 |
INTRODUCTION |
The epidermal growth factor receptor
(EGFR)1 plays an important
role in cell growth and development (1-3). Overexpression of the EGFR
can lead to epidermal growth factor-dependent
transformation (4, 5). High levels of the EGFR have been detected in
several types of cancers such as glioblastomas due to gene
amplification (6). Overexpression of EGFR transcripts in a variety of
tumors such as ovarian, cervical, and kidney tumors results from
transcriptional or post-transcriptional mechanisms (7). Also, a variety
of agents have been shown to increase EGFR gene expression (8-10). Repression of EGFR gene transcription by different agents has been also
reported (11, 12). Thus, transcriptional control plays a major role in
regulation of EGFR gene expression.
The promoter of the EGFR gene lacks a TATA box and CAAT box but
contains multiple GC boxes and multiple transcription initiation sites.
A number of regions in the promoter have been identified that bind
nuclear factors (13-15). Four repressor proteins, EGFR transcriptional
repressor, GC-binding factor, GC-binding factor 2, and the
Wilms' tumor suppressor bind to sites within the EGFR promoter
(16-19). Sp1, interferon-regulated factor-1, EGFR transcription factor, activator protein-1, and activator protein-2 have been shown to
increase EGFR gene transcription (20-24). Two groups have shown that
p53 can transactivate EGFR via binding to an upstream site of the EGFR
promoter between 
265 and 239 base pairs (25, 26).
p53 is the most frequently mutated tumor suppressor gene identified
thus far in human cancers (27, 28). p53 appears to induce cell cycle
arrest or apoptosis in response to cellular stresses such as DNA
damage, mitotic spindle misassembly, and hypoxia. The actions are
mediated through transcriptional regulation of p53 target genes (29,
30). Most functions of p53 involve its activity as a transcription
factor. p53 binds to its consensus binding sequence (two copies of the
10-base pair motif 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' separated by 0-13
base pairs) and transactivates expression of target genes (31). Several
biologically significant genes were found to contain this consensus
sequence and to be subjected to p53 regulation. Among those commonly
studied are p21Waf1/Cip1 (29), MDM2 (32), Gadd45 (33), BAX
(34), proliferating cell nuclear antigen (35), and cyclin G (36). The
p53 mutations found in many human cancers are clustered in the
DNA-binding domain of the p53 molecule (37). This leads to an
inactivation of p53 function through abolition of p53-specific DNA
binding and transactivation. By this means, p53 acts as a tumor
suppressor; its loss of function appears to confer selective advantages
to cells through deregulated growth and resistance to cell death (38,
39).
Two genes that are predicted to encode proteins with amino acid
sequence homologous to p53 have been recently identified (40-47). Each
of the p53 amino acid residues that are implicated in sequence-specific DNA binding is conserved in these proteins (40-47). One of these novel
genes, termed p73, is mapped to chromosome 1p36.33 and encodes a
protein with homology to p53 (40). Like p53, p73 activates the
transcription of p21Waf1/Cip1 and also induces apoptosis in
a p53-independent manner (40, 41). Another gene, termed
p63/p51/p73L/p40/KET/CUSP, maps to the long arm of chromosome 3 (42-47). Although the amino acid sequences and the molecular weights
were reported to be different, they have proven to be isotypes derived
from a single gene (43). Thus far, there have been six reported
isotypes of p63. Three of the variants are called TA (transactivation)
type and encode proteins with transactivation, DNA binding, and
oligomerization domains similar to p53. The three remaining variants,
which lack the acidic N-terminal domain, are called dN (
N terminus)
type. Both types, TAp63 and dNp63, have different C termini that are described as 
, 
, and 
(43). Thus, the TA types are designated TAp63, TAp63, and TAp63 and the dN types are designated
dNp63, dNp63, and dNp63. TAp63 has the shortest C terminus
and the potential to induce apoptosis and growth suppression in a
manner similar to p53. The mechanism is possibly through the p53
regulatory element and includes cellular responses similar to those
induced by p53 (42, 43). TAp63 transactivates several previously identified p53 target gene promoters such as p21Waf1/Cip1,
BAX, and MDM2 (42, 48). Thus, TAp63 is considered to be a tumor
suppressor gene and it may serve as an alternative tumor suppressor
gene whose expression is induced by the loss of p53 function (42).
On the other hand, variants that are truncated with the acidic N
terminus (dN type) or encode the longest C terminus ( type) are
thought to possess oncogenic properties (43). These variants also act
in a dominant-negative manner toward both p53 and transactivating versions of p63 through the reporter construct containing multiple copies of p53-binding sequence termed PG13 (43). In the present study,
we examined potential TAp63-dependent transactivation of the EGFR promoter using transfection assays and also TAp63 binding to
the EGFR promoter fragments by electrophoretic mobility shift assays.
Our results indicate a novel mechanism for regulation of EGFR gene
expression through interaction of TAp63 with Sp1.
| |
EXPERIMENTAL PROCEDURES |
Cell Lines and DNA Plasmids--
The human non-small cell lung
carcinoma cell line H1299, which is p53 deficient, was maintained in
RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented
with 10% fetal bovine serum and antibiotics. The human osteosarcoma
cell line Saos-2, which is also p53 deficient, was maintained in
McCoy's 5A medium (Life Technologies) with 15% fetal bovine serum.
Luciferase reporter constructs containing the EGFR promoter, pER1-luc,
pER9-luc, pER9A-luc, pER9C-luc, and pER10-luc, were prepared by
ligation of the HindIII promoter fragments from EGFR-CAT constructs into pGL3-Basic (Promega, Madison, WI) (21). The 3' end of
each of the following EGFR-luciferase constructs is at 16 relative to
the EGFR translational start site, while the 5' ends map to the
following positions: pER1-luc (1109), pER9-luc (388), pER9A-luc
(348), pER9C-luc (292), and pER10-luc (150) (Fig. 5A).
PG13-luc, which contains 13 copies of the p53-binding site, was kindly
provided by Dr. B. Vogelstein (29). TK-luc was generated by subcloning
of the EcoRI/HindIII thymidine kinase (TK)
promoter fragments from pRL-TK construct (Promega) into pGL3-basic. TK-TATA-luc was prepared by removal of the
EcoRI/BglII fragments from TK-luc and then
blunted and ligated. SV40-luc, pGL3-promoter, was purchased from
Promega. pNF-
B-luc and pCRE-luc were purchased from
CLONTECH (San Francisco, CA). The pERp53RE-luc was
constructed by subcloning of four p53-binding site sequences from the
EGFR promoter into the TK-TATA-luc construct using the XbaI
site (Fig. 3). This site is located upstream of the TK promoter TATA
region. The pER348-293-luc, pER348-293mt1-luc, pER348-293mt2-luc, or
pER348-293mt1,2-luc has the EGFR promoter region between 348 and 293 with or without the Sp1 site mutation as an insert instead of p53
consensus sequences (Fig. 6). The pER1p53mt-luc,
pER1Sp1mt1,2-luc, and pER1p53mtSp1mt1,2-luc constructs contain the same
mutations as the pER348-293mt constructs but are in the full EGFR
promoter context. The pCMV-p53 expression vector was purchased from
CLONTECH and the p53 cDNA was subcloned into
pcDNA3 (Invitrogen, Carlsbad, CA) with the EcoRI site to obtain pcDNA3-p53. TAp63 cDNA was cloned from murine testis
using PCR methods and subcloned into pcDNA3 vector.
Transfections and Luciferase Assays--
H1299 and Saos-2 cells
were seeded at 2.5 × 105 cells/35-mm dish and
incubated overnight at 37 °C in a 5% CO2 incubator. For each transfection, 0.2-1.0 µg of empty vector and/or expression vector plus 0.1 µg of promoter-luciferase DNA were mixed in 3.0 ml of
Opti-MEM (Life Technologies) and a precipitate was formed using
LipofectAMINE (Life Technologies) according to the manufacturer's recommendations. The cells were washed with Opti-MEM and complexes were
applied to the cells for 5 h. An equal volume of RPMI 1640 or
McCoy's 5A medium containing 20% (30% for McCoy's 5A medium) fetal
bovine serum was added, and cells were incubated for an additional
19 h. Cells were harvested and extracts were prepared with
luciferase cell lysis buffer (Pharmingen, San Diego, CA). Luciferase
activity was assayed in extracts in triplicate using the luciferase
assay kit (Pharmingen).
Western Blot Analysis--
H1299 cells were seeded at 2.5 × 106 cells/150-mm dish and incubated overnight at
37 °C and then transfected with 15 µg of either pcDNA3 empty
vector or constructs expressing TAp63 tagged at its N terminus with
influenza hemagglutinin (HA) peptide by the LipofectAMINE method as
described above. After 24 h, the media was changed to selective
media containing 700 µg/ml Geneticin (Life Technologies) to
reduce the number of untransfected cells. Cells were harvested 4 days
post-transfection and lysed on ice for 30 min in lysis buffer (10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 5 mM sodium fluoride, 0.1 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol), containing complete
protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis,
IN). The lysate was centrifuged at 14,000 rpm for 15 min and the
soluble fraction was collected. Protein concentrations were measured
with a Bio-Rad protein assay kit (Bio-Rad). Equal amounts of protein
extract (40 µg) were loaded onto a 4-12% SDS-polyacrylamide gel and
subjected to electrophoresis at 200 V for 50 min. The proteins were
transferred onto a polyvinylidene difluoride membrane and probed with
anti-EGFR antibodies (1005) (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-HA antibody (F-7) (Santa Cruz Biotechnology), and anti-actin
antibody (C4) (Roche Molecular Biochemicals). The blot was probed with
each antibody after stripping the membrane of the previous probe. EGFR
was detected by horseradish peroxidase-conjugated secondary antibody
coupled with enhanced chemiluminescence (ECL) Western blotting
detection reagents (Amersham Pharmacia Biotech). The intensity of each
EGFR band was normalized based on the intensity of the actin band.
RNA Isolation and Northern Analysis--
H1299 cells were seeded
at 2.5 × 106 cells/150-mm dish and incubated
overnight at 37 °C and then transfected with 15 µg of either
pcDNA3 empty vector or constructs expressing TAp63 by the
LipofectAMINE method as described above. After 24 h, the media was
changed to selective media containing 700 µg/ml Geneticin (Life
Technologies) to reduce the number of untransfected cells. Cells were
harvested 4 days post-transfection, and total cellular RNA was isolated
using TRIzol reagent (Life Technologies) and quantified by
A260/A280 measurement using an Ultraspec 3000 (Amersham Pharmacia Biotech). Total RNA samples (20 µg) were
subjected to Northern blot analysis. After electrophoresis in MOPS
electrophoresis buffer, the RNA was transferred to a nylon membrane in
10 × SSC buffer by capillary action. The RNA was fixed to the
nylon membrane by ultraviolet light exposure with a UV Stratalinker
(Stratagene, La Jolla, CA). The membrane was hybridized with
random-primed 32P-labeled probes in ExpressHyb
hybridization solution (CLONTECH) according to the
manufacturer's recommendation. After hybridization at 68 °C, the
membrane was washed twice in 2 × SSC containing 0.05% SDS at
room temperature and then washed twice at 50 °C using 0.1 × SSC containing 0.1% SDS. The filters were autoradiographed with Kodak
X-AR film for 24-72 h at 80 °C. The signal obtained from the
Northern blots was normalized to the signal for -actin.
In Vitro Transcription/Translation--
The p53 and TAp63
cDNAs were subcloned into pcDNA3 and tagged at their N termini
with HA peptide. To generate C-terminal truncated proteins, polymerase
chain reaction was used to amplify the regions encoding HA and amino
acids 1-363 for p53. This region was also subcloned into pcDNA3
and checked for fidelity of DNA sequence. Protein was synthesized
in vitro in the presence of unlabeled amino acids with the
coupled transcription/translation system (TNT) from Promega. Translated
products were analyzed by Western blotting using anti-HA antibody (F-7)
(Santa Cruz Biotechnology). For immunoprecipitation,
35S-labeled p53 and TAp63 were synthesized in the
presence of 40 µCi of 35S-labeled methionine and other
unlabeled amino acids with TNT system.
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were performed as described previously (26).
Briefly, a double-stranded oligonucleotide containing the p53 consensus
DNA-binding site (PG) was prepared by annealing two complementary
oligonucleotides, 5'-AGCTTAGACATGCCTAGACATGCCTA-3' and
5'-TAGGCATGTCTAGGCATGTCTAAGCT-3', in a buffer containing 10 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 1 mM EDTA. Equimolar amounts of the complementary
oligonucleotides were mixed in a 1.5-ml microcentrifuge tube and placed
in a heat block at 95 °C. The heat block was allowed to cool to room
temperature, and the sample was desalted on a G-25 microspin column
(Amersham Pharmacia Biotech). The double-stranded oligonucleotide was
end-labeled with 32P using T4 polynucleotide kinase and
[-32P]ATP. For electrophoretic mobility shift
analysis, the end-labeled double-stranded oligonucleotide 5000 cpm was
incubated with 2 µl of p53 and TAp63 at room temperature
(22 °C) for 30 min in the presence of a binding buffer (10%
glycerol, 20 mM HEPES-KOH (pH 7.5), 25 mM KCl,
2 mM dithiothreitol, 2 mM MgCl2,
0.4% Nonidet P-40, and 1 µg of salmon sperm DNA). When competition
assays were performed, an unlabeled p53 consensus sequence
oligonucleotide from p21Waf1/Cip1 or the EGFR promoter was
incubated with protein and buffer for 5 min prior to the addition of
the labeled oligonucleotide. Each binding site oligonucleotide was
purchased as two single-stranded DNAs from Genosys Biotechnologies
(Woodlands, TX) and annealed as described above. Samples (20 µl) were
loaded onto a 5% nondenaturing polyacrylamide gel and subjected to
electrophoresis at 150 V for 1 h using 0.33 × TBE (1 × TBE: 89 mM Tris-HCl, 8 mM boric acid, and 2 mM EDTA, pH 8.3) as running buffer.
For Sp1 binding, the Sp1 consensus oligonucleotide (Promega) was
labeled with 32P as described above. The end-labeled Sp1
consensus oligonucleotide and 5 µg of HeLa nuclear extract (Promega)
were incubated with or without the in vitro translated p53
or TAp63 for 30 min in binding buffer. For competition or supershift
assays, 1.75 pmol of unlabeled Sp1 oligonucleotide or 0.2 µg of
anti-Sp1 antibody (PEP2) (Santa Cruz Biotechnology) was incubated with
HeLa nuclear extract and the binding buffer for 5 min prior to the
addition of labeled Sp1 oligonucleotide. Samples were subjected to
electrophoresis with the same conditions as described above. After
electrophoresis, gels were transferred to Whatman 3MM paper and exposed
to Kodak X-AR film with intensifying screens at 80 °C.
Immunoprecipitation--
Sixty µg of HeLa nuclear extract
(Promega) and 35S-labeled p53 or TAp63 were incubated in
500 µl of Nonidet P-40 buffer (20 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 0.1 M NaCl, 10% glycerol, 1 mM EDTA) for 30 min. Two µg of anti-Sp1 antibody (PEP2)
was added and incubated for an additional 1 h. Subsequently, each immune complex was mixed with 20 µl of protein A-agarose beads for
16 h and then washed seven times in Nonidet P-40 buffer and once
in RIPA buffer (50 mM Tris-HCl, pH 7.8, 1% Triton X-100, 0.15 M NaCl, 0.1% SDS, 1% sodium deoxycholate). As a
negative control, agarose-coupled normal rabbit antibody from Santa
Cruz Biotechnology was used. All steps were carried out at 4 °C.
Immune complexes were boiled in sample buffer and separated on a
SDS-polyacrylamide gel. The gels were washed and dried before being
exposed to x-ray films at 80 °C.
| |
RESULTS |
TAp63 Transrepresses EGFR Expression--
In this study, we
sought to determine whether p63 gene products, which are p53-related
molecules, had a similar activating function on EGFR expression. To
examine potential p53 and TAp63-dependent transactivation of the EGFR promoter, we co-transfected the EGFR luciferase reporters, pER1-luc, with wild-type p53, TAp63, or empty
vector into p53-deficient H1299 and Saos-2 cells. Similar to the effect
on PG13 (43), p53 activated pER1-luc reporter activity (Fig.
1, A and B). While
TAp63 also transactivated PG13-luc, TAp63 surprisingly repressed
pER1-luc reporter activity (Fig. 1, A and C). A
83 and 75% decrease in luciferase activity was observed when compared
with the empty vector in Saos-2 and H1299 cells, respectively.
Additional co-transfection analysis showed that TAp63, but not
dNp63, repressed pER1-luc reporter activity in a
dose-dependent manner (Fig. 1C).
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Fig. 1.
TAp63 represses EGFR
promoter activity. A, transactivation of PG13-luc by
p53 and TAp63. H1299 cells and Saos-2 cells were transfected with
the PG13-luc (0.1 µg) and 1.0 µg of p53, TAp63, or pcDNA3.
Luciferase assays were performed after 24 h. B,
transactivation of EGFR gene by p53. H1299 cells and Saos-2 cells were
transfected with the pER1-luc (0.1 µg) and 1.0 µg of the p53
expression constructs or empty vector. Luciferase assays were performed
after 24 h. C, dose-dependent repression of
EGFR promoter activity by TAp63. H1299 cells and Saos-2 cells were
co-transfected with 1.0 µg of pcDNA3, dNp63 or 0.2 and 1.0 µg
of the TAp63 expression plasmid and 0.1 µg of the pER1-luc
reporter plasmid. The empty vector, pcDNA3, was used to keep total
plasmid transfected constant. Luciferase assays were performed after
24 h. Error bars indicate standard deviation in
triplicate assays.
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To determine whether TAp63 represses endogenous EGFR expression,
EGFR mRNA and protein levels were examined by introducing the
exogenous TAp63 and parental vector plasmids, respectively, into
H1299 cells, followed by Northern and Western blot analysis. EGFR
mRNA level was decreased by 67% in the TAp63-transfected cells
as compared with the vector-transfected cells (Fig.
2A). KB cells which have
relatively high expression of EGFR was used as a positive control (Fig.
2A). Also, the EGFR protein level was decreased by 64% in
the TAp63-transfected cells as compared with the vector-transfected
cells (Fig. 2B). The overexpression of TAp63 was
confirmed by Western blot analysis using anti-HA antibody (Fig.
2B). These results indicate that TAp63 represses EGFR
expression at the transcriptional level in an endogenous context. Thus,
we focused on elucidating the mechanism(s) why TAp63, unlike p53,
represses the EGFR expression.
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Fig. 2.
TAp63 represses
endogenous EGFR expression. A, EGFR mRNA level in
H1299 cells transfected with TAp63. H1299 cells were transfected
with plasmid constructs expressing TAp63 and pcDNA3 by the
LipofectAMINE method. Total RNAs isolated after transfection were
subjected to Northern blot as detailed under "Experimental
Procedures." Equal amounts of RNAs (20 µg) were loaded. Total RNA
isolated from KB cells was used as a positive control. B,
EGFR level in H1299 cells transfected with TAp63. H1299 cells were
transfected with plasmid constructs expressing HA-tagged TAp63 and
pcDNA3 by the LipofectAMINE method. Cell lysates were subjected to
Western analysis as detailed under "Experimental Procedures." Equal
amounts of cellular protein (40 µg) were loaded.
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TAp63 Does Not Bind to the EGFR p53 Response Element--
Given
the high degree of sequence homology within the DNA-binding domains of
the p53 and p63 proteins (42-47), it is likely that TAp63 can bind
p53 DNA target sites. We generated a luciferase construct which has
four tandem repeat of the p53-binding site identified in the EGFR
promoter upstream of the thymidine kinase TATA region and named
pERp53RE-luc (Fig. 3). To examine
potential p53-dependent transactivation and
TAp63-dependent transrepression of this construct, we
co-transfected the pERp53RE-luc, with wild-type p53, TAp63, or an
empty vector into H1299 cells. p53 was able to transactivate this
construct by more than 70-fold (Fig. 3). Unexpectedly, TAp63 also
transactivated pERp53RE-luc reporter activity (Fig. 3). A 10-fold
increase in luciferase activity was observed when compared with the
empty vector. Similar results were seen in Saos-2 cells (data not
shown). As a control, a luciferase reporter containing the minimal
thymidine kinase promoter was used. TAp63 or p53 did not activate
this construct (data not shown). These results imply that TAp63 can
transactivate using the p53-binding site of EGFR promoter when
presented in an oligomeric context.
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Fig. 3.
Effect of TAp63
expression on pERp53RE-luc reporter gene activity. In the
upper panel, the sequence of the EGFR p53 response element
and a schematic of pERp53RE-luc are shown. In the lower
panel, H1299 cells were transfected with pERp53RE-luc (0.1 µg)
and 1.0 µg of the p53, TAp63, or pcDNA3. Luciferase assays
were performed after 24 h.
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To address this issue further, we asked whether TAp63 could interact
with p53 target DNA sites. We used HA-tagged p53 and TAp63 proteins
prepared in vitro which were approximately equal in
concentration as examined by Western blot (data not shown). Ludes-Meyers et al. (25) showed that p53 could bind the
oligonucleotide consisting of the p53-binding site identified in the
EGFR promoter (EGFRp53RE) (26). However, we could not detect binding of
TAp63 to the EGFRp53RE (data not shown) but could confirm binding of p53 and TAp63 to the p53-binding site (PG) (Fig.
4, lanes 2 and 5).
The binding of p53 and TAp63 to this element was sequence-specific since the binding was competed by 30-fold excess of the p53 response element of p21 (p21p53RE) (Fig. 4, lanes 3 and
6). Additionally, the binding could be supershifted by
anti-HA antibody (data not shown). The binding to p53 was only slightly
competed by 100-fold excess of cold EGFRp53RE (Fig. 4, lane
4), and the binding to TAp63 was not competed by cold EGFRp53RE
oligonucleotide (Fig. 4, lane 7). The band intensities were
quantified and the results of three independent experiments are plotted
in Fig. 4. Taken together, these results indicate that p53 binds to the
putative p53-binding site found in the EGFR promoter with relatively
low affinity and p63 binding is not detectable by our assays.
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Fig. 4.
Analysis of TAp63
interaction with the EGFR p53 response element. The DNA
sequence of PG, the p53-binding site from p21 promoter (p21p53RE), and
the p53-binding site from EGFR promoter (EGFRp53RE) are shown in the
top. The mismatches are depicted as lowercase
letters. The p53 consensus DNA-binding site (PG) was end-labeled
and used in binding reactions with p53 protein (3 µl) or TAp63
protein (3 µl). The thick arrow indicates p53 complex and
thin arrow indicates TAp63 complex. Rabbit reticulocyte
lysate devoid of in vitro expressed protein was used as a
control (the first lane). For the competition assays, 30-fold molar
excess of the p21p53RE and 100-fold molar excess of the EGFRp53RE were
used, respectively. In the lower panel, the band intensities
were quantified using BAS2000 image analyzer (Fuji film) and the
results of three independent experiments are plotted. Error
bars indicate standard deviation.
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Sp1-binding Sites in the EGFR Promoter Are Critical for
TAp63-mediated Repression--
We next aimed to determine the
responsible element(s) in the EGFR promoter for TAp63-mediated
repression. For this purpose, we generated serial deletion mutants of
the EGFR promoter region attached to the luciferase gene (Fig.
5A). Using these reporters, we
co-transfected H1299 cells with the TAp63 expression vector. The
results show that pER1-luc containing 1109 to 16, pER9-luc containing 381 to 16, and pER9A-luc containing 348 to 16 were repressed to the similar extent by co-transfection with TAp63, whereas pER9C-luc and pER10-luc that contain 292 to 16 and 150 to
16, respectively, were not repressed (Fig. 5B). A similar effect was also found in Saos-2 cells (data not shown). These results
suggest that nucleotides between 348 and 293 within the EGFR
promoter are critical for TAp63-mediated repression.
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Fig. 5.
Determination of the EGFR promoter
region necessary for TAp63 repression.
A, schematic of the series of 5' deletion derivatives of the
EGFR promoter. B, luciferase assays were performed after
co-transfection of H1299 cells with the indicated EGFR promoter
luciferase constructs (0.1 µg), along with either the pcDNA3
expression vector (1.0 µg) or the TAp63 expression plasmid (1.0 µg). Error bars indicate standard deviation in triplicate
assays.
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To confirm whether this EGFR promoter region was crucial for TAp63
repression, we generated a luciferase construct, which has this region
upstream of a minimal thymidine kinase promoter. Co-transfection
experiments in Saos-2 cells were performed using this reporter
construct, pER348-293-luc, and the results revealed that TAp63
repressed the activity by 67% (Fig. 6).
A similar effect was also found using H1299 cells (data not shown).
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Fig. 6.
Mutation of Sp1 site abrogates
transcriptional activity of pER348-293-luc. Saos-2 cells were
transfected with the pER348-293-luc (0.1 µg), pER348-293mt1-luc (0.1 µg), pER348-293mt2-luc (0.1 µg), or pER348-293mt12-luc (0.1 µg)
and 1.0 µg of the indicated TAp63 expression constructs. The
sequences of each wild-type Sp1 site are ATCCCTCCTC and GTCCCTCCTC and
those of each mutated Sp1 site are ATCaaTaaTC and GTCaaTaaTC,
respectively. Lowercase indicates mutations. Luciferase
assays were performed after 24 h. Error bars indicate
standard deviation in triplicate assays.
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In the EGFR promoter region between nucleotides 348 and 293, there
are two Sp1-binding sites, 323 and 314 and 304 and 296 (13). It
has been shown that Sp1 transactivates EGFR promoter activity (13) and
that Sp1 interacts with p53 and may interact with p73 (49-52). Since
this region seems to be crucial for EGFR repression by TAp63, we
tested whether TAp63 could bind to this region by electrophoretic
mobility shift assay. Our results showed no interaction of TAp63
with this region (data not shown). We next examined the possible
interaction between TAp63 and Sp1. To test whether the TAp63
repression activity could be mediated through Sp1-binding sites, we
mutated these sites in the pER348-293-luc. We transfected the
pER348-293mt1-luc, pER348-293mt2-luc, or pER348-293mt1,2-luc into
Saos-2 cells along with TAp63 or p53 expression plasmids. TAp63
repressed pER348-293-luc activity to a similar extent as pER1-luc and
mutations in the Sp1-binding sites diminished this repression (Figs. 1
and 6). Also, p53 repressed the activity of this reporter construct.
Neither TAp63 nor p53 was able to repress the activity when both Sp1
sites were mutated. Similar results were observed in H1299 cells (data
not shown). These results suggest that the Sp1-binding sites are
critical for TAp63-mediated repression of EGFR expression. To
further investigate the role of this region in regulating EGFR promoter
activity, we generated mutations in the p53-binding site and the
Sp1-binding sites in the full promoter. These pER1 mutant constructs
were tested for their ability to be regulated by p53 and TAp63
(Fig. 7). The pER1p53mt construct was not
induced by p53 but was repressed by TAp63. The pER1Sp1mt construct
was not repressed by TAp63 but induced by p53. The induction by p53
was not as strong as with pER1 luc. Mutation of both the p53 and Sp1
sites resulted in loss of p53 induction and TAp63 repression. These
results confirm that nucleotides between 348 and 293 are critical
for TAp63 mediated repression.
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Fig. 7.
Mutation of Sp1 sites prevents
transcriptional repression by TAp63.
Saos-2 cells were transfected with the pER1-luc (0.1 µg),
pER1p53mt-luc (0.1 µg), pER1Sp1mt1,2-luc (0.1 µg), or
pER1p53mtSp1mt1,2-luc (0.1 µg) and 1.0 µg of the indicated p53 or
TAp63 expression constructs. The mutations refer to the same ones as
described in the legend to Fig. 6. Luciferase assays were performed
after 24 h. Error bars indicate standard deviation in
triplicate assays.
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How does TAp63 repress EGFR gene expression? The hypothesis that we
pursued was that TAp63 physically interacted with Sp1 and abrogated
binding to the Sp1 elements in the EGFR promoter. To investigate
whether there was protein-protein interaction between Sp1 and TAp63,
35S-labeled TAp63 was incubated with nuclear extracts
from HeLa cells and Sp1 antibody used to immunoprecipitate
complexes. As shown in Fig. 8, anti-Sp1
antibody precipitated TAp63·Sp1 and p53·Sp1 complexes. These
results indicated that the TAp63 repressive activity on EGFR
promoter may be mediated through the Sp1 sites and suggested a
potential interference of Sp1-DNA binding by TAp63 through
protein-protein interaction.
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Fig. 8.
Analysis of TAp63
interaction with Sp1 in vitro.
35S-Labeled p53 or TAp63 was analyzed in
immunoprecipitation assays as described under "Experimental
Procedures." No binding was observed using rabbit normal IgG instead
of anti-Sp1 antibody. The input represents 10% of the amount of
labeled protein used in each immunoprecipitation assay.
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TAp63 Represses Several Promoters Containing Sp1-binding
Sites--
To examine the TAp63 effect on additional promoters that
have Sp1-binding sites, co-transfection assays were performed using these reporter constructs and TAp63. In H1299 cells, co-transfection of TK-luc which has five Sp1-binding sites along with the empty vector
pcDNA3 or TAp63 revealed that TAp63 could repress TK-luc more
than 86%, whereas the minimal TK-luc construct, which contains only
one Sp1-binding site, was repressed only 33% (Fig.
9, A and B).
SV40-luc which has several Sp1-binding sites was repressed as much as
83%, whereas pCRE-luc and pNF-B-luc which have only one Sp1 site
were not repressed or repressed less than 33% (Fig. 9A).
These results indicate that TAp63 may generally compromise Sp1-mediated transcription.
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Fig. 9.
Effect of TAp63
expression on promoter activity. A, H1299 cells
were co-transfected with 1.0 µg of pcDNA3 or the TAp63
expression plasmid and 0.1 µg of the each promoter reporter plasmid.
Luciferase assays were performed after transfection. B,
transcriptional repression by TAp63 was decreased by deletion of Sp1
sites. The HSV-thymidine kinase promoter and minimal TK promoter were
assayed for TAp63 repression. Error bars indicate
standard deviation in triplicate assays.
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TAp63 Impairs Sp1 Binding to DNA--
It has been reported that
p53 impairs Sp1 binding to its consensus sequence through
protein-protein interaction (49-51). To determine if TAp63
interferes with Sp1 binding to DNA, we performed EMSA experiments using
HeLa nuclear extract and TAp63. As shown in Fig. 9, a 22-base pair
oligonucleotide consisting of the Sp1-binding site, bound to Sp1 in
HeLa nuclear extract (Fig. 10,
lane 2). This binding was confirmed by supershifting with
anti-Sp1 antibody (Fig. 10, lane 3). When TAp63 protein
was added, the binding of Sp1 was reduced in a
dose-dependent manner similar to the effect of p53 (Fig.
10, lanes 7-12). These results indicate that TAp63 impairs the binding of Sp1 to the promoter region through its direct
interaction with Sp1.
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Fig. 10.
TAp63 impairs the
Sp1 binding to DNA. Electrophoretic mobility shift assays were
performed with the end-labeled Sp1 consensus oligonucleotide. Sp1 from
HeLa nuclear extract (NE) bound to Sp1 consensus
oligonucleotide. For the supershift assay, anti-Sp1 antibody (0.2 µg)
was added into binding reaction. For the competition assays, 100-fold
molar excess of the unlabeled Sp1 consensus oligonucleotide was used.
To examine the TAp63 effect on Sp1 binding to DNA, different amounts
of TAp63 were added into each reaction. p53 was used as a positive
control and rabbit reticulolysate (RL) devoid of in
vitro expressed protein was used as a negative control.
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DISCUSSION |
In the present study, we investigated the roles of p53-related p63
gene products in the regulation of EGFR expression. We demonstrated
reporter assays that showed TAp63-mediated repression of EGFR
promoter activity (Fig. 1C). The results from Northern blot
and Western blot analyses revealed that TAp63 repressed endogenous
EGFR expression (Fig. 2, A and B). This was
surprising since TAp63 has been shown to possess p53-like
transcriptional activating activity (43). To clarify such a discrepancy
between p53 and TAp63 in the regulation of EGFR promoter, we
demonstrated another set of reporter assays using pERp53RE-luc, which
harbors four copies of the p53-responsible element found in the EGFR
promoter region (Fig. 3). The results showed that TAp63 did not
transactivate pERp53RE-luc activity to the same level as p53 (Fig. 3).
EMSAs using EGFRp53RE as a probe showed that TAp63, in
contrast to p53, did not bind to the EGFRp53RE (Fig. 4 and data not
shown). This is not surprising because p53 and TAp63 showed
different profiles of DNA binding affinity to the same p53-responsible
elements such as p21p53RE, mdm2p53RE, cyclinGp53RE, and
BAXp53RE.2 It is, therefore,
possible to speculate that binding affinity of p53 and TAp63 to the
EGFRp53RE is above and below detection at our level, respectively.
We determined by deletion mutants of pER1-luc that the responsible
region of TAp63-mediated repression was between nucleotides 348
and 293 (Fig. 5B). Previous studies have shown that there are two Sp1-binding sites in this region (13). Mutation analysis in
these two Sp1-binding sites showed that TAp63-mediated repression was largely dependent on the intact Sp1-binding sites (Figs. 6 and 7).
Sp1 is a well known basal transcription factor that is involved
in the regulation of several important genes (53). Therefore, we next
examined possible association between Sp1 and TAp63 by
immunoprecipitation analysis. The results show that TAp63 could
physically associate with Sp1 as does p53 (Fig. 8). In this point of
view, p53, as well as TAp63 in this study, might act as a negative
regulator of certain gene promoters. In fact, p53 literally represses
the transcription of HIV long terminal repeat, SV40 and telomerase
reverse transcriptase, through forming a Sp1·p53 complex which no
longer can mediate gene transcription (49-51). Additionally, p73,
another p53 family member, has been reported to repress vascular
endothelial growth factor transcription through interference
with Sp1 binding (52). These observations propelled us to investigate
whether TAp63 compromises the ability of Sp1 to bind to the target
sites in the EGFR promoter region. The results showed that
TAp63, as well as p53, inhibited Sp1 binding to the target DNA
fragment in a dose-dependent manner (Fig. 10). Altogether,
it seems possible to generalize that p53/p73/TAp63 transactivates
genes harboring their binding site(s) in the promoter region and that
p53/p73/TAp63 can act as a negative regulator when the
promoter lacks their binding site(s) and is regulated by Sp1. The
results in Fig. 9, a finding that repression by TAp63 depended on
the copy number of Sp1-binding site in the promoter region, also
supports this speculation.
We have clearly demonstrated that TAp63 represses EGFR promoter
activity through a mechanism that involves the Sp1-binding sites. One
initially could speculate that the interaction of Sp1 and TAp63
could result in a complex that inhibits the role of Sp1 in EGFR
activation. However, p53, which interacts with Sp1 with a similar
affinity, is able to activate EGFR promoter activity through
direct binding to the promoter region. On the other hand, in the
absence of a binding site, p53 leads to repression of promoter activity
to a similar extent as TAp63 (Fig. 6). Thus, the mechanism(s) by
which changes in EGFR promoter activity are mediated by TAp63 may
involve its interaction with Sp1 and/or another as yet unidentified factor.
Overexpression of EGFR has been implicated in a number of malignant
tumors (4-7). Despite the extensive studies on the regulation of EGFR
gene expression, little is known so far concerning the negative
regulator(s) of its expression. Such negative regulators are potential
explanations for the elevated level of EGFR gene expression, when they
are inactivated, in human cancer cells. Our present results suggest
that TAp63 might be a good candidate as a negative regulator of EGFR
expression. p63 is necessary for limb and craniofacial development and
a mutation has been found in an autosomal dominant disorder (54-56).
Up-regulation of the EGFR has been linked to defects in development
(57). Although the p63 gene was originally isolated as a p53 relative,
it is not frequently mutated in human cancers examined to date
(58-61). However, we and Park et al. (61) have reported
that expression of TAp63 was frequently lost in certain epithelial
cancer cells, but was constantly expressed in normal epithelial cells
(61-63).
In summary, we have shown that TAp63 represses EGFR gene expression
through a mechanism involving factors associated with the Sp1-binding
site. These findings, showing that TAp63 can act as a
transcriptional repressor of the EGFR promoter, may suggest an
anti-oncogenic role of TAp63 in human epithelial cells. The ability
of TAp63 to prevent malignant transformation of epithelial cells by
repressing EGFR gene expression, and the correlation between TAp63
and EGFR expressions in human tumors are under investigation.
,
TOP
ABSTRACT
INTRODUCTION
