Regulation of the c-met Proto-oncogene Promoter by p53*

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-metpromoter 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 −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-metgene product and p21 WAF1/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 p21 WAF1/CIP1 was noted by UV irradiation. These results suggest that the c-met gene is also a target of p53 gene regulation.

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 tumorderived 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 ؊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 wildtype p53 increased the level of the endogenous c-met gene product and p21 WAF1/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 p21 WAF1/CIP1 was noted by UV irradiation. These results suggest that the c-met gene is also a target of p53 gene regulation.
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)(12)(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)(18)(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 Ϫ278 and Ϫ78 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 Ϫ278 to Ϫ78 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 p21 WAF1/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 p21 WAF1/CIP1 is observed. These findings shed new light on the regulation of c-met gene expression.

EXPERIMENTAL PROCEDURES
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(ϩ) 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 (Ϫ1390, ϩ184) 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 0.5mcmet-CAT vector (Ϫ278, ϩ184), the 1.6mc-met-CAT construct was cut with HindIII and ApaI, blunt-ended, and religated. The 0.4mc-met-CAT vector (Ϫ209, ϩ184) 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 (Ϫ278 to Ϫ216) with an added XhoI site at the 5Ј 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.
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 g/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.
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 [␥-32 P]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 MgCl 2 , 6% glycerol, 2% Ficoll, and 50 ng of nonspecific DNA (poly(dI⅐dC)). 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 0.5ϫ TBE at room temperature. The gels were dried and exposed to x-ray film.
Western Blot Analysis-Total cell lysate was separated by SDSpolyacrylamide 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 p21 WAF1/CIP1 were detected with anti-p53 and anti-p21 WAF1/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 5Ј-flanking region (24). Nucleotide sequence analysis of the mouse c-met gene promoter region identified a potential p53 binding site at Ϫ278 to Ϫ216 (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 (Ϫ1390 to ϩ184, 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 wildtype 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).
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 5Ј 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 5Ј end to Ϫ279 did not alter the stimulating effect of wild-type p53 on the c-met gene promoter. However, deletion of 70 base pairs from Ϫ278 to Ϫ209 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 (Ϫ278 to Ϫ216) 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 Ϫ278 to Ϫ216 in the c-met promoter region contains an element(s) responsible for p53-mediated transactivation.
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 5Ј-PuPuPuC(A/T)(A/T)GPyPyPy-3Ј (32). For high affinity binding, two 10-bp sites are required. Nucleotide sequence analysis indicated that the c-met p53 response element within Ϫ278 to Ϫ216 region contains two 10-bp p53 binding sites, 5Ј-GGACAAACCT-3Ј from Ϫ261 to Ϫ252 and 5Ј-AGACACGTGC-3Ј from Ϫ233 to Ϫ224 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 (Ϫ278 to Ϫ216) 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 con-structs 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 (Ϫ278 to Ϫ216) containing the putative c-met p53 binding element is responsi- 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 g 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 g 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 g 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 g 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.).

FIG. 2.
Mapping of the p53 regulatory region of the c-met gene promoter. A, schematic representation of the 5Ј end deletion of the c-met gene promoter. B, representative CAT assay results. Two g of various 5Ј end deletion mutants of the c-met gene promoter were cotransfected with 2 g 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 Ϯ S.D. of three separate experiments performed in duplicate.
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 (Ϫ278 to Ϫ216) was internally deleted from the 1.6mc-met-CAT construct was prepared as shown in A. 2 g of 1.6mc-met-CAT or 1.6Dp53mc-met-CAT construct were cotransfected with 2 g 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.
ble 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).
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 (Ϫ278 to Ϫ216) 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.

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 upregulated after UV irradiation, resulting in the induction of its down stream target genes such as p21 WAF1/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, p21 WAF/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 p21 WAF1/CIP1 proteins. These results suggest that wild-type p53 is important in the up-regulation of c-Met under these experimental conditions. DISCUSSION 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 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 (Ϫ278 to Ϫ216) 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 cmet p53 response element. 2 g of RGC-W-3X-CAT construct, two independent clones of the p53-E1BCAT construct, or the cloning vector E1BCAT were cotransfected with 2 g 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. 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 (5Ј-PuPuPuC(A/T)(A/T)GPyPyPy-3Ј) (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, p21 WAF1/CIP1 , epidermal growth factor receptor (EGFR), Bax, human transforming growth factor-␣ (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 g of p53 expression plasmid led to a continuous increase of p53-mediated stimulation. Maximal stimulation was reached at a dose of 1.5 g 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.
p53-mediated stimulatory activity in the mouse c-met promoter was mapped to the region Ϫ278 to Ϫ216 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 Ϫ278 to Ϫ216 contains two 10-bp p53 binding sites (5Ј-GGACAAACCT-3Ј and 5Ј-AGACACGTGC-3Ј) 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, 5Ј-PuPuPuC(A/T)(A/ T)GPyPyPy-3Ј (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 (Ϫ278 to Ϫ216) 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).
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 FIG. 5. Nucleotide sequence comparison of the mouse and human cmet 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.
GC-rich. We identified two Sp1 binding sites (5Ј-GGGCGG-3Ј) 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 p21 WAF1/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 p21 WAF1/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 transacti-vates the genes encoding EGFR and TGF-␣, 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. FIG. 6. Direct binding of p53 protein to the c-met p53 response element. EMSA was performed with the 63-bp DNA fragment from Ϫ278 to Ϫ216 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.

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 p21 WAF1/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 p21 WAF1/CIP1 proteins. B, the bar graph shows the induction of c-Met, p53, and p21 WAF1/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 Ϯ S.D. of three separate experiments after normalization of the data for protein loading, as described under "Experimental Procedures."