4 days, aggregates were dissociated by 0.25%
trypsin-0.53 mM EDTA (Invitrogen), and re-plated in
poly-L-lysine-coated tissue culture dishes and then allowed
to differentiate 6 more days. For endodermal differentiation, P19 and
F9 cells were cultured with 10 and 100 nM RA, respectively,
for 4 days.
Wnt-3a-expressing L cells (kindly provided by Dr. Roel Nusse, Stanford
University) were cultured in DMEM containing 10% FBS and 200 µg/ml
G418 (Invitrogen). For preparation of Wnt-3a-conditioned medium (CM),
the Wnt-3a-expressing L cells were grown in serum-free DMEM in
humidified 5% CO2, and Wnt-3a CM was harvested after
24 h. Wnt-3a CM was concentrated to 30 times by using Centricon
(Millipore). P19 cells were incubated with 10× Wnt-3a CM supplemented
with 10% FBS.
Immunochemical Staining--
For immunofluorescent labeling,
cells were fixed with 4% paraformaldehyde and washed in
phosphate-buffered saline (PBS), then permeabilized with 0.05% Triton
X-100 in PBS. The fixed cells were incubated with blocking solution
(1% normal goat serum (Jackson ImmunoResearch Laboratories, Inc.), 2%
bovine serum albumin (Sigma) in PBS) for 1 h. The cells were then
incubated overnight at 4 °C with mouse monoclonal anti-SSEA-1
(Development Studies Hybridoma Bank), anti- Northern Blot and RT-PCR--
Total RNA was isolated using
TRIzol (Invitrogen) from undifferentiated or differentiated P19 cells
at different time periods. For Northern blot analysis, ~20 µg of
total RNA was separated on a 1.2% formaldehyde-agarose gel and
transferred to NC membranes (Amersham Biosciences) by using a
TurboBlotter kit (Schleicher & Schuell). The inserts for the
preparation of probes for mouse Wnt-1, Wnt-3a,
and Wnt-5a were generated by PCR, using the pLNCX/Wnt-1, pLNCX/Wnt-3a, and pLNCX/Wnt-5a plasmid DNA (kind gift from Dr. Jan
Kitajewski, Columbia University) as templates with primers derived from
sequences in GenBankTM. The insert for the mouse
The following primers were used for PCR: for Wnt-1,
5'-CAGTAGTGGCCGATGGTG-3' and 5'-ATCGATGTTGTCACTGC-3'; for
Wnt-3a, 5'-TAGTGCTCTGCAGCCTGAA-3' and
5'-CCACAGATAGCAGCTGAT-3'; for Wnt-5a,
5'-ATTGGAATATTAAGCCCG-3' and 5'-GTGACCATAGTCGATGTT-3'; for
Western Blot--
Cells were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 0.1% SDS,
0.5% deoxycholic acid, 10% glycerol, 100 mM sodium
orthovanadate, and protease inhibitor mixture (1 mM EDTA, 1 mM PMSF, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin). Brains of 13.5 dpc Balb/c mouse embryos and 1-month-old postnatal mice were dissected and homogenized in lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 10%
glycerol, 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 2 mM Na3VO4, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin). Homogenates were clarified by centrifugation at
14,000 × g for 15 min at 4 °C, and total extracts
were obtained in the supernatant. Protein concentration was measured
using Bradford reagent (Bio-Rad). About 20 µg of lysate was subjected
to SDS-PAGE, and Western blot using following antibodies. Mouse
monoclonal antibodies for c-myc (for the detection of
myc-tagged Axin, Oncogene), Preparation of Cytoplasmic Fraction--
Cells were washed twice
in PBS and scraped in physiological buffer containing 10 mM
Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM EDTA, 2 mM dithiothreitol, 0.5 mM PMSF, 2 µg/ml
aprotinin, 1 µg/ml leupeptin. Cells were lysed by 30 strokes in a
chilled Potter-Elvehjem homogenizer (Wheaton) at 4 °C. The lysates
were centrifuged at 500 × g for 10 min to remove
unbroken cells and nuclei. The cleared lysates were subject to
ultracentrifugation at 100,000 × g for 90 min at
4 °C. The supernatants were collected as the cytoplasmic fraction. Protein concentration of the cytoplasmic fraction was measured by using
Bradford reagent (Bio-Rad).
Construction of Plasmids--
To construct pBI-EGFP-Axin,
pCS2-MT-mAxin (125-956) was digested with ClaI and
NotI, and both ends were filled-in with Klenow fragment
(Invitrogen). The 3-kb fragment was inserted into pBI-EGFP (Clontech), which was digested with NheI
and blunted with Klenow fragment. The SpeI and
HindIII digested fragment of the pTet-On (Clontech) was replaced with
SpeI/HindIII fragment of the
pUHDrtTA2S-M2 (kind gift from Dr. Wolfgang Hillen, Erlangen
University (52)) to construct CMV-rtTA2S-M2, which has a
neomycin resistance gene and improved version of rtTA. Mouse ICAT
cDNA was cloned by RT-PCR using total mouse RNA as template from a
published sequence (53). Cloned ICAT cDNA was inserted into the
pCS2-MT vector (13) for myc tagging on the amino terminus
(pCS2-MT-ICAT). pBI-EGFP-ICAT was constructed from pCS2-MT-ICAT with
the same methods that were described for the generation of
pBI-EGFP- Axin.
Generation of Stable Cell Lines and Induction by
Doxycycline--
P19 cells were cotransfected with the
CMV-rtTA2S-M2 and pBI-EGFP-Axin, or pBI-EGFP using the
Lipofectin reagent (Invitrogen) according to the manufacture's
protocol. Two days after transfection, transfectants were subcultured
and selected in culture media supplemented with 400 µg/ml G418. Each
selected clone was analyzed by fluorescence microscopy and Western blot
analysis. To induce expression of Axin or green fluorescent protein
(GFP), P19 cells were treated with 500 ng/ml doxycycline (Sigma).
Luciferase Assay--
To measure the inhibitory effect of an
ICAT-expressing construct on XTT Assay--
Cell viability was analyzed using an XTT
cell proliferation kit (Roche Molecular Biochemicals) according
to the manufacturer's instructions. P19 cells exposed to RA during
aggregation were trypsinized, and 5 × 103 cells were
re-plated onto 96-well plates. Cells were incubated with or without Dox
for 6 days, and tetrazolium salt XTT was added. The plates were
incubated further for 4 h at 37 °C, and the optical density
at 450 nm (A450) was measured with a spectrophotometer.
Differential Expression of Wnt Signaling Components during Neuronal
Differentiation of P19 EC Cells--
P19 embryonal carcinoma (EC)
cells can be differentiated into neuronal cells upon retinoic acid (RA)
treatment during aggregation followed by plating on tissue culture
plates (47, 48). This process can be detected with the
neuron-specific marker proteins,
It was shown that Axin is down-regulated upon Wnt-3a treatment of C57MG
and L cells (41, 54). Because the endogenous Wnt-3a level increased
during neuronal differentiation of P19 EC cells, we examined the levels
of Axin mRNA (Fig. 2A) and
protein (Fig. 2B), and found that they were both
down-regulated during neuronal differentiation (3.2 ± 0.9-fold
reduction in protein level for the 6-day-differentiated compared with
undifferentiated samples (n = 3)). We predicted that
down-regulation of Axin might lead to stabilization of cytoplasmic
Inducible, Ectopic Expression of Axin in RA-treated P19 Cells
Blocks Formation of Neurite-like Structures--
To test whether the
reduction in Axin levels is important for neuronal differentiation, we
used the Tet-On-inducible system to force the continued expression of
Axin in RA-treated P19 cells. Axin was cloned into the bi-directional
pBI-EGFP vector allowing EGFP to be used to infer the level of Axin
expression in the transfected cells (Fig.
3A). Cells transiently
cotransfected with CMV-rtTA and pBI-EGFP-Axin showed clear induction of
Axin and EGFP after Dox treatment (Fig. 3B), Axin was
clearly induced in pBI-EGFP-Axin-transfected clones after 1 day of Dox
treatment and disappeared in a day after removal of Dox, whereas the
EGFP level was maintained (Fig. 3C), possibly reflecting an
intrinsic difference in the stability of the two proteins in these
cells. The rapid reduction in Axin levels after withdrawal of Dox was
useful, in that it allowed us to induce Axin expression in transient
manner.
P19 cell clones stably transfected with
pBI-EGFP-Axin/CMV-rtTA2S-M2 or
pBI-EGFP/CMV-rtTA2S-M2 were treated with Dox.
This resulted in clear induction of myc-tagged Axin in
pBI-EGFP-Axin/CMV-rtTA2S-M2 (Fig.
4A, lanes 2 and
4). In the absence of RA treatment (i.e. in
undifferentiated cells), there was no apparent change in the level of
Previously we and others have shown that overexpression of
Axin caused apoptosis in transgenic mice and certain cell lines (56,
57). However, we could not see any obvious cell death after transient
induction of Axin in undifferentiated P19 cells (data not shown). We
also tested whether the failure of Dox-induced pBI-EGFP-Axin/CMV-rtTA2S-M2 cells to form neurite-like
structures upon RA treatment was due to excessive cell death.
pBI-EGFP-Axin/CMV-rtTA2S-M2- or
pBI-EGFP/CMV-rtTA2S-M2-expressing stable cell clones were
aggregated and differentiated in the presence of RA and different
concentrations of Dox. The XTT assay revealed no clear difference in
viable cell numbers between pBI-EGFP-Axin/CMV-rtTA2S-M2 and
pBI-EGFP/CMV-rtTA2S-M2 clones upon ectopic Axin induction
(Fig. 4D). Therefore, the effect of Axin on neuronal
differentiation of P19 cells is not due to cell death.
Ectopic Induction of a The Later Phase of Neuronal Differentiation by P19 Cells Is the
Sensitive Period for Blockage by Axin--
The ability to transiently
induce the expression of Axin by a short treatment with Dox (Fig.
3C) allowed us to examine the stage of P19 neuronal
differentiation that is sensitive to Axin expression. Neuronal
differentiation of pBI-EGFP-Axin/CMV-rtTA2S-M2 cells (with
no Dox treatment) resulted in induction of Ectopic Expression of Axin Has No Effect on the Initiation of
Differentiation but Blocks the Maturation of Neurite-like
Structures--
We have shown that ectopic induction of Axin blocks
neuronal differentiation, such as the formation of neurite-like
structures. Next, we tested whether induction of Axin blocks other
differentiation processes. It is known that the level of the embryonic
antigen SSEA-1 and E-cadherin are reduced upon RA-induced
differentiation of P19EC cells (59, 60). Undifferentiated
pBI-EGFP-Axin/CMV-rtTA2S-M2 cell clones, and those induced
to differentiate in the absence or presence of Dox, were
immunochemically stained with antibodies specific for SSEA-1,
Wnt3a Enhances Neuronal Differentiation of P19 EC
Cells--
Smolich and Papkoff (49) showed that overexpression of
Wnt-1 could not induce normal neuroectodermal differentiation of P19 EC
cells, although it could enhance certain aspects of that process. They
suggested that correct timing of Wnt expression is necessary for proper
neural differentiation. Wnt expression patterns, as shown by others
(49) and in Fig. 1B, suggest that Wnt-3a may have a more
important role than Wnt-1 in late neuronal differentiation. Because
Wnt-3a is induced during the late neuronal differentiation period, we
tested whether Wnt-3a could enhance neuronal differentiation.
Consistent with several published reports, incubation of P19 EC cells
with Wnt-3a-conditioned media (CM) caused a reduction in the Axin level
and an increase in the cytoplasmic While the evidence for the significance of Wnt signaling in
overall neural development was accumulated in many model organisms, such as in mouse, zebrafish, and frog, specific roles of different Wnts
in neuronal cell differentiation were not well studied. Only a few
studies, which revealed determination of neuronal cell fate by Wnt3a and regulation of presynaptic axon structure by Wnt7a, have
been published (10, 46). The involvement of Wnt signaling in the
neuronal differentiation of P19 embryonic carcinoma cells has been
suggested by the differential expression of various Wnts during that
process (49). Here, we provide evidence for the role of Wnt/canonical
Several groups have used P19 cells to test the role of Wnt signaling in
neuronal differentiation by simple overexpression of specific Wnts (49,
50). However, the dynamic changes in the expression of diverse Wnts
during neuronal differentiation (Fig. 1B) lead us to test
the significance of canonical Wnt signaling by blocking it, through the
induced expression of Axin, rather than by overexpression of specific
Wnts. Although Wnt1 and Wnt3a show similar spatial and temporal
expression patterns in vivo and are considered to belong to
the same group of Wnts, which signal through We found that the Wnt1 and Wnt3a mRNA levels are increased and
To determine whether the blockage of neuronal differentiation by
ectopic Axin induction was due to down-regulation of canonical In addition to the inhibition experiments, we used Wnt-3A-conditioned
media to confirm that Wnt/ Although induction of Axin during the neurite extension period (on
tissue culture plates after aggregation) blocked the formation of
neurite-like structures, induction of Axin during the aggregation period did not (Fig. 6). Furthermore, induction of Axin throughout the
aggregation and differentiation periods did not block the initiation of
differentiation (i.e. the disappearance of markers of
undifferentiated cells) (Fig. 7). One possible explanation is that
endogenous Axin is already highly expressed during the aggregation
stage, so that induction of exogenous Axin did not further enhance the
down-regulation of The findings, that the Axin level was lower in the brains of
1-month-old postnatal mice than in 13.5 dpc mouse embryos and that the
abnormal induction of Axin blocked the formation of neurite-like structures in P19 cells, suggest that Wnt signaling has a similar function in vivo. We are currently testing that possibility
by using a neuron-specific promoter to direct Tet-inducible
expression in transgenic mice.
-catenin. To test the role of Wnt signaling in
neuronal differentiation, embryonal carcinoma P19 cells (P19 EC), which
can be stimulated to differentiate into a neuron-like phenotype in
response to retinoic acid (RA), were used. Reverse transcription-PCR
and Western blot analysis showed that Axin is expressed in
undifferentiated cells, whereas the level is clearly reduced during
RA-induced neuronal differentiation. Interestingly, Axin levels were
not reduced during endodermal differentiation of P19 EC cells and F9 EC
cells by RA, suggesting that the reduction of the Axin level is a
specific property of neuronal differentiation. Western analysis showed that the cytoplasmic level of
-catenin increased during neuronal differentiation of P19 EC cells. Indirect immunofluorescence with
-catenin antibody showed that the localization of
-catenin was changed from membrane in undifferentiated cells to nuclei in neuronal P19 EC cells. Induced expression of Axin during endodermal and early
neuronal differentiation, using the Tet-On system, did not block
normal differentiation. However, maintenance of the Axin level blocked
neuronal differentiation and inhibited expression of a neuron-specific
marker protein,
III-tubulin. Also, ectopic induction of a
-catenin signaling inhibitor, ICAT, inhibited expression of
III-tubulin. In contrast, addition of Wnt-3A-conditioned medium
during the neuronal differentiation period enhanced the expression of
III-tubulin. Overall, our data show that Wnt-3a/canonical
-catenin signaling through the down-regulation of Axin may play an
important role in neuronal differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin degradation
complex, which contains adenomatous polyposis coli,
GSK-3
,1 and Axin, as well
as other proteins (13-25).
-Catenin that escapes from the
degradation complex enters into nuclei and interacts with Tcf/LEF
factors to regulate expression of downstream target genes (26-34).
-catenin pathway (37),
a finding that has been confirmed by genetic analysis in
Drosophila (38, 39). The accumulating data have shown that
Axin acts as a scaffolding protein, containing several domains that
interact with adenomatous polyposis coli, GSK-3
, CKI,
-catenin,
and other proteins (13-25). In the absence of a Wnt signal, Axin
itself is phosphorylated and enhances phosphorylation of
-catenin by
GSK-3
by bringing those proteins together, and the
ubiquitin-proteasome-mediated pathway leads phosphorylated
-catenin
to degradation (26-30). However, antagonizing GSK-3
activity upon
binding of Wnts to Frizzled leads to an increase in hypophosphorylated
Axin, which has lower affinity to interact with
-catenin. This
results in the release of hypophosphorylated
-catenin from the
degradation complex (40, 41). The released hypophosphorylated
-catenin is accumulated in the cytoplasm, translocated into nuclei,
and then interacts with Tcf/LEF factors to regulate downstream target
gene expression. Currently, about 50 target genes have been identified.
The known function of several identified target genes explains some of
the phenotypes that are caused by abnormal Wnt signaling. For example,
induction of c-myc or cyclin D1 expression by
Wnt signaling causes abnormal cell proliferation and results in tumors,
whereas ectopic Xenopus axis induction by the injection of
Wnts is due to enhanced expression of the dorsalizing homeobox gene
siamois, brachyury gene, and others (42-45).
-catenin signaling is involved in the neuronal
differentiation of P19 cells (49, 50).
-catenin
accumulates in the cytoplasm and the nucleus, during the neuronal differentiation of P19 cells. These data imply that canonical
-catenin signaling is involved in the neuronal differentiation of
P19 cells. To test the significance of canonical
-catenin signaling
for this process, the expression of Axin was induced during RA-induced
neuronal differentiation, using the Tet-On-inducible system (51, 52).
This treatment blocked the formation of neurite-like structures. In
contrast, neuronal differentiation of P19 cell was enhanced in the
presence of Wnt-3a-conditioned media. Overall, our data lead us to
conclude that Wnt-3a/canonical
-catenin signaling through the
down-regulation of Axin may play an important role in neural differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
III-tubulin (BAbCO),
anti-MAP2 (Sigma), anti-
-catenin, or anti-E-cadherin antibodies
(Transduction laboratories). After a rinse with PBS, the cells were
incubated with rhodamine, or fluorescein isothiocyanate-conjugated
(Jackson ImmunoResearch Laboratories) secondary antibodies at room
temperature for 1 h, mounted, and examined using a fluorescence
microscope (Zeiss).
-actin was obtained by RT-PCR with total RNA of P19 EC
cells. The sequences of all PCR products were confirmed by automated
sequencing. All probes were labeled with [
-32P]dCTP
using a Random Primed DNA Labeling kit (Roche Molecular Biochemicals)
and hybridized to the membrane with ExpressHyb solution (Clontech) according to the manufacturer's
protocol. All Northern blots were stripped and hybridized to
-actin
cDNA probe for even RNA loading control.
-actin, 5'-AGGCCAACCGCGAAGATGACC-3' and
5'-GAAGTCCAGGGCGACGTAGCAC-3'; for Axin,
5'-CAGGGTTTCCCCTTGGACC-3' and 5'-GGTCAAACATGGCAGGATC-3'; and for
ICAT, 5'-GAATTCGATGAACCGCGAGGGAGCAC-3' and
5'-CTCGAGCTACTGCCTCCGGTCTTCCGT-3'.
III-tubulin (BAbCO), MAP2, actin,
-tubulin (Sigma), GFP (Clontech), GSK-3
,
-catenin antibodies (Transduction Laboratories), and a rabbit
polyclonal antibody for Axin (kind gift from Dr. Paul Polakis,
Genentech Inc.) were used to detect the corresponding proteins.
Peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit secondary
antibodies (Sigma) were used, and then the proteins were detected by
using an enhanced chemiluminescence (ECL) reagent (Santa Cruz
Biotechnology, Inc.).
-catenin/Tcf signaling, we performed
luciferase assays as described previously (40).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
III-tubulin and MAP2
(microtubule-associated protein 2) (Fig. 1A). Two days after plating,
neurite-like structures began to appear, and after 6 days these
structures were obvious and clearly detected by immunochemical staining
with
III-tubulin and MAP2 antibodies. It has been shown that several
Wnts were differentially expressed during neuronal differentiation of
P19 EC cells (49). We observed a similar pattern of Wnt expression
during neuronal differentiation (Fig. 1B). Wnt-1 mRNA is
transiently expressed upon plating on tissue culture plates after
aggregation and the early stages of neuronal differentiation have
occurred. Interestingly, during the later stages of differentiation (4 and 6 days after plating), the level of Wnt-1 mRNA expression
rapidly diminished, whereas the expression of Wnt-3a was induced. In
contrast to either Wnt-1 or Wnt-3a, the level of Wnt-5a increases
steadily during differentiation. These observations suggest that
Wnt-1, Wnt-3a, and Wnt-5a may have different roles in P19 EC cell
differentiation. The occurrence of proper neuronal
differentiation was confirmed by Western blot analysis using anti
III-tubulin and MAP2 antibodies (Fig. 1C).
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Fig. 1.
Differential Wnt expression during neuronal
differentiation of P19 embryonal carcinoma (EC) P19 EC cells.
A, to induce neuronal differentiation, P19 EC cells were
plated on bacterial Petri dishes and allowed to aggregate for 4 days in
the presence of 1 µM RA and then re-plated on tissue
culture dishes. 2 and 6 days after plating, the cells were stained with
anti- III-tubulin or MAP2 antibodies. Rhodamine-conjugated secondary
antibody was used to detect the expression of
III-tubulin
(b and c, phase contrast (PC);
e and f, indirect immunofluorescence
(IF)). Fluorescein isothiocyanate-conjugated secondary
antibody was used to visualize the expression of MAP2 (h and
i, PC; k and l, IF). Undifferentiated
cells (UD) are shown as a control (a and
g, PC; d and j, IF). B,
Northern blot analysis of several Wnts during neuronal differentiation.
At an early stage of neuronal differentiation the Wnt-1 level is
transiently elevated. At a later stage, Wnt-1 is down-regulated while
the Wnt-3a level is up-regulated. The Wnt-5a level increases steadily
throughout differentiation. C, induction of
III-tubulin
and MAP2, as revealed by Western blot analysis, confirms the proper
neuronal differentiation of P19 EC cells.
-catenin. As shown in Fig. 2C, cytoplasmic
-catenin is
steadily accumulated during neuronal differentiation (4.1 ± 1.2-fold induction in undifferentiated versus in
6-day-differentiated samples (n = 3)), whereas the
actin and total
-catenin levels are unchanged. It is well accepted that accumulated cytoplasmic
-catenin is translocated into nuclei to
regulate the expression of downstream target genes. Immunochemical staining with anti
-catenin antibody revealed that
-catenin is
mainly localized in the nuclei of differentiated cells, in contrast to
its membrane localization in undifferentiated cells (Fig.
2D). These data suggest that down-regulation of Axin and induction of
-catenin signaling is important for neuronal
differentiation. To test whether the down-regulation of Axin occurs
in vivo as well as during P19 EC cell differentiation, the
Axin level was compared in the brains of 13.5 dpc mouse embryos (which
contain many undifferentiated neuronal precursor cells)
versus brains of 1-month-old postnatal mice (which contain
more differentiated neurons). The level of the neuron-specific marker
III-tubulin was increased during that period, as shown by others
(55), whereas the Axin level was reduced (Fig. 2E). RA
treatment of F9 EC, or treatment of P19 EC cells with a low
concentration of RA (10 nM), can lead to endodermal
differentiation (48). During endodermal differentiation of both cell
lines, as monitored by morphological changes (data not shown) the Axin
level was not changed (Fig. 2F). These in vivo
and in vitro data imply that the down-regulation of Axin
occurs specifically during neuronal differentiation but not endodermal
differentiation.
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Fig. 2.
Axin mRNA and protein levels are reduced,
whereas the cytoplasmic -catenin level is
increased during neuronal differentiation. RT-PCR (A)
and Western blot analysis (B) illustrate that the Axin level
is reduced while the
III-tubulin level is increased during neuronal
differentiation. An equal level of
-tubulin was used for the loading
control. C, the level of
-catenin in the cytoplasm is
induced during neuronal differentiation, whereas the total
-catenin
level is unchanged. Actin was used as a loading control. D,
the localization of
-catenin was changed from plasma membrane in
undifferentiated P19 EC cells to nuclei in neuronally differentiated
cells. Bar, 50 µm. E, the level of Axin protein
is lower in the brain of a 1-month-old mouse than in 13.5 dpc embryonic
brain, whereas the expression pattern of a neuronal marker,
-III
tubulin, is increased. F, axin protein level is not reduced
during endodermal differentiation of P19 EC and F9 embryonal carcinoma
cells.
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Fig. 3.
Use of the Tet-On-inducible system to express
Axin. A, diagram for the scheme of the Tet-On-inducible
system. B, induction of Axin and EGFP by Dox treatment of
P19 EC cells, which were stably transfected with pBI-EGFP/CMV-rtTA or
pBI-EGFP-Axin/CMV-rtTA. C, Axin was detectable after 1 day
of induction by Dox and lost within 1 day after removal of Dox, whereas
EGFP was still detectable 2 days after removal of Dox. Actin was used
for the loading control.
-catenin following Axin induction (Fig. 4B, lanes
1 and 2), which might be due to a very low level of
cytoplasmic
-catenin in undifferentiated P19 cells. When the P19
cells were induced to differentiate with RA, there was an increase in
cytoplasmic
-catenin (Fig. 4B, lanes 1 and
3), consistent with the data of Fig. 2C. However,
this accumulation of cytoplasmic
-catenin was reduced by the
Dox-induced expression of Axin (Fig. 4B, lanes 3 and 4). Interestingly, the expression of Axin in these cells also blocked the induction of the neuron-specific marker
III-tubulin (5.2 ± 1.4-fold reduction (n = 4),
Fig. 4A, lanes 3 and 4). We took
advantage of the bi-directional inducible vector system to examine
morphological changes in pBI-EGFP-Axin/CMV-rtTA2S-M2- or
pBI-EGFP/CMV-rtTA2S-M2-expressing stable cell clones. When
pBI-EGFP/CMV-rtTA2S-M2-expressing stable cell clones were
aggregated and differentiated by RA treatment, neurite-like
structures formed (arrows in Fig. 4C, top
panel, 76.3 ± 5.5% of EGFP-positive cells). However, the Dox-induced expression of Axin in
pBI-EGFP-Axin/CMV-rtTA2S-M2-expressing stable cell clones
significantly reduced the formation of those structures (Fig.
4C, bottom panel, 34.0 ± 13.5% of
EGFP-positive cells).
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Fig. 4.
Induction of Axin blocks neuronal
differentiation of P19 EC cells. A and B:
lanes 1 and 2, P19 EC cells stably
transfected with CMV-rtTA2SM2 and pBI-EGFP-Axin were treated with Dox
for 4 days (lane 2) without RA. III-tubulin was not
induced, and the cytoplasmic
-catenin level was not changed by the
forced expression of Axin. Lanes 3 and 4, P19 EC
cells were aggregated in the presence of RA and re-plated on tissue
culture dishes without (lane 3) or with Dox (lane
4) for 4 days. The induction of cytoplasmic
-catenin
(lane 4 in B), and the neuronal marker
III-tubulin (lane 4 in A) was clearly reduced.
C, induction of Axin blocks formation of the neurite-like
structures (bottom panel), whereas P19 EC clones carrying
the control vector pBI-EGFP and CMV-rtTA formed neurites in the
presence of Dox (arrows in top panel).
D, XTT assay for the measurement of viable cells.
pBI-EGFP/CMV-rtTA- or pBI-EGFP-Axin/CMV-rtTA-expressing stable cell
lines were aggregated and differentiated in the presence of different
Dox concentrations. Six days after differentiation, tetrazolium salt
XTT was added to those cultures, incubated for 4 h and measured
for formation of XTT formazan.
-Catenin Signaling Inhibitor, ICAT,
Inhibited Expression of
III-tubulin during Neuronal Differentiation
of P19 Cells--
To determine whether reduction of
-catenin
signaling is the main reason for the inhibition of neuronal
differentiation when Axin is induced, we used a different approach to
block
-catenin signaling. ICAT is known to inhibit Wnt/
-catenin
signaling by blocking the interaction between
-catenin and Tcf/LEF
factors (53, 58). We tested whether ectopic induction of ICAT acted similarly to Axin. Western analysis showed that the ICAT level was
maintained consistently throughout neuronal differentiation (data not
shown). Luciferase reporter assay by transient transfection of ICAT
plasmid suggested that our myc-tagged ICAT works as an inhibitor of
Wnt/
-catenin signaling (Fig. 5,
A-C). Myc-tagged ICAT was cloned into pBI-EGFP vector (Fig.
5A), and P19 cell clones stably transfected with
pBI-EGFP-ICAT/CMV-rtTA2S-M2 were treated with Dox.
Induction of myc-ICAT resulted in the reduction of neuron-specific
marker
III-tubulin (2.3 ± 0.3-fold (n = 3))
compared with control cells in which neuronal differentiation was
induced by RA in the absence of Dox (Fig. 5D,
lanes 3 and 4). This result supports our
conclusion that the blocking of neuronal differentiation by ectopic
Axin induction is mediated through the canonical Wnt pathway.
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Fig. 5.
Induction of ICAT down-regulates the
expression of the neuronal marker
III-tubulin. A, diagram of
pCS2-MT-ICAT and pBI-EGFP-ICAT constructs. B, expression of
myc-GFP and myc-ICAT after transient transfection. C,
luciferase reporter assay after transfection of 293T cells with
indicated plasmids showed that myc-ICAT blocked
-catenin mediated
induction of Tcf signaling. D: lanes 1 and
2, P19 EC cells stably transfected with CMV-rtTA2SM2 and
pBI-EGFP-ICAT were treated with Dox for 4 days (lane 2)
without RA. Lanes 3 and 4, P19 EC cells were
aggregated in the presence of RA and re-plated on tissue culture dishes
without (lane 3) or with Dox (lane 4) for 4 days. The induction of the neuronal marker
III-tubulin (lane
4 in D) was clearly reduced upon induction of
myc-ICAT.
III-tubulin expression
(Fig. 6, lanes 1 and
2), as shown above for P19 cells (Fig. 2). Dox-induced
expression of Axin during the entire 10-day culture period caused a
reduction in
III-tubulin induction (Fig. 6, lane 7).
However, Dox-induced expression of Axin during the first 4 days
(aggregation), followed by withdrawal of Dox for the next 6 days
(differentiation), had no effect on
III-tubulin expression (Fig. 6,
compare lanes 2 and 6). Axin expression was eliminated within 2 days after withdrawal of Dox (Fig. 6, lane 4). These data suggest that induction of Axin during the 4-day aggregation period is not sufficient to block neuronal differentiation. Induction of Axin for the first 6 days has no greater effect on
III-tubulin expression than does induction for the first 4 days (Fig. 6, compare lanes 4 and 5). In Fig. 4
(A and C), neuronal differentiation was blocked
when Axin was induced only during the neuronal differentiation period
and not during aggregation. Overall, these experiments suggest that the
later stages of RA-induced neuronal differentiation by P19 EC cells are
sensitive to the level of Axin expression.
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Fig. 6.
The late stage of neuronal differentiation is
sensitive to the inhibitory effects of Axin. Proteins were
isolated from pBI-EGFP-Axin/CMV-rtTA-expressing stable cell clones
after different periods of Dox treatment and withdrawal. Lane
1, undifferentiated cells; lane 2, cells differentiated
for 10 days without Dox; in lanes 3-7, Dox was added during
the 4-day aggregation period, and then either continued or withdrawn at
different times after plating; lane 3, 4 days Dox;
lane 4, 4 days Dox plus 2 days withdrawal; lane
5, 6 days Dox; lane 6, 4 days Dox plus 6 days
withdrawal; lane 7, 10 days Dox. The level of -III
tubulin was used to monitor neuronal differentiation. Constitutive
induction of Axin during later neuronal differentiation blocked the
induction of
-III tubulin (lanes 6 and
7).
III-tubulin, MAP-2, and E-cadherin. Consistent with the above data,
the Dox-induced expression of Axin greatly reduced the expression of
the neuronal markers
III-tubulin and MAP-2 (Fig.
7; compare with Fig. 4). However, this
treatment had no effect on the down-regulation of SSEA-1 or E-cadherin
(Fig. 7). These data suggest that ectopic induction of Axin does not block the initiation of differentiation by P19 cells but blocks a
later step in the neuronal differentiation pathway.
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Fig. 7.
Induced expression has no significant effect
on the initiation of differentiation but blocks formation of
neurite-like structures. pBI-EGFP-Axin/CMV-rtTA-containing P19 EC
clones were differentiated by RA treatment in the presence or absence
of Dox. Cells were stained with either markers for undifferentiated EC
cells (SSEA-1 and E-cadherin) or neuronal markers ( -III tubulin and
MAP2) and rhodamine-conjugated secondary antibodies. Regardless of Axin
induction, markers for undifferentiated EC cells were lost upon RA
treatment (a, e, and i for SSEA-1;
d, h, and l for E-cadherin). Neuronal
differentiation markers
-III tubulin and MAP2 were barely detectable
in the presence of Axin induction (b, f, and
j for
-III tubulin; c, g, and
k for MAP2).
-catenin level (Fig.
8). Incubation of undifferentiated P19 EC cells in the presence of Wnt-3a CM did not induce expression of the
neuronal marker protein
III-tubulin. These data suggest that Wnt-3a
does not have the ability to induce neuronal differentiation. However,
when P19 EC cells were aggregated and differentiation was induced by RA
in the presence of Wnt-3a-conditioned media, the level of
III-tubulin was obviously enhanced (Fig. 8). These data suggest that
Wnt-3a signaling through the down-regulation of Axin and up-regulation
of cytoplasmic
-catenin may play an important role in neural
differentiation.
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Fig. 8.
Wnt-3a-conditioned medium down-regulates Axin
level and results in clear induction of the neuronal differentiation
marker -III tubulin. Undifferentiated P19
EC cells were grown in control or Wnt-3a-conditioned media
(CM). Incubation with Wnt-3a CM led to down-regulation of
Axin and induction of cytoplasmic
-catenin. However, it did not
induce
-III tubulin expression in undifferentiated P19 EC cells
(lanes 1 and 2). P19 EC cells were aggregated for
4 days in the presence of 1 µM RA and then re-plated on
tissue culture dishes in the presence of control or Wnt-3A CM for 4 more days. The Axin level was reduced in cells differentiated in the
presence of Wnt-3a CM and the level of neuronal differentiation marker
-III tubulin was clearly enhanced.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin signaling specifically in neurite extension/maturation. We
report that Axin, a negative regulator of the canonical Wnt signaling
pathway, is down-regulated while the
-catenin level is increased
during neuronal differentiation of P19 cells. Furthermore, the forced
expression of either Axin or ICAT, a
-catenin signaling inhibitor,
during the neuronal differentiation process resulted in blockage of
neurite formation and the induction of the neuron-specific marker
III-tubulin. In addition, the enhanced expression of
III-tubulin
after treatment with Wnt3A-conditioned medium suggests that
Wnt/
-catenin signaling has a positive role in neuronal differentiation.
-catenin, the temporal
expression of these two Wnts do not seem to overlap during neuronal
differentiation of P19 cells (Fig. 1B). These differing
expression profiles imply that Wnt1 and Wnt3a may have different roles
in the regulation of the neuronal differentiation process, which might
account for the failure of Wnt1 to induce normal differentiation when
overexpressed in P19 cells (49). Recently emerging data suggest that
Ca2+ signaling is involved in learning and memory, and a
non-canonical protein kinase C/Ca2+ pathway is also
important in transducing certain Wnt signals (for review, see Refs. 2
and 61). The steady increase of Wnt5a, which is believed to
regulate the protein kinase C/Ca2+ pathway, during neuronal
differentiation (Fig. 1B) suggests a role for non-canonical
Wnt signaling in neuronal differentiation of P19 cells (although it was
not examined in the current work).
-catenin is accumulated in nuclei during neuronal differentiation (Fig. 2). It is generally considered that Wnt1 and 3a have mitogenic activity rather than a role in differentiation. However, recently it
has been shown that Wnt3a/
-catenin signaling is necessary and
sufficient for myogenic differentiation in P19 cells, and Wnt1 is also
known to have role in melanocyte expansion and differentiation during
mouse embryogenesis (62, 63). This raises the interesting question of
how the same
-catenin accumulation in nuclei directs two opposite
outputs: enhancement of mitogenic activity and differentiation.
-catenin signaling, rather than activation other signaling pathways, such as JNK activation, we used two different approaches:
down-regulation of
-catenin by Axin (Fig. 4) and inhibition of
-catenin-Tcf complex interaction by ICAT (Fig. 5). When either Axin
or ICAT was induced during the period of neuronal differentiation, the expression of the neuron-specific marker
III-tubulin was clearly reduced, although the effect of ICAT was weaker (5.2-fold reduction in
Axin induction versus 2.3-fold reduction in ICAT induction, Figs. 4 and 5). This may be due to the relatively high level of endogenous ICAT (data not shown), which could reduce the impact of
induced ICAT expression. In any case, the results support the conclusion that at least part of the activity of Axin is due to its
effects on the
-catenin pathway.
-catenin signaling has a positive role in
neuronal differentiation. Although the results were consistent with
such an effect, we cannot rule out the possibility that the enhanced
expression of the neuron-specific marker
III-tubulin was caused by
other molecules secreted by the Wnt-3A-expressing cells rather
than Wnt-3A itself.
-catenin in that period. Another plausible
explanation is that Wnt signaling is not involved in the initiation of
differentiation. In all our experiments we compared undifferentiated
versus differentiated P19 cells after aggregation, as shown
in the diagram of Fig. 6. The weak level of Wnt1 expression right after
aggregation (Fig. 1B, lane 2) suggests that
aggregation in the presence of RA is itself sufficient to initiate
differentiation stage without Wnt signaling.
![]() |
FOOTNOTES |
---|
* This study was supported by the Biomedical Brain Research Center, Ministry of Health & Welfare, Republic of Korea (Grant 01-PJ8-PG6-01NE01-0003 to E. J.) and the National Institutes of Health (to F. C.).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.
To whom correspondence may be addressed. Tel.: 822-2210-2681;
Fax: 822-2210-2888; E-mail: ej70@uos.ac.kr.
** To whom correspondence may be addressed. Tel.: 822-590-2613; Fax: 822-533-3801; E-mail: ckjoo@catholic.ac.kr.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M300591200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GSK-3, glycogen
synthase kinase 3
;
EC, embryonal carcinoma;
RA, retinoic
acid;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine
serum;
CM, conditioned medium;
PBS, phosphate-buffered saline;
RT, reverse transcriptase;
PMSF, phenylmethylsulfonyl fluoride;
MAP2, microtubule-associated protein 2;
GFP, green fluorescence protein;
EGFP, enhanced GFP;
CMV, cytomegalovirus;
Dox, doxycycline;
Tcf/LEF, T
cell factor/lymphoid enhancer factor;
CKI, casein kinase I;
dpc, days
post-coitum;
rtTA, reverse tetracycline controlled transactivator;
ICAT, inhibitor of
-catenin and Tcf-4.
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