From the Department of Pathology, Division of
Cellular and Molecular Pathology, University of Pittsburgh School of
Medicine, Pittsburgh, Pennsylvania 15261 and the ¶ Indiana
University Cancer Center, Indianapolis, Indiana 46202
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ABSTRACT |
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In the present study, we have investigated the
possible involvement of p53 in the transcriptional regulation of the
c-met gene. Cotransfection of various c-met
promoter reporter vectors with p53 expression plasmids demonstrated
that only wild-type p53 but not tumor-derived mutant forms of p53
resulted in a significant enhancement of c-met promoter
activity. Functional assays revealed that the p53 responsive element in
the c-met promoter region is located at position Hepatocyte growth factor
(HGF)1 receptor (c-Met) is
the product of the c-met proto-oncogene (1, 2), which was
originally described as an activated oncogene in a chemically treated
human osteosarcoma cell line (3, 4). c-Met is a transmembrane tyrosine
kinase receptor, which is expressed in a wide variety of adult and
embryonic tissues and transmits multiple biological responses such as
mitogenesis (1), motogenesis (5), morphogenesis (6, 7), and
anti-apoptotic activity (8) elicited by HGF (for a detailed review, see
Refs. 9 and 10). Animal experiments demonstrated that HGF/c-Met are
important in organ regeneration in adults, and studies using HGF or
c-met gene knock-out mice have shown that this
receptor-ligand system plays a pivotal role in embryonic development
and normal growth (11-13). Dysregulated c-met gene
expression is observed in a variety of human carcinomas (14, 15) and
sarcomas (16). It also mediates the movement and invasiveness of
neoplastic cells and promotes metastasis (6). Transgenic mouse models
that overexpress HGF or in which an autocrine loop between HGF and
c-Met was established show accelerated tumor formation (17-19). The
HGF/c-Met signaling system has also been shown to relay tumor
suppressor activities, as activation of this signaling pathway results
in growth inhibition of some tumor cells, and the expression of c-Met
is reduced or lost in other tumor tissues (20-22). Moreover, in
c-myc/HGF double transgenic mice, HGF behaves as a tumor
suppressor gene antagonizing the tumorigenic effect of c-myc
(23). Thus, elucidation of the molecular mechanisms governing the
transcriptional regulation of the c-met gene is crucial to
understand the role of HGF/c-Met in normal and neoplastic growth.
The molecular mechanisms regulating c-met gene transcription
are largely unknown. Previously, our laboratory reported the cloning
and functional characterization of the mouse c-met gene promoter (24) and demonstrated that the region between Plasmid Vector Construction--
1.6-, 0.5-, and 0.4mc-met-CAT
constructs were made as described previously (24). Briefly, the
NotI-EcoRI genomic DNA fragment containing the
mouse c-met gene promoter region was subcloned into the
pBluescript II SK(+) plasmid. The plasmid containing the
c-met insert was cut with SalI, and then the
2-kilobase pair DNA fragment was subcloned into the SalI
site of the promoterless pCAT-Basic plasmid (Promega, WI) to construct
the 2.0mc-met-CAT. The vector 1.6mc-met-CAT ( Cell Lines--
The p53 mutant cell line C-33A obtained from the
American Type Culture Collection (Rockville, MD) was maintained at
37 °C in Dulbecco's modified Eagle's medium supplemented with 10%
calf serum and gentamycin (100 µg/ml). The RKO cell line is a colon carcinoma cell line that contains the wild-type p53 gene. The RKOmp53
cell line is an RKO derivative stably transfected with a mutant p53
encoding gene. The RKO-E6 cell line is a clone isolated after RKO cells
were transfected with human papillomavirus E6 oncoprotein gene driven
by the CMV promoter (27). The cells were cultured and treated with UV
light for 3 h as described previously (27).
Transfection and CAT Assay--
Transient transfection using the
calcium phosphate precipitation method was carried out as described
previously (28, 29). Cells were harvested 24 h after transfection
and analyzed for CAT activity. Normalization for CAT activity was
performed based on the protein concentration of each cell lysate.
Oligonucleotides--
A 63-base pair DNA fragment containing the
c-met p53 binding site
(5'-CCAGGAGGGACCGTTGGGGACAAACCTAGAGCGACAGGGACGAACAGACACGTGCTGGGGCGG-3', from Electrophoretic Mobility Shift Assay (EMSA)--
A purified p53
core domain protein (amino acids 102-292) was a generous gift of Dr.
Nikola P. Pavletich (Memorial Sloan-Kettering Cancer Center, New York,
NY) (30). The oligonucleotides described above were end-labeled with
[ Western Blot Analysis--
Total cell lysate was separated by
SDS-polyacrylamide gel electrophoresis under reducing conditions as
described previously (31). The proteins were transferred to
polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) and
Ponceau S (Sigma) staining was performed to confirm the proper loading
and transfer of proteins and to normalize the signals. Nonspecific
binding to the membrane was blocked by 5% nonfat milk in Tris-buffered
saline/Tween buffer, and then specific antibodies were added. c-Met
protein was detected by addition of a polyclonal anti-c-Met antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by
horseradish peroxidase-conjugated goat anti-rabbit antibody (Sigma).
p53 and p21WAF1/CIP1 were detected with anti-p53
and anti-p21WAF1/CIP1 monoclonal antibody (Santa
Cruz Biotechnology, Inc.), respectively, followed by addition of
horseradish peroxidase conjugated goat anti-mouse antibody (Sigma). The
signals were visualized by enhanced chemiluminescence system (ECL)
solution (NEN Life Science Products).
Transcriptional Activation of the c-met Gene Promoter by Wild-type
p53 in a Dose-dependent Manner--
Previously, our
laboratory cloned and functionally characterized the mouse
c-met 5'-flanking region (24). Nucleotide sequence analysis
of the mouse c-met gene promoter region identified a potential p53 binding site at Identification of the c-met Promoter Region Responsible for
Conferring p53-mediated Stimulation--
To functionally identify the
region in the c-met gene promoter that is responsible for
p53-mediated stimulation, a series of 5' end deletion mutants were
constructed and cotransfected with wild-type p53 expression plasmid
(Fig. 2A). Fig. 2B
shows the results of CAT assays. Deletion from the 5' end to Ability of a Nucleotide Sequence Containing the c-met p53 Response
Element to Confer Stimulation by Wild-type p53 to a Heterologous
Minimal Promoter--
The consensus p53 binding site is the 10-bp
element of 5'-PuPuPuC(A/T)(A/T)GPyPyPy-3' (32). For high affinity
binding, two 10-bp sites are required. Nucleotide sequence analysis
indicated that the c-met p53 response element within p53 Protein Binds to the c-met Promoter p53 Response Element in
Vitro--
To directly show the interaction of p53 protein and the
c-met p53 response element, EMSAs were performed. The 63-bp
DNA fragment ( Induction of Endogenous c-met Gene Expression by p53--
We were
interested to know whether p53 plays a role in regulating the
expression of the endogenous c-met gene. We used RKO cells
that express wild-type p53 and RKO cells that have been stably
transfected either with a dominant negative mutant form of p53 or with
viral E6 protein to impair p53 function. In this system, it is well
documented that p53 expression is up-regulated after UV irradiation,
resulting in the induction of its down stream target genes such as
p21WAF1/CIP1 only in the parental RKO cell line,
which has a functional wild-type p53 (27). As shown in Fig.
7, c-Met expression is up-regulated in
RKO cells having a functional wild-type p53 protein within 3 h
after UV light exposure. Similarly, the well known target of p53,
p21WAF/CIP1 protein, is also induced. In contrast, RKO
cells transfected with mutant p53 or the viral E6 gene, which
inactivates wild-type p53 protein, fail to show induction of c-Met and
p21WAF1/CIP1 proteins. These results suggest
that wild-type p53 is important in the up-regulation of c-Met under
these experimental conditions.
p53 has been demonstrated to function as an important regulator of
cell proliferation in response to certain stimuli such as DNA damage
(34, 35). Despite the progress achieved toward understanding p53
functions, the molecular mechanisms by which p53 acts as a key
regulator of cell growth and tumorigenesis are still unclear. Studies
have demonstrated that p53 functions as a transcription factor and
regulates a number of target genes at the transcriptional level. The
central region of the p53 protein interacts with the promoter of target
genes in a sequence-specific manner, binding to two copies of a
consensus element (5'-PuPuPuC(A/T)(A/T)GPyPyPy-3') (32). While
wild-type p53 is a transactivator of the promoters containing a p53
binding motif, various tumor-derived mutant forms of p53 protein are
defective in sequence-specific transactivation. Among the genes that
are positively influenced by p53, p21WAF1/CIP1,
epidermal growth factor receptor (EGFR), Bax, human transforming growth
factor- p53-mediated stimulatory activity in the mouse c-met
promoter was mapped to the region p53 has been shown to interact with other transcription factors such as
Sp1 and MDM-2 (44, 45). As shown by nucleotide sequence analysis (24),
the c-met gene promoter is highly GC-rich. We identified two
Sp1 binding sites (5'-GGGCGG-3') in the c-met gene promoter
and demonstrated that Sp1 transcription factor is critically involved
in transcriptional regulation of the c-met gene promoter
(24). Thus, we cannot rule out the possibility that p53 also regulates
the c-met gene promoter activity by cooperatively interacting with Sp1.
As the "guardian of the genome," p53 is an important component of
the DNA damage-inducible response. It is activated by genotoxic agents
such as UV irradiation and then transactivates other genes to induce
DNA repair and cell survival (27). In our experiment, we observed that
the endogenous c-met gene product is induced after UV
exposure, correlating with the expression of p53 and its target
p21WAF1/CIP1. However, when wild-type p53
function was impaired by a mutant form of p53 or by viral E6
protein, induction of the p53 target gene
p21WAF1/CIP1 as well as c-Met was
abolished. These results demonstrate for the first time that c-Met is
induced by UV light and that p53 plays a role in this activation process.
Although p53 is a transcription factor that serves a dual role in the
regulation of cell proliferation, it is most recognized for its cell
cycle arrest and apoptotic activity. As stated previously, HGF/c-Met is
a multifunctional system that is involved in growth control and
differentiation. HGF/c-Met inhibits the growth of some normal and tumor
cells (20-22), and the level of expression of HGF and c-Met correlates
with the degree of cell differentiation (31). c-Met is underexpressed
in human breast carcinomas that harbor a mutant form of p53 (46). These
results suggest that c-Met may cooperate with wild-type p53 to
negatively regulate cell growth and induce differentiation. Experiments
have demonstrated that wild-type p53 transactivates the genes encoding
EGFR and TGF-278 to
216 and confers p53 responsiveness not only in the context of the
c-met promoter but also in the context of a heterologous
promoter. Electrophoretic mobility shift assays using purified
recombinant p53 protein showed that the p53 binding element identified
within the c-met promoter specifically binds to p53
protein. Induction of p53 by UV irradiation in RKO cells that express
wild-type p53 increased the level of the endogenous c-met
gene product and p21WAF1/CIP1, a known target
of p53 regulation. On the other hand, in RKO cells in which the
function of p53 is impaired either by stable transfection of a dominant
negative form of p53 or by HPV-E6 viral protein, no induction of the
endogenous c-met gene or
p21WAF1/CIP1 was noted by UV irradiation. These
results suggest that the c-met gene is also a target of p53
gene regulation.
INTRODUCTION
Top
Abstract
Introduction
References
278 and
78
of the promoter contains positive regulatory elements, including two
Sp1 binding sites that are essential for promoter function. In that
study, we also identified a putative p53 binding site within the
278
to
78 region of the promoter by computer analysis. Therefore, we
focused on the functionality of the potential p53 binding site and its
involvement in the transcriptional regulation of the c-met
gene. Using various c-met gene promoter constructs and p53
expression vectors, our current study demonstrates that mouse
c-met gene promoter activity is transactivated by wild-type p53 but not by several tumor-derived mutant forms of p53; the p53-mediated transactivation of the c-met gene promoter is
dependent upon direct binding of p53 to the cognate binding site
identified in the c-met gene promoter; induction of p53 by
UV irradiation in RKO cells that express wild-type p53 increased the
level of the endogenous c-met gene product and
p21WAF1/CIP1, a known target of p53 regulation,
and in RKO cells in which the function of p53 is impaired either by
stable transfection of a dominant negative form of p53 or by HPV-E6
viral protein, no induction of the endogenous c-met gene or
p21WAF1/CIP1 is observed. These findings shed
new light on the regulation of c-met gene expression.
EXPERIMENTAL PROCEDURES
1390, +184) was prepared
by cutting the 2.0mc-met-CAT with XbaI and BstXI,
blunt-ending with DNA polymerase I, followed by religation. To generate
the 0.5mc-met-CAT vector (
278, +184), the 1.6mc-met-CAT construct was
cut with HindIII and ApaI, blunt-ended, and
religated. The 0.4mc-met-CAT vector (
209, +184) was obtained by
digestion of the 1.6mc-met-CAT construct with ApaI and
KpnI, followed by self-ligation. RGC-W-3X-CAT and RGC-M-3X-CAT vectors, which are the positive and negative control vectors, respectively, for p53-mediated stimulation, have been described previously (25). To construct the p53-E1BCAT plasmid, a
63-base pair synthetic DNA fragment (
278 to
216) with an added XhoI site at the 5' end was inserted into the
XhoI site of E1BCAT vector which contains only a minimal
promoter element. The internal deletion mutant vector 1.6Dp53mc-met-CAT
was prepared by digestion of the 1.6mc-met-CAT construct with
ApaI and KpnI, followed by self-ligation.
Wild-type and mutant p53 expression vectors have been described
previously (26). They contain a full-length human cDNA sequence for
wild-type or various mutant p53 proteins inserted downstream of the
cytomegalovirus (CMV) promoter/enhancer in the pCMV-Neo-Bam vector. The
p53 expression vectors and the RGC-W-3X-CAT, RGC-M-3X-CAT plasmids were
kindly provided by Dr. Paul Robbins, Department of Molecular Genetics
and Biochemistry, University of Pittsburgh, Pittsburgh, PA.
278 to
216; p53 binding half-sites are underlined) and a
consensus p53 binding site (5'-AGGCATGCCTAGGCATGCCT-3') (26) were
chemically synthesized and used as probes and competitors.
-32P]dATP and used as probes. Protein-DNA binding
analysis was carried out in a buffer described previously (29)
consisting of 10 mM HEPES (pH 7.9), 100 mM KCl,
1 mM dithiothreitol, 0.05 mM EDTA, 2.5 mM MgCl2, 6% glycerol, 2% Ficoll, and 50 ng
of nonspecific DNA (poly(dI·dC)). For competition experiments, 100- or 200-fold molar excess of specific or nonspecific oligonucleotide
competitors were added to the reaction mixture. After incubation for 10 min at room temperature, reaction mixtures were loaded onto 4%
polyacrylamide gels, and run in 0.5× TBE at room temperature. The gels
were dried and exposed to x-ray film.
RESULTS
278 to
216 (Fig.
1A), which indicates the
possible involvement of the p53 tumor suppressor gene product in the
transcriptional regulation of the c-met gene. To determine if p53 regulates c-met gene promoter activity, C-33A
carcinoma cells, which harbor a mutated p53, were cotransfected with a
reporter vector containing the c-met gene promoter region
(
1390 to +184, relative to the transcription start site) (24) fused
to the CAT reporter gene, and expression vectors for wild-type or
tumor-derived mutant forms of p53. Fig. 1B is a
representative CAT assay result. c-met gene promoter
activity was enhanced significantly by coexpression of wild-type p53 as
compared with the c-met promoter construct cotransfected
with an expression vector lacking p53 encoding sequence (labeled
vector in Fig. 1). However, cotransfection of expression plasmids encoding three different mutant forms of p53 did not affect
c-met gene promoter activity in parallel experiments. The reporter construct RGC-W-3X-CAT containing three wild-type p53 binding
sites was also stimulated by cotransfection with expression plasmid
encoding wild-type p53, and, as expected, promoter activities of the
RGC-M-3X-CAT plasmid containing three copies of mutant p53 binding site
and the pCAT-Basic plasmid (which is promoterless) were not affected by
forced expression of wild-type p53. Stimulation of the c-met
gene promoter by wild-type p53 was dependent upon the input dose of
wild-type p53 (Fig. 1C).
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Fig. 1.
Transcriptional activation of the
c-met gene promoter by wild-type p53 but not by
tumor-derived mutant forms of p53. A, schematic
representation of the c-met gene promoter reporter vector.
The numbers designate the nucleotide position relative to
the transcription start site, which is marked by an arrow.
Boxed regions I and II display the
potential p53 binding half-sites identified by nucleotide sequence
analysis. Asterisks mark the mismatched nucleotides from the
consensus p53 sites. RGC-W-3X-CAT and RGC-M-3X-CAT reporter vectors
contain three copies of wild-type binding site (W) or the
mutant (M) p53 binding site known as RGC, respectively.
B, representative CAT assay result. Using the calcium
phosphate precipitation method, 2 µg of each reporter construct
(1.6mc-met-CAT, promoterless cloning vector pCAT-Basic, RGC-W-3X-CAT,
or RGC-M-3X-CAT) was cotransfected with 2 µg of the plasmid
expressing wild-type or tumor-derived mutant form of p53 protein. Cells
were harvested 24 h after transfection and analyzed for CAT
activity. Transfection and CAT assays were performed in duplicate in at
least three independent experiments, and consistent results were
obtained. C, p53 activates the c-met gene
promoter in a dose-dependent manner. Two µg of the
1.6mc-met-CAT construct was cotransfected with increasing amounts of
wild-type p53 expression plasmid using the calcium phosphate
precipitation method. The total amount of DNA for each reaction was
adjusted to 4 µg with pCMV vector. Cells were harvested 24 h
after transfection and analyzed for CAT activity. CAT assays were
performed at least three times in duplicate, and the results are
depicted as relative CAT activity (-fold increase over the activity of
the 1.6mc-met-CAT construct, which did not receive p53 expression
plasmid). The bar indicates the standard deviation
(S.D.).
279 did not alter the stimulating effect of wild-type p53 on the
c-met gene promoter. However, deletion of 70 base pairs from
278 to
209 completely eliminated responsiveness to p53. To assess
functionality of the identified c-met p53 response element
in the context of the c-met gene promoter, an internal
deletion mutant of the c-met promoter (1.6Dp53mc-met-CAT)
was constructed in which the identified c-met p53 response
element (
278 to
216) was deleted (Fig.
3A). Cotransfection of the
wild-type c-met construct (1.6mc-met-CAT) with the wild-type
p53 expression plasmid resulted in stimulation of c-met gene
promoter activity. In contrast, p53-mediated stimulation of
c-met promoter activity was completely abolished in the
mutant construct when cotransfected with wild-type p53 expression
plasmid (Fig. 3B). These results indicate that the
nucleotide sequence from
278 to
216 in the c-met
promoter region contains an element(s) responsible for p53-mediated
transactivation.
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Fig. 2.
Mapping of the p53 regulatory region of the
c-met gene promoter. A, schematic
representation of the 5' end deletion of the c-met gene
promoter. B, representative CAT assay results. Two µg of
various 5' end deletion mutants of the c-met gene promoter
were cotransfected with 2 µg of wild-type p53 expression plasmid
using the calcium phosphate precipitation method. Cells were harvested
24 h after transfection and analyzed for CAT activity. The
relative CAT activity in the presence or absence of wild-type p53
expression plasmid is plotted in the bar graph.
Values are means ± S.D. of three separate experiments performed
in duplicate.
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Fig. 3.
Dependence of p53-mediated stimulation of the
c-met gene promoter on the c-met p53
response element. The 1.6Dp53mc-met-CAT construct in which the
identified p53 response element ( 278 to
216) was internally deleted
from the 1.6mc-met-CAT construct was prepared as shown in A.
2 µg of 1.6mc-met-CAT or 1.6Dp53mc-met-CAT construct were
cotransfected with 2 µg of wild-type p53 expression plasmid using the
calcium phosphate precipitation method. Cells were harvested 24 h
after transfection and analyzed for CAT activity. Transfection
experiments and CAT assays were performed three times in duplicate, and
the results are depicted as relative CAT activity (-fold increase over
the corresponding met-CAT construct that did not receive p53 expression
plasmid) (B). The bar indicates the standard
deviation.
278
to
216 region contains two 10-bp p53 binding sites, 5'-GGACAAACCT-3'
from
261 to
252 and 5'-AGACACGTGC-3' from
233 to
224 as shown
in Fig. 4A. These two sites
are separated by an 18-nucleotide spacer (Fig. 4A). Each
site has only one and two mismatched nucleotides, respectively, compared with the published consensus sequence. To confirm whether this
p53 binding element is responsible for conferring stimulation to the
c-met gene promoter, a copy of the 63 base pair DNA fragment (
278 to
216) containing the p53 binding motif was inserted upstream of the minimal promoter element of the E1BCAT plasmid (Fig.
4A) and cotransfected with wild-type p53 expression plasmid.
Cotransfection of the wild-type p53 expression vector dramatically
stimulated promoter activity of two independently prepared plasmid
constructs by more than 20-fold (p53-E1BCAT clones 1 and 2) (Fig.
4B). The extent of stimulation of the heterologous promoter
by the 63-bp DNA fragment containing the c-met p53 binding
site was comparable to that of the RGC-W-3X sequence containing three
copies of a p53 consensus binding site (positive control) (Fig.
4B). The promoter activity of the E1BCAT control vector
lacking the p53 binding site (negative control) was not affected by
expression of wild-type p53 (Fig. 4B). Taken together, these
results demonstrate that the DNA fragment (
278 to
216) containing
the putative c-met p53 binding element is responsible for
p53-mediated stimulation of mouse c-met gene promoter
activity, and the identified p53 binding element is functional.
Alignment of the mouse c-met promoter nucleotide sequence
with that of the human indicates that the p53 binding site is well
conserved between the two species (Fig. 5).
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Fig. 4.
Ability of the c-met p53
response region to confer activation of a heterologous promoter by
p53. A, schematic representation of the p53-E1B-CAT
vector. A copy of the c-met p53 response region ( 278 to
216) containing the p53 binding element was inserted into the E1BTCAT
plasmid, which contains a minimal promoter element to generate the
p53-E1BCAT construct. The RGC-W-3X-CAT vector containing three copies
of the wild-type consensus p53 binding element was used as a positive
control. B, a representative CAT assay showing the
activation of a heterologous promoter by c-met p53 response
element. 2 µg of RGC-W-3X-CAT construct, two independent clones of
the p53-E1BCAT construct, or the cloning vector E1BCAT were
cotransfected with 2 µg of wild-type p53 expression plasmid using the
calcium phosphate precipitation method. Cells were harvested 24 h
after transfection and analyzed for CAT activity. Shown is a
representative CAT assay result. The experiment was performed at least
three independent times in duplicate with similar results.
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Fig. 5.
Nucleotide sequence comparison of the mouse
and human c-met 5'-flanking region. Nucleotide
sequence alignment of the mouse and human c-met promoter
regions (GenBank accession numbers AF030200 and AC002080, respectively)
were carried out using the DNASTAR alignment software. The 10-bp p53
binding sites are boxed and marked as I and
II, respectively. The asterisks indicate
identical nucleotides shared between species (m, mouse;
h, human). The arrow indicates the transcription
start site.
278 to
216) containing the c-met p53
binding element was used as a probe, and the purified recombinant p53
core domain protein was used in the binding reaction. It is known that
p53 contains 393 amino acids and is divided into three functional
domains: amino acids 1-101 for transactivation by interacting with the basal transcriptional machinery, amino acids 102-292 for
sequence-specific DNA binding, and amino acids 293-393 for
oligomerization. The DNA binding specificity of the core domain protein
is comparable to the full-length wild-type p53 protein (33). As we
expected, the mobility of the labeled probe DNA was shifted by p53 core domain protein (Fig. 6, lane
2) and formed a binding complex. The shifted complex was
diminished by increasing amounts of self-competitor as well as a
wild-type consensus p53 binding site (lanes 3,
4, and 7) but not by nonspecific competitor
(lanes 5 and 6). When the wild-type
consensus p53 binding site was used as a probe, it specifically bound
to the p53 core domain protein (lanes 9 and
10 as positive control). These results demonstrate that p53 protein directly binds to the identified c-met p53 response
elements to exert p53-mediated stimulation of the c-met gene
promoter.
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Fig. 6.
Direct binding of p53 protein to the
c-met p53 response element. EMSA was performed
with the 63-bp DNA fragment from 278 to
216 containing the
c-met p53 response element (lanes
1-7) or with the wild-type p53 binding element
(lanes 8-10) as a probe, respectively. Probes
were end-labeled with T4 kinase and incubated with 50 ng of purified
recombinant p53 core domain protein (lanes 1 and
8, free probe without p53 protein) in the presence or
absence of specific or nonspecific competitors and subjected to EMSA.
The arrow indicates the specific probe DNA-protein complex.
F stands for free probe.
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Fig. 7.
Induction of endogenous c-Met expression by
UV irradiation depends on a functional p53. A, Western
blot analysis shows the induction of c-Met, p53, and
p21WAF1/CIP1 proteins after UV irradiation.
Human RKO cells having different p53 statuses were exposed to UV light
for 20 s and cultured for 3 h. The protein extracts were
separated by SDS-polyacrylamide gel electrophoresis and subjected to
Western blot analysis using specific antibodies against c-Met, p53, and
p21WAF1/CIP1 proteins. B, the
bar graph shows the induction of c-Met, p53, and
p21WAF1/CIP1 protein. Western blot results were
analyzed by densitometry, and the results are plotted as -fold
induction over corresponding control cultures without UV exposure. The
experiment was repeated three times, and the results are consistent.
Values are means ± S.D. of three separate experiments after
normalization of the data for protein loading, as described under
"Experimental Procedures."
DISCUSSION
(TGF-
), insulin-like growth factor-binding protein 3 (IGF-BP 3), and cyclin G are well known regulators of cell growth and
differentiation (36-41). c-Met is the protein tyrosine kinase cell
surface receptor for HGF and transmits its multiple signals such as
induction of cell growth, differentiation, and the
apoptotic/antiapoptotic processes (8, 42). c-Met also plays an
important role in tumor growth and progression (14-16). Studies of
c-met gene expression regulation are important in
understanding its biological functions in normal and abnormal tissue
growth. Previously, we have cloned and characterized the
c-met gene promoter (24). Nucleotide sequence analysis
identified a potential p53 regulatory element in the promoter. To
determine whether p53 regulates expression of the c-met
gene, we examined the ability of p53 to regulate c-met gene promoter activity by cotransfection of the c-met gene
promoter construct and expression vectors encoding wild-type or
tumor-derived mutant forms of p53 in a p53 mutant cell line, C-33A.
Cotransfection assays demonstrated that c-met gene promoter
activity is stimulated by wild-type p53 (Fig. 1). Unlike some p53
target gene promoters, which have been shown to be transactivated by
tumor-derived mutant forms of p53 ("gain of function" activity)
(37), c-met gene promoter activity was not affected by
various p53 mutants (Fig. 1). In addition, the stimulatory effect of
p53 on the c-met gene promoter was dependent upon the input
dose of p53 expression plasmid (Fig. 1C). Doses ranging from
0.25 to 1.5 µg of p53 expression plasmid led to a continuous increase
of p53-mediated stimulation. Maximal stimulation was reached at a dose
of 1.5 µg of p53 expression plasmid and was slightly repressed at
higher doses. It has been reported that p53 interacts with TATA
box-binding protein (TBP) and interferes with the binding of TBP to the
TATA box (43). Thus, it seems likely that, at a higher dose, p53 may
sequester TBP and prevent its interaction with TFIID, which is required for the initiation of RNA polymerase II-dependent transcription.
278 to
216 by functional analysis (Figs. 2 and 3). These functional analysis results are in agreement with results of nucleotide sequence analysis of the c-met
gene promoter. The potential p53 binding element identified within
278 to
216 contains two 10-bp p53 binding sites (5'-GGACAAACCT-3' and 5'-AGACACGTGC-3') separated by 18 base pairs (Fig. 1A).
These sites contain only one and two nucleotide mismatches,
respectively, compared with the published consensus p53 binding site,
5'-PuPuPuC(A/T)(A/T)GPyPyPy-3' (32). Previous studies demonstrated that
at least two copies of the 10-base pair p53 binding site, separated by
0-13 base pairs, are required for high affinity p53 binding (32), and
the number of intervening nucleotides is not absolutely crucial. It is
of interest to note that the p53 binding element is well conserved between the mouse and human c-met promoters (Fig. 5). The
extent of stimulation of the heterologous promoter by one copy of the DNA fragment (
278 to
216) containing the c-met p53
binding element was highly comparable to that produced by the
RGC-W-3X-CAT containing three copies of the consensus p53 binding
element (Fig. 4). Moreover, the purified DNA binding domain of p53
protein strongly and specifically bound to the c-met p53
response element in EMSA (Fig. 6).
, which are known to positively modulate cell
proliferation (37, 39). A number of studies have reported
overexpression of EGFR (47, 48) and TGF-
(49, 50) in a wide variety of human cancers. It is also interesting to note that the HGF promoter
was recently reported to be transcriptionally activated by p53 (51).
These findings suggest that wild-type p53 plays a role in controlling
the expression of both c-Met and its ligand, HGF, ultimately leading to
regulation of cell growth and differentiation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Paul Robbins for RGC-W-3X-CAT, RGC-M-3X-CAT, and p53 expression plasmids, and Dr. Nikola P. Pavletich for purified p53 core domain protein. We also thank Dr. Marie C. DeFrances and Aaron Bell for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant CNE-97736 (to R. Z.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF030200 and AC002080.
§ These authors contributed equally to this work.
To whom all correspondence should be addressed: Dept. of
Pathology, Div. of Cellular and Molecular Pathology, BST S 419, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Tel.: 412-648-8657; Fax: 412-648-1916; E-mail:
rezazar+{at}pitt.edu.
The abbreviations used are:
HGF, hepatocyte
growth factor; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; bp, base pair(s); TBP, TATA box-binding protein; EMSA, electrophoretic mobility shift assay; EGFR, epidermal growth factor
receptor; TGF-, transforming growth factor-
.
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REFERENCES |
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