p53 Homologue p63 Represses Epidermal Growth Factor Receptor Expression
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m101241200
ISSN1083-351X
AutoresHirotaka Nishi, Makoto Senoo, Katsura Nishi, Barbara A. Murphy, Toshiki Rikiyama, Yasuko Matsumura, Sonoko Habu, Alfred C. Johnson,
Tópico(s)Molecular Biology Techniques and Applications
ResumoTumor suppressor p53 has been shown to transactivate epidermal growth factor receptor (EGFR) expression through binding to a putative p53 responsive element in the EGFR promoter between nucleotides −265 and −239 (EGFRp53RE). Isotypes of p63 gene products, recently identified as p53 relatives, have a similar function to transactivate several p53 target gene promoters. However, our results indicate that TAp63γ has a very low ability to bind to the EGFRp53RE and surprisingly represses both basal EGFR promoter activity and endogenous EGFR expression. Transient transfection assays show that the EGFR promoter region between −348 and −293, containing two Sp1 sites, is crucial for the repression of the EGFR expression by TAp63γ. Mutations in these Sp1 sites in the reporter constructs result in loss of the TAp63γ repression effect. We further show that TAp63γ directly interacts with Sp1 by immunoprecipitation analysis and that TAp63γ impairs Sp1 binding to the target DNA site in electrophoretic mobility shift assays. These results suggest that TAp63γ is involved in the regulation of the EGFR gene expression through interactions with basal transcription factors. Tumor suppressor p53 has been shown to transactivate epidermal growth factor receptor (EGFR) expression through binding to a putative p53 responsive element in the EGFR promoter between nucleotides −265 and −239 (EGFRp53RE). Isotypes of p63 gene products, recently identified as p53 relatives, have a similar function to transactivate several p53 target gene promoters. However, our results indicate that TAp63γ has a very low ability to bind to the EGFRp53RE and surprisingly represses both basal EGFR promoter activity and endogenous EGFR expression. Transient transfection assays show that the EGFR promoter region between −348 and −293, containing two Sp1 sites, is crucial for the repression of the EGFR expression by TAp63γ. Mutations in these Sp1 sites in the reporter constructs result in loss of the TAp63γ repression effect. We further show that TAp63γ directly interacts with Sp1 by immunoprecipitation analysis and that TAp63γ impairs Sp1 binding to the target DNA site in electrophoretic mobility shift assays. These results suggest that TAp63γ is involved in the regulation of the EGFR gene expression through interactions with basal transcription factors. epidermal growth factor receptor transactivation type of p63 subtype γ δN terminus stimulatory protein 1 p53 response element luciferase mutant hemagglutinin thymidine kinase 3-(N-morpholino)propanesulfonic acid The epidermal growth factor receptor (EGFR)1 plays an important role in cell growth and development (1Carpenter G. Annu. Rev. 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Targoff I.N. Kaufman K.M. Chorzelski T.P. Jablonska S.J. Invest. Dermatol. 1999; 113: 146-151Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). One of these novel genes, termed p73, is mapped to chromosome 1p36.33 and encodes a protein with homology to p53 (40Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1538) Google Scholar). Like p53, p73 activates the transcription of p21Waf1/Cip1 and also induces apoptosis in a p53-independent manner (40Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1538) Google Scholar, 41Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (902) Google Scholar). Another gene, termed p63/p51/p73L/p40/KET/CUSP, maps to the long arm of chromosome 3 (42Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar, 43Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar, 44Senoo M. Seki N. Ohira M. Sugano S. Watanabe M. Inuzuka S. Okamoto T. Tachibana M. Tanaka T. Shinkai Y. Kato H. Biochem. Biophys. Res. Commun. 1998; 248: 603-607Crossref PubMed Scopus (119) Google Scholar, 45Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. Nat. Med. 1998; 4: 747-748Crossref PubMed Scopus (225) Google Scholar, 46Augustin M. Bamberger C. Paul D. Schmale H. Mamm. Genome. 1998; 9: 899-902Crossref PubMed Scopus (56) Google Scholar, 47Lee L.A. Walsh P. Prater C.A. Su L.J. Marchbank A. Egbert T.B. Dellavalle R.P. Targoff I.N. Kaufman K.M. Chorzelski T.P. Jablonska S.J. Invest. Dermatol. 1999; 113: 146-151Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 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 (43Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar). 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 (δN terminus) type. Both types, TAp63 and dNp63, have different C termini that are described as α, β, and γ (43Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar). Thus, the TA types are designated TAp63α, TAp63β, and TAp63γ and the dN types are designated dNp63α, dNp63β, and dNp63γ. TAp63γ 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 (42Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar, 43Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar). TAp63γ transactivates several previously identified p53 target gene promoters such as p21Waf1/Cip1, BAX, and MDM2 (42Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar, 48Shimada A. Kato S. Enjo K. Osada M. Ikawa Y. Kohno K. Obinata M. Kanamaru R. Ikawa S. Ishioka C. Cancer Res. 1999; 59: 2781-2786PubMed Google Scholar). 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 (42Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar). On the other hand, variants that are truncated with the acidic N terminus (dN type) or encode the longest C terminus (α type) are thought to possess oncogenic properties (43Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar). 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 (43Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar). 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. 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) (21Rubinstein Y.R. Proctor K.N. Bergel M. Murphy B. Johnson A.C. FEBS Lett. 1998; 431: 268-272Crossref PubMed Scopus (18) Google Scholar). 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. 5 A). PG13-luc, which contains 13 copies of the p53-binding site, was kindly provided by Dr. B. Vogelstein (29El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar). 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-κB-luc and pCRE-luc were purchased fromCLONTECH (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 fromCLONTECH and the p53 cDNA was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) with the EcoRI site to obtain pcDNA3-p53. TAp63γ cDNA was cloned from murine testis using PCR methods and subcloned into pcDNA3 vector.Figure 3Effect of TAp63γ 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, TAp63γ, or pcDNA3. Luciferase assays were performed after 24 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Mutation 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 TAp63γ 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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). 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 TAp63γ 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. 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 TAp63γ 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 β-actin. 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 TAp63γ were synthesized in the presence of 40 μCi of 35S-labeled methionine and other unlabeled amino acids with TNT system. Electrophoretic mobility shift assays were performed as described previously (26Sheikh M.S. Carrier F. Johnson A.C. Ogdon S.E. Fornace A.J. Oncogene. 1997; 15: 1095-1101Crossref PubMed Scopus (40) Google Scholar). 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 [γ-32P]ATP. For electrophoretic mobility shift analysis, the end-labeled double-stranded oligonucleotide 5000 cpm was incubated with 2 μl of p53 and TAp63γ 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 w
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