From the Section on Growth Factors and
§ Laboratory of Developmental and Molecular Immunity,
National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892,
Division of Growth Regulation, Department
of Medicine, Beth Israel-Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215, and
§§ Laboratory of Molecular Biology, Division of
Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland
20892
Received for publication, September 26, 2000
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ABSTRACT |
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Recently, we characterized the rat epidermal
growth factor receptor (EGFR) promoter and demonstrated that TCC repeat
sequences are required for the down-regulation of EGFR by nerve
growth factor (NGF) in PC12 cells. In this study, we report that the
Wilms' tumor gene product WT1, a zinc finger transcription factor, is able to enhance the activity of the rat EGFR promoter in cotransfection assays. Gel mobility shift assays demonstrate that WT1 binds to the TCC
repeat sequences of the rat EGFR promoter. Overexpression of WT1
resulted in up-regulation of the expression levels of endogenous EGFR
in PC12 cells. Interestingly, NGF down-regulated the expression levels
of WT1 and EGFR in PC12 cells, but not in the
p140trk-deficient variant PC12nnr5 cells or in
cells expressing either dominant-negative Ras or dominant-negative Src.
Most importantly, we evaluated the inhibitory effect of antisense WT1
RNA on EGFR expression, and we found that antisense WT1 RNA could
substantially reduce EGFR repression in either histochemical staining
study or immunoblot analysis. These results indicate that NGF-induced down-regulation of the EGFR in PC12 cells is mediated through WT1 and
that WT1 may play an important role in the differentiation of
nerve cells.
The epidermal growth factor receptor
(EGFR)1 is a member of the
ErbB family of ligand-activated tyrosine kinase receptors, which play a
central role in the proliferation, differentiation, and/or oncogenesis
of epithelial cells, neural cells, and fibroblasts (1). Some years ago,
we reported that PC12 cells respond to both epidermal growth factor
(EGF) and nerve growth factor (NGF), but that the response to epidermal
growth factor is mitogenic, whereas the response to nerve growth factor
leads to differentiation (2). It was of interest to determine what
would happen if the cells were exposed to both stimuli at the same
time. The answer was that the cells simply did not respond to EGF after
they had been treated with NGF, and the reason that they did not
respond is that the EGFR is markedly down-regulated by treatment of the cells with NGF (3).
Our studies have focused on the molecular mechanism by which NGF
down-regulates the EGFR. Previous studies have demonstrated that
NGF-induced down-regulation of the EGFR was at the transcriptional level (4) and that the down-regulation is p140trk-, Ras-,
and Src-dependent (5). Recently, we isolated and
characterized the rat EGFR promoter region and found that TCC repeat
sequences in the promoter region of the rat EGFR are required for the
down-regulation of rat EGFR by NGF during the differentiation of PC12
cells (6).
WT1, the Wilms' tumor suppressor gene, is located at chromosome locus
11p13 and encodes a zinc finger protein that is one of several
transcription factors that have been found to interact with TCC repeat
sequences (7-9). Experimental evidence has indicated that WT1 not only
plays a role during kidney development but is also involved in the
development and homeostasis of other tissues. The products of WT1 have
been implicated in various cellular processes such as proliferation,
differentiation, and apoptosis (10). To better understand the molecular
mechanisms of down-regulation of EGFR by NGF in PC12 cells, it is
important to study the possible interaction between WT1 and EGFR during
the differentiation of PC12 cells.
Here, we report that the Wilms' tumor gene product WT1 activates the
activity of the rat EGFR promoter through binding to the TCC repeat
sequences. Treatment of PC12 cells with NGF decreases the expression
levels of WT1 and EGFR. However, the down-regulation of WT1 by NGF,
like the EGFR, is not seen in PC12nnr5 cells or in cells expressing
either dominant-negative Ras or dominant-negative Src. As another
approach, we have studied the inhibitory effect of antisense WT1 RNA on
EGFR expression and found that antisense WT1 RNA could substantially
reduce EGFR repression. These data strongly suggest that WT1 is
involved in the NGF-induced down-regulation of the expression levels of
EGF receptor in PC12 cells.
Materials--
NGF and rat type I collagen were purchased from
Becton Dickinson (Bedford, MA). Monoclonal antibody against the EGFR
(6F1) was obtained from Medical and Biological Laboratories, Co., Ltd. (Nagoya, Japan). Anti-WT1 antibody (C-19) and anti-Sp1 antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LipofectAMINE
and LipofectAMINE 2000 were purchased from Life Technologies, Inc.
(Gaithersburg, MD). Cy3-linked donkey anti-mouse antibody was purchased
from Jackson Immunoresearch (West Grove, PA). The Mammalian
Transfection Kit was purchased from Stratagene (La Jolla, CA). Stable
dominant-negative Src PC12 variant cells (SrcDN2) were the kind gift of
Dr. Simon Halegoua (State University of New York-Stoneybrook,
Stoneybrook, NY), stable dominant-negative Ras PC12 variant cells
(M-M17-26) were generously provided by Dr. Geoffrey Cooper (Dana
Farber Cancer Center, Boston, MA), and PC12nnr5 (a pheochromocytoma
clone unresponsive to nerve growth factor) cells were graciously
contributed by Dr. Lloyd Greene (Columbia University, New York, NY).
Cell Culture--
PC12 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 5% fetal bovine serum, 10% horse
serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. For NGF
treatment, 100 ng/ml NGF was added to the culture medium. In all
experiments involving extended treatment with NGF, the medium was
changed, and fresh NGF was added every other day. PC12nnr5 cells (11) were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum and 10% horse serum. The PC12 cell variants M-M17-26 expressing the Ha-ras Asn-17 gene under the transcriptional control of the mouse metallothionein-I promoter (12)
and srcDN2 expressing the K295R mutant (kinase-inactive) form of
chicken Src under the control of the cytomegalovirus (CMV) promoter
(13) were grown, as were PC12 cells, in Dulbecco's modified Eagle's
medium supplemented with 5% fetal bovine serum and 10% horse serum.
In all experiments, cells were cultured on collagen-coated dishes (5 µg/cm2).
Western Blot Analysis--
Cells were lysed in a buffer
containing 20 mM HEPES, pH 7.4, 100 mM NaCl,
0.1% deoxycholic acid, 10% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM sodium vanadate, and 50 mM
sodium fluoride at 4 °C for 30 min. Insoluble material was removed
by centrifugation, and equal amounts of lysates (25 µg) were used for
immunoblotting. Protein concentration was estimated using the Bio-Rad
protein assay system. Samples were resolved on a 7.5%
SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking with nonfat dry milk, the blot was incubated with either 6F1 anti-EGFR antibody (0.6 µg/ml) or anti-WT1 antibody (C-19; 1:1,000 dilution). Bound antibodies were detected by sheep anti-mouse immunoglobulin or
donkey anti-rabbit immunoglobulin antibody conjugated with horseradish
peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ) and analyzed
with the Supersignal Chemiluminescent Substrate (Pierce, Rockford, IL).
Plasmid Constructs--
The rat EGFR promoter-luciferase pRE
constructs and the chimeric EGFR reporter pRE-TK-Luc plasmids have been
described previously (6). The original WT1 expression vector pCMV-WT1
consisted of the full-length protein coding regions under the control
of the CMV immediate-early promoter (14). pCMV-WT1 was cut with EcoRI and subcloned into pTracer-CMV2 (Invitrogen, Carlsbad,
CA), which contains a SV40 early promoter that drives expression of the
green fluorescent protein (GFP) gene fused to a Zeocin resistance gene
and a CMV immediate-early promoter driving expression of the second
cDNA. WT1 expression plasmid pCMV2-WT1-GFP and antisense WT1
expression plasmid pCMV2-antiWT1-GFP were confirmed by DNA sequencing.
Plasmid DNAs were prepared from these constructs using Maxi-prep kits
(Qiagen, Valencia, CA) and quantitated by UV spectroscopy. The plasmid
pRL-TK (Promega, Madison, WI), which contained the Renilla
luciferase gene under the control of the thymidine kinase promoter, was
prepared in a similar way and used as an internal control.
Reporter Gene Assay of the WT1 Expression Plasmid with the Rat
EGFR Promoter Constructs in PC12 Cells--
PC12 cells cultured on
collagen-coated 6-well plates (Nunc, Naperville, IL) were transfected
using LipofectAMINE with 5 µg of pCMV-WT1 or CMV empty vector, 1 µg
of each reporter gene plasmid, and 0.05 µg of the internal control
pRL-TK. Five h after transfection, the solution was removed, and
culture medium was added. Twenty-four h after transfection, cell
lysates were prepared with the Dual-Luciferase Reporter Assay system
(Promega), and both firefly and Renilla luciferase
activities were measured in a LB 9507 luminometer (Berthold, Wildbad,
Germany). The transfection efficiency was normalized according to the
Renilla luciferase activity. The data are expressed as the
means ± S.D.
Overexpression of WT1 Protein in PC12 Cells--
Seventy to
eighty percent confluent cultures of PC12 cells were transiently
transfected with 30 µg of pCMV2-WT1-GFP expression vector or GFP
empty vector, 1 µg of pRE318 reporter gene plasmid, and 0.05 µg of
the internal control pRL-TK using the calcium phosphate coprecipitation
method according to the protocol for the Mammalian Transfection Kit
(Stratagene). Each transfection in duplicate was performed in 150-mm
dishes. Forty-eight h after transfection, cells were sorted by GFP
fluorescence using a FACScan flow cytometer (Becton Dickinson, San
Jose, CA). Cells with GFP expression or untransfected cells were
harvested, and cell lysates were prepared separately for Western blot
analysis and reporter assay as indicated above.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from PC12 cells as described previously (15). The TCC repeat
sequences corresponding to the rat EGFR promoter region from Evaluation of Endogenous WT1 Effects on EGFR Expression in PC12
Cells--
For histochemical staining, PC12 cells grown on
poly-L-lysine (Sigma, St. Louis, MO)-coated LAB-TEK chamber
slides (Nalge Nunc, Naperville, IL) were transiently transfected with 1 µg of pCMV2- antiWT1-GFP or GFP alone for 24 h using
LipofectaMINE 2000 according to the manufacturer's protocol. Cells
were fixed in PBS containing 4% paraformaldehyde for 10 min and then
washed three times in PBS. EGFR was detected with anti-EGFR antibody 6F1 at a dilution of 1:50 and with Cy3-linked donkey anti-mouse antibody at a dilution of 1:100. Cells were imaged on a Nikon ECLIPSE
E800 microscope with a ×40 immersion lens. Fifteen fields were
examined, and representative fields were photographed.
For immunoblot analysis, PC12 cells transfected with antisense
pCMV2-antiWT1-GFP or GFP alone using the calcium phosphate coprecipitation method as described above were cultured for 14 additional days in medium supplemented with 500 µg/ml Zeocin. The
Zeocin-resistant transfectants were pooled, and the expression level of
EGFR and WT1 was examined by immunoblot analysis in pooled cell lysates.
WT1 Activates the Promoter Activity of the Rat EGFR
Promoter--
Recently, we isolated and characterized the promoter
region of the rat EGFR promoter, and we found that TCC repeat sequences in the promoter region are required for NGF-induced transcriptional down-regulation of EGFR (6). The TCC repeat sequences in the human EGFR
promoter have been reported to be a DNA-binding sequence for the
Wilms' tumor repressor, WT1 (16). Thus, it is possible that WT1 may
bind to this TCC-rich sequence and modulate the transcription of the
rat EGFR gene. To test this hypothesis, we cotransfected different
reporter plasmids containing sequential or internal deletions of rat
EGFR promoter into PC12 cells with pCMV-WT1 expression vectors or empty
control vectors. Interestingly, expression of WT1 activated the
promoter activity of the reporter plasmid about 2-fold. These results
suggest that the rat EGFR gene is one of the genes transcriptionally
activated by WT1 (17).
Sequence analysis of the 5'-flanking region of the rat EGFR gene shows
that the TCC repeat sequences were found at
To further determine whether the TCC repeat sequences in the EGFR
promoter are functionally responsible for the activation of the EGFR by
WT1, we cloned oligonucleotides containing the 59-base pair DNA
fragments ( WT1 Up-regulates the Expression Levels of Endogenous EGFR in PC12
Cells--
To determine whether WT1 up-regulates EGFR expression, we
transiently transfected PC12 cells with the pCMV2-WT1-GFP expression vector or with the GFP empty vector as a control and prepared GFP
fluorescence-sorted cell lysates that were then tested for the levels
of EGFR. Western analysis indicated that WT1 increased the protein
level of EGFR in PC12 cells (Fig.
2A). Additionally, WT1 was
found to lead to a 6-fold activation of the rat EGFR promoter construct
pRE318 (Fig. 2B). These results indicate that expression of
exogenous WT1 in PC12 cells results in an increase of the expression levels of endogenous EGFR.
WT1 Binds Directly to the TCC Repeat Sequences of the EGFR
Promoter--
To determine whether WT1 binds directly to the TCC
repeat sequences within the rat EGFR promoter ( WT1 Expression Level Is Down-regulated by NGF in PC12
Cells--
We have shown that the region between NGF-induced Down-regulation of WT1 Is p140trk-,
Ras-, and Src-dependent--
Signal transduction pathways in
PC12 cells have been studied thoroughly, providing the opportunity to
define the pathway mediating WT1 down-regulation. We have previously
shown that down-regulation of EGFR in PC12 cells caused by NGF
treatment is dependent on p140trk, Ras, and Src pathways
(5). It is quite reasonable to ask whether these pathways are involved
in the down-regulation of WT1. To test this possibility, three cell
lines were used: PC12nnr5, a cell line that expresses very little or no
p140trk, the high affinity receptor for NGF (11); and two
PC12 cell variants that express dominant-negative forms of Ras
(M-M17-26) and Src (SrcDN2), respectively (12, 13). When cells were
treated with NGF for 5 days, expression levels of both WT1 and EGFR
were decreased dramatically in wild-type PC12 cells. However, no
reduction of WT1 and EGFR was observed upon NGF treatment in M-M17-26,
SrcDN2, and PC12nnr5 cells (Fig. 4C). These data strongly
suggest that NGF-induced down-regulation of EGFR involves WT1 in a
p140trk-, Ras-, and Src-dependent manner.
Endogenous WT1 Effects on EGFR Expression in PC12 Cells--
To
further determine the effects of endogenous WT1 on EGFR expression, we
evaluated the inhibitory effect of an antisense WT1-GFP construct in
PC12 cells. Histochemical staining revealed that expression of both WT1
and EGFR in pCMV2-antiWT1-GFP-transfected cells was decreased
compared with that in GFP control cells and wild-type PC12 cells (Fig.
5A). The decrease in EGFR
expression was also detected by immunoblot analysis comparing extracts
from pCMV2- antiWT1-GFP-transfected cells with GFP control
cells and wild-type PC12 cells (Fig. 5B). This result
indicates that endogenous WT1 contributes to the regulation the EGFR
expression.
EGFR, the prototypical member of the superfamily of receptors with
intrinsic tyrosine kinase activity, is widely expressed on many cell
types. Upon activation by its ligand, the intrinsic kinase
phosphorylates its own tyrosines and numerous intermediary effector
molecules, including closely related c-erbB receptor family members.
This initiates numerous signaling pathways. The integrated biological
responses to EGFR signaling are pleiotropic, including mitogenesis,
apoptosis, enhanced cell motility, and differentiation. In addition to
being implicated in organ morphogenesis, unregulated EGFR signaling has
been correlated in a wide variety of tumors with invasion and
metastasis. Thus, regulation of EGFR signaling including its own
expression has potential impact on the integrity of cells.
The Wilms' tumor gene (WT1) is one of at least three genes that are
involved in the development of Wilms' tumor, a pediatric kidney
cancer. Wilms' tumor has been the subject of intense clinical and
basic scientific research because it presents a model for cancer
treatment and for the relationship between development and cancer, and
it illustrates the impact of genetic alterations on development and
tumorigenesis. The products of WT1 have been implicated in various
cellular processes such as proliferation, differentiation, and
apoptosis. In agreement with the diverse functions of WT1, the number
of candidate target genes is rapidly mounting (10). EGFR has been shown
to be one of potential targets of WT1 (17).
The PC12 cell line is a good model for the study of NGF action and
responds to NGF by differentiating into a postmitotic cell type with
neuronal characteristic (18, 19). Although both EGF and NGF stimulate a
common set of signaling pathways, NGF induces differentiation in PC12
cells, whereas EGF induces proliferation in PC12 cells. We have
previously shown that EGFR is down-regulated by NGF treatment and
controlled transcriptionally (4, 6). Here we provide evidence that the
WT1 protein is directly involved in the down-regulation of EGFR in PC12
cells. First, rat EGFR promoter activity is enhanced by expression of
WT1. Second, deletion and mutation of TCC repeat sequences within the
EGFR promoter abolish the enhancing effect of WT1. Third, WT1 binds to
TCC repeat sequences in electrophoretic mobility shift analysis, and
mutations in TCC repeat sequences diminish the binding, consistent with the result of the reporter gene assays. Fourth, overexpression of the
WT1 protein increases the expression level of endogenous EGFR. Fifth,
the expression level of endogenous WT1 is concomitantly decreased along
with NGF treatment in PC12 cells, suggesting that WT1 regulates the
transcription of EGFR. Supportive evidence is that neither the EGFR nor
WT1 expression level is altered when NGF is added to the culture of
cells lacking p140trk and expressing dominant-negative
forms of Ras and Src. Finally, antisense WT1 RNA substantially reduces
WT1 and EGFR expression, indicating that endogenous WT1 is contributing
to regulation of EGFR expression. These data strongly demonstrate that
down-regulation of rat EGFR by NGF in PC12 cells is mediated by WT1. It
is likely that the down-regulation of EGFR is a consequence of
down-regulation of WT1 by NGF. Both events are dependent on
p140trk, Ras, and Src, which is consistent with this possibility.
Although it had been shown that WT1 protein could activate or repress
the expression of a number of promoters in cotransfection experiments
(17, 20-23), endogenous forms of many of these putative target genes
were later shown not to be regulated by WT1 in biological systems (24).
However, our data include not only cotransfection assays of the
reporter plasmids and an increased expression of endogenous EGFR in
PC12 cells but also the endogenous WT1 effect on EGFR repression.
Englert et al. (21) have reported that WT1 suppresses
synthesis of the human EGFR and induces apoptosis by binding to two
TC-rich repeat sequences in certain cell lines (Saos-2 and U2OS), but
not in all types of cells. Our result supports the notion that cellular
environment is a key aspect for the function of WT1 in
vivo.
Because WT1 protein is expressed mainly in developing kidney, testis,
and ovary, indicating that it is involved in the differentiation of
genitourinary tissues, it is of interest that WT1 protein is decreased
along with neuronal differentiation of cells. Once neurotrophin is
provided and cells are committed to being differentiated into neurons,
there is suppression of a specific set of genes. Given that EGFR is a
crucial factor for proliferation of cells, WT1 could have the potential
to regulate differentiation. Studies using a variety of leukemic cell
lines suggest that wild-type WT1 is acting to promote cellular
proliferation rather than differentiation (24). Recently, amphiregulin,
a member of the epidermal growth factor family, was found to be a major
target of WT1 and activated by WT1 (25). This also indicates that genes
whose transcription is activated by WT1 may be critical for biological activity.
In summary, down-regulation of WT1 by NGF appears to mediate
down-regulation of EGFR during the differentiation of PC12 cells induced by NGF. The molecular mechanism for the down-regulation of WT1
by NGF in PC12 cells will help us to understand details about the
down-regulation of EGFR and cell differentiation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISSCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISSCUSSION
REFERENCES
318 to
260 and its complement were labeled with 32P and used as
the probe. Aliquots of nuclear extracts (10 µg) were preincubated
with the binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 4% glycerol, 1 mM MgCl2,
2 µg of poly(deoxyinosinic-deoxycytidylic acid), and appropriate double-stranded competitor oligonucleotide) for 10 min at
room temperature. After the addition of labeled probe, samples were
incubated for an additional 20 min. The DNA-protein complexes were
subjected to electrophoresis using 4% polyacrylamide, 0.5×
Tris-Glycine EDTA gels, followed by autoradiography. To
determine specific binding, unlabeled specific competitor
oligonucleotides were added in 50-fold molar excess over labeled probe.
To identify proteins in the DNA-protein complex, supershift experiments
were performed with either an anti-WT1 C-19 antibody or an anti-Sp1 antibody.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISSCUSSION
REFERENCES
343,
317, and
298
relative to the translation starting site (6). Significant transcriptional activation of the rat EGFR promoter by WT1 was observed
in the constructs pRE1102, pRE1102D260, pRE318, and pRE318D260, all of
which contain two or three TCC repeat sequences, whereas the construct
pRE1102D318-260, which contains only one TCC repeat sequence, was
activated slightly by WT1 (Fig.
1A). Also, the promoter activities of the constructs pRE260 and pRE181, both of which lack TCC
repeat sequences, were not activated by WT1. Thus, we concluded that
WT1 activated the promoter activity of the EGFR gene through the TCC
repeat sequences in the promoter region.
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Fig. 1.
Transcriptional activation of the rat EGFR
promoter by WT1. A, successive and internal deletions
of the rat EGFR 5'-region were ligated to firefly luciferase reporter
gene. The 3' terminus of each deletion construct is nucleotide 2
relative to the rat EGFR translation initiation codon. Individual
reporter plasmids were cotransfected into PC12 cells together with
either pCMV-WT1 expression vector or empty vector. Twenty-four h after
transfection, luciferase activity was measured. Transfection efficiency
was normalized by Renilla luciferase activity derived from
pRL-TK. Error bars, ±S.D. The values are the means
of triplicate results. B, the 59-base pair duplex fragments
extending from
318 to
260 bearing wild-type, deletion, or mutant
elements (for TCC motif) were cloned in front of the herpes simplex
virus thymidine kinase luciferase promoter reporter plasmid. The
sequences of the promoter fragments are shown at the left,
and the mutated sites are underlined. Individual reporter
plasmids were cotransfected with pCMV-WT1 expression plasmid or empty
plasmid, and luciferase activity was measured as described above.
318 to
260 of the EGFR promoter) upstream of the herpes
simplex virus thymidine kinase luciferase reporter plasmid. This
reporter plasmid was cotransfected with pCMV-WT1 expression vector into
PC12 cells (Fig. 1B). WT1 produced a 2- to 4-fold
activation of the reporter constructs containing TCC repeat sequences
and single site mutation of TCC repeat sequences. However, the
activation was abolished in the reporter constructs containing no TCC
repeat sequences (292D260Tk-Luc) or multiple mutation of TCC repeat
sequences (M1TK-Luc). These data support the observation that the TCC
repeat sequences are responsible for the transcriptional activation of
the rat EGFR mediated by WT1.
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Fig. 2.
Exogenous expression of WT1 increases the
level of endogenous EGFR in PC12 cells. PC12 cells were
cotransfected with either pCMV2-WT1-GFP expression vector or empty GFP
vector, together with pRE318 reporter plasmid and pRL-TK, by the
calcium phosphate method. Forty-eight h after transfection, cells were
sorted by GFP fluorescence using a FACScan flow cytometer. Cells with
GFP expression and untransfected cells were harvested, and expression
levels of EGFR and WT1 (A) and luciferase activity
(B) were measured. A, immunoblot analysis of EGFR
and WT1 detected by anti-EGFR antibody 6F1 and anti-WT1 antibody,
respectively. B, luciferase activity induced by WT1
expression. Error bars, ±S.D. Each experimental
point was performed in triplicate.
318 to
260),
electrophoretic mobility shift analysis was performed (Fig.
3). Two major protein-DNA complexes (Fig.
3, complexes A and B) were observed with
nuclear extracts from PC12 cells. To determine whether WT1 was present in the protein-DNA complexes, polyclonal antibody against WT1 was used.
The addition of anti-WT1 antibody decreased the intensity of complex A
dramatically, whereas the addition of anti-Sp1 antibody did not,
indicating that WT1 binds to the region of EGFR promoter (Fig. 3,
lanes 3 and 4). In support of this result,
mutations in TCC repeat sequences abolished the binding of WT1 in
electrophoretic mobility shift analysis as described previously (6).
Thus, some of complex A contains WT1 that reacts with anti-WT1. These results suggest that WT1 transcriptionally triggers EGFR promoter activity through direct binding to the TCC repeat sequences within the
EGFR promoter.
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Fig. 3.
The TCC repeat sequences appeared to be
responsible for the WT1-mediated down-regulation of the EGFR by NGF in
PC12 cells. The 59-base pair radiolabeled duplex probe (1 ng)
extending from 318 to
260 was incubated with 10 µg of untreated
PC12 cell nuclear extracts in the absence (lane 1) or
presence of 50-fold molar excesses of unlabeled cold probe (lane
2), depicted at the top of each lane. Supershift
experiments were performed with either anti-WT1 C-19 antibody or
anti-Sp1 antibody (lanes 3 and 4), and
electrophoresis was performed on nondenaturing 4%
polyacrylamide gels. Two major protein-DNA complexes (A and
B) were observed with nuclear extracts from PC12
cells..
318 and
260 in
rat EGFR promoter contains a NGF response element (6). Transcriptional activation of the EGFR promoter through direct binding of WT1 to TCC
repeat sequences described above led us to examine whether the WT1
expression level is altered in PC12 cells upon NGF treatment (Fig.
4, A and B).
Immunoblot analysis revealed that reduction in the expression levels of
the WT1 and EGFR proteins in PC12 cells is detectable 1 day after the
addition of NGF, and this reduction is gradual and progressive. The
correlation coefficient (r) between the relative protein level of EGFR
and WT1 is 0.9758 (p < 0.001).
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Fig. 4.
Down-regulation of EGFR and WT1 expression
levels induced by NGF in PC12 cells. A, expression
level of EGFR and WT1 was examined by immunoblot analysis. Cell lysates
were prepared at each time point after NGF addition. B,
relative protein level of EGFR and WT1 at each time point was estimated
by analyzing the band intensities obtained by Western blots using the
ImageQuant (Molecular Dynamics) program. Values were calculated as
percentages of untreated cells (0 h) and represent the means ± S.D. from three different experiments. The correlation coefficient (r)
between the relative protein level of EGFR and WT1 is 0.9758. C, PC12 cells and their variants were cultured with either
control medium (C) or NGF (N) for 5 days. Cell
lysates were prepared, and expression levels of EGFR and WT1 were
examined by immunoblot analysis.
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Fig. 5.
Effect of antisense WT1 RNA on EGFR
expression in PC12 cells. A, PC12 cells were
transiently transfected with pCMV2-antiWT1-GFP or GFP alone for
24 h. After fixation, the cellular distribution of antisense WT1
or GFP alone was detected by the GFP signal and staining with anti-EGFR
antibody. Left, phase-contrast images; middle,
cells transfected with GFP or pCMV2-antiWT1-GFP plasmids;
right, cells expressing EGFR were stained with anti-EGFR
antibody and visualized with Cy-3 fluorescence. Arrowheads
indicate the decreased level of EGFR expression in PC12 cells
expressing antisense WT1 RNA. B, PC12 cells transfected with
pCMV2-antiWT1-GFP or GFP alone were cultured for 14 additional days in
medium supplemented with 500 µg/ml Zeocin. The Zeocin-resistant
transfectants were pooled, and the expression level of EGFR and WT1 was
examined by immunoblot analysis in pooled cell lysates.
DISSCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISSCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Simon Halegoua for dominant-negative Src cells, Dr. Geoffrey Cooper for dominant-negative Ras cells, and Dr. Lloyd Greene for PC12nnr5 cells. We are grateful to Dr. D. Jing for help with fluorescence microscopy and Dr. Q. Yu for the fluorescence-activated cell-sorting experiments. We are very grateful to Dr. B. Lu for support of the revised manuscript.
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FOOTNOTES |
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* 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.
This article is dedicated to the memory of Dr. Gordon Guroff, Chief
of Section on Growth Factors and Deputy Scientific Director of the
National Institute of Child Health and Human Development, who died on
July 9, 1999. During his long career, he enriched the field of
neuroscience and all of us who had the privilege to know and work with him.
¶ Present address: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Present address: American Red Cross, Holland Laboratory,
Biochemistry Department, 15601 Crabbs Branch Way, Rockville, MD 20855.
** Present address: William T. Gossett Neurology Laboratories, Henry Ford Health Sciences Center, 1 Ford Place, 4D Research, Detroit, MI 48202.
¶¶ To whom correspondence should be addressed: Bldg. 37, Rm. 2D18, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-3224; Fax: 301-402-1344; E-mail: aj2e@nih.gov.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M008776200
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; NGF, nerve growth factor; CMV, cytomegalovirus; GFP, green fluorescent protein.
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