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• W1999494313 abstract "The bradykinin type 2 receptor (BK2) is a developmentally regulated G protein-coupled receptor that mediates diverse actions such as vascular reactivity, salt and water excretion, inflammatory responses, and cell growth. However, little is known regarding regulation of the BK2 gene. We report here that the rat BK2 receptor is transcriptionally regulated by the tumor suppressor protein p53. The 5′-flanking region of the rat BK2 gene contains two p53-like binding sites: a sequence at −70 base pairs (P1 site) that is conserved in the murine and human BK2 genes; and a sequence at −707 (P2) that is not. The P1 and P2 motifs bind specifically to p53, as assessed by gel mobility shift assays. Transient transfection into HeLa cells of a CAT reporter construct driven by 1.2-kilobases of theBK2 gene 5′-flanking region demonstrated that theBK2 promoter is dose dependently activated by co-expression of wild-type p53. Co-expression of a dominant negative mutant p53 suppresses the activation of BK2 by wild-type p53. Promoter truncation localized the p53-responsive element to the region between −38 and −94 base pairs encompassing the p53-binding P1 sequence. Furthermore, p53-mediated activation of the BK2 promoter is augmented by the transcriptional co-activators, CBP/p300. Interestingly, removal of the P2 site by truncation or site-directed deletion amplifies p53-mediated activation of the BK2promoter. These results demonstrate that the rat BK2promoter is a target for p53-mediated activation and suggest a new physiological role for p53 in the regulation of G protein-coupled receptor gene expression. The bradykinin type 2 receptor (BK2) is a developmentally regulated G protein-coupled receptor that mediates diverse actions such as vascular reactivity, salt and water excretion, inflammatory responses, and cell growth. However, little is known regarding regulation of the BK2 gene. We report here that the rat BK2 receptor is transcriptionally regulated by the tumor suppressor protein p53. The 5′-flanking region of the rat BK2 gene contains two p53-like binding sites: a sequence at −70 base pairs (P1 site) that is conserved in the murine and human BK2 genes; and a sequence at −707 (P2) that is not. The P1 and P2 motifs bind specifically to p53, as assessed by gel mobility shift assays. Transient transfection into HeLa cells of a CAT reporter construct driven by 1.2-kilobases of theBK2 gene 5′-flanking region demonstrated that theBK2 promoter is dose dependently activated by co-expression of wild-type p53. Co-expression of a dominant negative mutant p53 suppresses the activation of BK2 by wild-type p53. Promoter truncation localized the p53-responsive element to the region between −38 and −94 base pairs encompassing the p53-binding P1 sequence. Furthermore, p53-mediated activation of the BK2 promoter is augmented by the transcriptional co-activators, CBP/p300. Interestingly, removal of the P2 site by truncation or site-directed deletion amplifies p53-mediated activation of the BK2promoter. These results demonstrate that the rat BK2promoter is a target for p53-mediated activation and suggest a new physiological role for p53 in the regulation of G protein-coupled receptor gene expression. bradykinin chloramphenicol acetyltransferase base pair(s) cytomegalovirus electrophoresis mobility shift assay cAMP response element-binding protein The type 2 bradykinin (BK)1 receptor, BK2, is a G protein-coupled receptor that mediates diverse actions, such as vascular relaxation, urinary sodium excretion, inflammatory responses, and cellular growth (1.Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). The rat BK2 gene is composed of 4 exons, the last of which contains the entire open reading frame (2.Pesquero J.B. Lindsey C.J. Zeh K. Paiva A.C.M. Ganten D. Bader M. J. Biol. Chem. 1994; 269: 26920-26925Abstract Full Text PDF PubMed Google Scholar). Initial characterization of the rat BK2 gene by Pesquero and co-workers (2.Pesquero J.B. Lindsey C.J. Zeh K. Paiva A.C.M. Ganten D. Bader M. J. Biol. Chem. 1994; 269: 26920-26925Abstract Full Text PDF PubMed Google Scholar) revealed that this gene lacks a typical TATA module and identified potential binding sites for a number of transcription factors, including cyclic AMP response element-binding protein, AP-1, NF-κB, SP-1, and Egr-1. Although traditionally thought to be expressed in a constitutive manner, the BK2 gene is developmentally regulated in the kidney and cardiovascular system (3.El-Dahr S.S. Figueroa C.D. Gonzalez C.B. Müller-Esterl W. Kidney Int. 1997; 51: 739-749Abstract Full Text PDF PubMed Scopus (63) Google Scholar), and is induced by mitogenic growth factors (4.Dixon B.S. Sharma R.V. Dennis M.J. J. Biol. Chem. 1996; 271: 13324-13332Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and inflammatory cytokines (5.Schmidlin F. Scherrer D. Daeffler L. Bertrand C. Landry I. Gies J-P Mol. Pharmacol. 1998; 53: 1009-1015PubMed Google Scholar, 6.Lung, C-C., Jagels, M. A., Daffern, P. J., Tan, E. M., and Zuraw, B. L. (1998) 39, 243–253Google Scholar). Furthermore, BK2 receptors are overexpressed inras-transformed fibroblasts (7.Hembree T.N. Leeb-Lundberg L.M.F. J. Cell. Physiol. 1996; 169: 248-255Crossref PubMed Scopus (6) Google Scholar) and are coupled with mitogenic signaling pathways (8.Owen N.E. Villereal M.L. Cell. 1983; 32: 979-985Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 9.El-Dahr S.S. Dipp S. Baricos W.H. Am. J. Physiol. 1998; 275: F343-F352Crossref PubMed Google Scholar). The nuclear factors regulating BK2 gene expression are largely unknown. The tumor suppressor protein, p53, is present at low levels in normal cells but is activated in response to cellular stress such as hypoxia, oncogene activation, and DNA damage (10.Agarwal M.L. Taylor W.R. Chernov M.V. Chernova O.B. Stark G.R. J. Biol. Chem. 1998; 273: 1-4Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 11.Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2292) Google Scholar). Tight regulation of p53 activity appears to be essential for normal development as overexpression of p53 in transgenic mice produces abnormal kidneys (12.Godley L.A. Kopp J.B. Eckhaus M. Paglino J.J. Owens J. Varmus H.E. Genes Dev. 1996; 10: 836-850Crossref PubMed Scopus (115) Google Scholar) and knockout of its negative regulator, mdm2, is fatal (13.De Oca Luna R.M. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1207) Google Scholar). The consensus DNA sequence specific for p53 binding consists of two copies of the inverted repeat sequence (RRRC(A/T)(T/A)GYYY) separated by 0–13 nucleotides (14.El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1748) Google Scholar, 15.Funk W.D. Pak D.T. Karas R.H. Wright W.E. Shay J.W. Mol. Cell. Biol. 1992; 12: 2866-2871Crossref PubMed Scopus (678) Google Scholar). p53 binds DNA as a tetramer with a stoichiometry of one p53 molecule per consensus pentamer (16.Nagaich A.K. Zhurkin V.B. Durell S.R. Jernigan R.L. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1875-1880Crossref PubMed Scopus (99) Google Scholar, 17.McLure K.G. Lee P.W.K. EMBO J. 1998; 17: 3342-3350Crossref PubMed Scopus (204) Google Scholar). Recent studies have also shown the importance of protein-protein interactions between p53 and the co-activators, cyclic AMP response element-binding protein/p300, in mediating the transcriptional effects of p53 (18.Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 19.Gu W. Shi X.-L. Roeder R.G. Nature. 1997; 387: 296-299Crossref PubMed Scopus (522) Google Scholar, 20.Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (596) Google Scholar, 21.Liu L. Scolnick M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (654) Google Scholar). p53 transactivates a number of genes important for cell cycle control (e.g. p21 Waf-1), DNA repair (e.g. GADD45) and apoptosis (e.g. IGF-BP3, Bax) (22.Buckbinder L. Talbott R. Velasco-Moguel S. Takenaka I. 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Spillare E.A. Weinberg W.C. Felley-Bosco E. Wang X.W. Geller D.A. Tzeng E. Billiar T.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2442-2447Crossref PubMed Scopus (395) Google Scholar). In addition to its transactivation function, p53 can directly repress transcription by interfering with basal promoter function (35.Agoff S.N. Hou J. Linzer D.I.H. Wu B. Science. 1993; 259: 84-87Crossref PubMed Scopus (263) Google Scholar, 36.Farmer G. Colgan J. Nakatani Y. Manley J.L. Prives C. Mol. Cell. Biol. 1996; 16: 4295-4304Crossref PubMed Google Scholar, 37.Farmer G. Friedlander P. Colgan J. Manley J.L. Prives C. Nucleic Acids Res. 1996; 24: 4281-4288Crossref PubMed Scopus (62) Google Scholar, 38.Wang Q. Zambetti G.P. Suttle D.P. Mol. Cell. Biol. 1997; 17: 389-397Crossref PubMed Google Scholar, 39.Wang X.W. Forrester K. Yeh J. Feitelson M.A. Gu J.-R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (635) Google Scholar) or by sequence-specific DNA binding and exclusion of atrans-acting factor (40.Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (151) Google Scholar). We report here that the promoter region of the rat BK2 gene contains a p53-binding site and that p53 activates BK2promoter-driven transcription of a reporter gene. We further found an upstream p53-binding DNA element which down-modulates p53-mediated activation of the BK2 promoter. These data may have important implications regarding the regulation of BK2 gene expression in early development and in response to cellular stress. A DNA fragment spanning from −1184 to +93 of BK2 5′-flanking region was amplified by polymerase chain reaction from rat liver genomic DNA. The primers 5′-GTCCTTCGTTTTGAGTCTGG-3′ and 5′-GGTTCTGTGTTGTAGGGAGT-3′ were selected from the gene sequence published by Pesquero et al. (2.Pesquero J.B. Lindsey C.J. Zeh K. Paiva A.C.M. Ganten D. Bader M. J. Biol. Chem. 1994; 269: 26920-26925Abstract Full Text PDF PubMed Google Scholar). The DNA fragment was inserted into the SmaI site in the polylinker region of a T/A cloning vector, pCR2.1 (Invitrogen). Following digestion withKpnI (in the polylinker of pCR2.1, upstream to theSmaI site) and NheI (at position +55 of the insert), the DNA fragment was ligated into the reporter plasmid pCAT3Basic (Promega) upstream from the chloramphenicol acetyltransferase (CAT) open reading frame. For the generation of promoter constructs with serial deletions in the 5′-upstream region, the original construct (−1184+55/CAT) was digested withXhoI (−1087), NcoI (−827), BglII (−635), and EcoRI (−563). The −384, −200, −94, and −38/CAT promoter constructs were generated by polymerase chain reaction using the −1184+55/CAT construct as a DNA template. Partial (15/20 bp) or complete (20/20 bp) deletion of the p53-binding site at −707 to −688 was performed by the QuickChangeTMSite-directed Mutagenesis System (Stratagene, La Jolla, CA) following the manufacturer's recommendations. All constructs were sequenced to verify the sequence and orientation by manual DNA sequencing using T7 Sequenase (Amersham Pharmacia Biotech) or by automated DNA sequencing (Applied Biosystems, Model 373A). HeLa cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Life Technologies, Inc.) at 37 °C in a humidified incubator with 5% CO2. Cells were plated in duplicates in six-well plates at 1.8 × 104 cells/well in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum 1 day prior to transfection. Cells were transfected with 1.2 μg of DNA per well of pCAT vectors, driven by various BK2 promoter fragments. A control β-galactosidase vector pSVZ (Promega, 0.5 μg of DNA/well) was co-transfected with BK2-pCAT plasmids to correct for transfection efficiency. CMV-driven expression vectors for wild-type or mutant p53 (33.Morris G.F. Bischoff J.R. Mathews M.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 895-899Crossref PubMed Scopus (133) Google Scholar) were co-transfected using 10–500 ng of DNA/well. Transfections were performed using LipofectAMINE PLUS Reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. Four hours after transfection, fresh medium was replaced and cell extracts were prepared 24 h later using a reporter lysis reagent (Promega). After normalization for β-galactosidase activity in the lysate, aliquots of cell lysates were analyzed for CAT activity by the addition to the aliquot of 20 μl of 4 mm acetyl-CoA, 70 μl of 1 m Tris-Cl (pH 7.8), 5 μl of 25 μCi/ml [14C]chloramphenicol (40–60 mCi/mmol), and H2O to a final volume of 150 μl, and incubated at 37 °C for 1 h. The reaction was extracted with 1 ml of ethyl acetate and the ethyl acetate evaporated in a Speedvac evaporator. Samples were suspended in 25 μl of ethyl acetate and spotted 2 cm above the edge of a plastic-backed TLC sheet. Thin layer chromatography was run in 19:1 chloroform/methanol, and the sheet was air-dried and placed on film for autoradiography. The optical density of the radioactive spots was measured by densitometry and CAT activity was calculated as percent acetylated product. The oligonucleotide sequences used were as follows (double-stranded): consensus p53, 5′-TAGGCATGTCTAGGCATGTCTAAGCT-3′ (14.El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1748) Google Scholar); p21, 5′-ATCAGGAACATGTCCCAACATGTTGGAACATGTCCCAACATGTTGAGCTC-3′ with the p53 consensus in the p21 gene; BK2-P1 5′-AGGGGGGAGGTGCCCAGGAGAGTGATGA-3′ with the p53 site in the rat BK2 receptor gene (positions −70 to −50 bp) and BK2-P2 5′-ACTCTTGCCTGGTCTTCCCT-3′ (positions −707 to −688) (2.Pesquero J.B. Lindsey C.J. Zeh K. Paiva A.C.M. Ganten D. Bader M. J. Biol. Chem. 1994; 269: 26920-26925Abstract Full Text PDF PubMed Google Scholar). Nuclear extracts containing wild-type p53 were prepared from the embryonic fibroblast cell line, SVT-2 (from ATCC). In addition, we used purified C terminus truncated and constitutively active p53 (gift from M. Barton, University of Cincinnati). Double-stranded oligonucleotides were 5′-end labeled with [32P]dATP with T4 kinase for use as probes in the gel mobility shift assays. The labeled probe was incubated for 20 min at room temperature with nuclear extracts and the binding buffer (5 mm Hepes, pH 7.9, 5% glycerol, 0.2 mmdithiothreitol, 5 mm spermidine, 1.5 μg of poly(dI-dC)). Reactions containing a p53-specific activating antibody, pAb421 (Oncogene Science), were preincubated on ice for 15 min before adding the radiolabeled probe. Specific competitor oligonucleotides were added in 100–300-fold molar excess 15 min before addition of the radioactive probe. Based on homology alignment, two putative binding sites for p53 were identified at positions −70 to −50 (BK2-P1): 5′-GGAGGTGCCCAGGAGAAGTGA-3′ and −707 to −688 (BK2-P2): 5′-ACTCTTGCCTGGTCTTCCCT-3′ of the rat BK2 receptor promoter (Fig.1 A). Sequence variation from the tandem 10-bp motif, RRRC(A/T)(T/A)GYYY, described as a consensus p53 recognition sequence (14.El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1748) Google Scholar) is underlined (see also Fig.1 B). Whereas the P1 sequence is conserved in the human (41.Ma J-X. Wang D-Z. Ward D.C. Chen L. Dessai T. Chao J. Chao L. Genomics. 1994; 23: 362-369Crossref PubMed Scopus (87) Google Scholar,42.Kammerer S. Braun A. Arnold N. Roscher A.A. Biochem. Biophys. Res. Commun. 1995; 211: 226-233Crossref PubMed Scopus (70) Google Scholar) and mouse 2Z. Saifudeen and S. S. El-Dahr, unpublished data. BK2 genes, the P2 site is not (Fig. 1 C). We next examined whether p53 binds to these putative p53-binding sites (P1 and P2) using EMSA. As a source of p53, we used nuclear extract from SVT-2 embryonic fibroblasts as well as recombinant p53. Western blots using the p53 antibodies, PAb421 (Oncogene Science), and FL-393 (Santa Cruz), showed that SVT-2 cells contain p53 (data not shown). EMSA assays utilizing a 32P-labeled BK2-P1 oligonucleotide and SVT-2 nuclear extracts revealed two major DNA-protein complexes (Fig.2 A, lane 2). The addition of the p53-activating antibody, PAb421 (lane 1, 2 μl) resulted in enhanced DNA binding activity (Fig. 2 A,lanes 3 and 4), demonstrating the presence of p53 in the complexes. To test the specificity of the DNA-protein interactions, EMSA competition experiments were performed (Fig.2 B). Incubation of the consensus p53 oligoduplex probe with SVT-2 nuclear extracts produced two major DNA-protein complexes (Fig.2 A, lane 1). The addition of 100-fold molar excess of unlabeled consensus p53 oligoduplex eliminated both band shifts (Fig. 2 B, lane 2). An unlabeled oligonucleotide corresponding to the p53-binding site in the p21 gene prevented the formation of the higher mobility complex when added at 100-fold molar excess (Fig. 2 B, lane 3) and both low and high mobility complexes when added at 200- and 300-fold excess (Fig. 2 B, lanes 4 and 5). An 100–200-fold molar excess of unlabeled BK2-P1 oligoduplex also competed efficiently with binding to the consensus p53 probe (Fig.2 B, lanes 6 and 7). In contrast, a P1 probe containing point mutations in the p53 BK2-P1 sequence did not produce any specific band shifts (Fig. 2 B, lanes 8–10, see figure legend). Finally, the band shift produced by binding of nuclear proteins to the P1 probe was greatly diminished upon addition of 100-fold molar excess of unlabeled p53 consensus sequence (Fig.2 B, lanes 11, 12). To further document the specific binding of p53 to the p53-like motif in the P1 site, we examined the binding of the P1 duplex to purified p53 (Fig. 2 C). Binding of p53 to the P1 oligoduplex (Fig.2 C, lane 1) is competed, albeit not completely, by 100-fold excess of unlabeled oligoduplexes corresponding to the p53-binding site in the p21 promoter (lane 2) or the p53 consensus (lane 3). However, when added at 200-fold excess, the p53 consensus sequence and the p53 motif in the p21 gene compete efficiently with the BK2-P1 for binding to purified p53 (Fig.2 D). Binding of the p53 consensus oligoduplex to purified p53 (Fig. 2 C, lane 4) is abolished or attenuated by 100-fold excess of unlabeled p53 consensus sequence (lane 5), p53 motif in the p21 gene (lane 6) or BK2-P1 (lane 7). These cross-competition experiments demonstrate that p53 binds specifically to the P1 sequence in the BK2promoter. In addition, the results suggest that the binding affinity of the p53 site in BK2-P1 to p53 may be higher than that of the consensus p53 sequence or the p21 gene p53 site. We speculate that the difference in mobility of the DNA-protein complexes in SVT-2 nuclear extracts (two major bands) as compared with recombinant p53 (one major band) may be related to the presence of p53-interacting proteins in the former but not the latter. In HeLa cells, p53 is bound to the human papillomavirus E6 protein, which targets p53 for degradation (43.Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1991; 63: 1129-1136Abstract Full Text PDF Scopus (3464) Google Scholar) and renders these cells functionally deficient in p53. Other studies have shown that these cells can be used to study p53-mediated transcriptional responses (33.Morris G.F. Bischoff J.R. Mathews M.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 895-899Crossref PubMed Scopus (133) Google Scholar). HeLa cells were transiently transfected with CAT vectors containing segments of the BK2 promoter with and without a CMV promoter-driven p53 expression plasmid. As shown in Fig.3 A, cells co-transfected with pCMV-p53(wt) and seven different BK2 promoter-CAT constructs, pBK2 (−1184), (−827), (−635), (−563), (−384), (−200), and (−94), showed a clear induction of CAT activity only in the presence of wild-type p53. In contrast, no increase in CAT expression was observed in cells co-transfected with pCMV-p53(wt) and pBK2(−38) (Fig.3 A). Wild-type p53 stimulated pBK2(−1184) or pBK2(−94)-driven CAT expression in a dose-dependent manner (Fig. 3 B). These results suggest that the p53 response element in the BK2 promoter is located in the region between −38 to −94 bp, which is consistent with the location of the P1 p53-binding site. To test the specificity of the stimulatory effect of wild-type p53 onBK2 promoter-driven CAT transcription, HeLa cells were co-transfected with a CMV promoter-driven expression plasmid, pCMVp53E258K, which encodes a dominant negative mutant of p53, with a Glu → Lys change at amino acid 258 (33.Morris G.F. Bischoff J.R. Mathews M.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 895-899Crossref PubMed Scopus (133) Google Scholar). Co-transfection with this p53 mutant which lacks the ability to bind DNA failed to induceBK2 promoter-driven CAT expression (data not shown and Fig.5 B), and in experiments in which both pCMV-p53(wt) and pCMVp53258K were co-transfected, the mutant p53 repressed the activation of the BK2 promoter by wild-type p53 in a dose-dependent manner (Fig. 3, C andD). CBP/p300 are transcriptional co-activators that bind and activate a number of transcription factors including the p53 protein (18.Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 19.Gu W. Shi X.-L. Roeder R.G. Nature. 1997; 387: 296-299Crossref PubMed Scopus (522) Google Scholar, 20.Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (596) Google Scholar, 21.Liu L. Scolnick M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (654) Google Scholar, 44.Utley R.T. Ikeda K. Grant P.A. Côté J. Steger D.J. Eberharter A. John S. Workman J.L. Nature. 1998; 394: 498-502Crossref PubMed Scopus (446) Google Scholar, 45.Berger S.L. Curr. Opin. Cell Biol. 1999; 11: 336-341Crossref PubMed Scopus (141) Google Scholar). Accordingly, we examined the effect of CBP/p300 on p53-mediated transcriptional responses of the BK2 promoter. As shown in Fig. 4, A andB, co-transfection of viral promoter driven expression plasmids for CBP/p300 (0, 10, and 50 ng of DNA) into HeLa cells augmented the p53-mediated activation of BK2 promoter in a dose-dependent manner. The transactivation by CBP/p300 most likely occurs via the p53-like motif at the P1 site (located at −70), rather than via the NF-κB site which overlaps with the upstream BK2-P2 motif, for two reasons. First, CBP/p300 activates CAT transcription from the pBK(−94) construct, which lacks the NF-κB site (Fig. 4 C). Second, deletion of the BK2-P2 site (see below) destroys the NF-κB site, yet this mutant construct (with an intact P1 site) remains responsive to CBP/p300 (data not shown). In addition to the proximal P1 p53-like binding site, the rat BK2 5′-flanking region contains a second p53-like motif at positions −707 to −688 (Fig. 1 B). Unlike P1, P2 is not conserved in the human BK2 gene. This DNA sequence binds specifically to p53 present in SVT-2 cells' nuclear extracts as indicated by the enhanced DNA binding activity following treatment with the PAb421 antibody (Fig. 5 A). Specificity of the complex was demonstrated by competition by unlabeled p21 and BK2-P2 (Fig. 5 A) but not by irrelevant oligoduplex (Jun/AP-1) (data not shown). To determine the functional relevance of the P2 site, we compared the effect of wild-type p53 on the activity ofBK2 promoter constructs either containing (pBK-1184) or lacking (pBK-635) the P2 site. As shown in Fig. 5B, p53-mediated transactivation is approximately 2-fold higher in pBK2-635 than pBK2-1184. To validate this finding, we utilized a deletion mutagenesis approach in which we removed either 3/4 (ΔP215bp) or 4/4 (ΔP220bp) pentamers of the P2 site. As shown in Fig. 5,C and D, pCMV-p53(wt) activates the activity of the BK2(−1184) promoter construct in a dose-dependent manner. When co-transfection was performed using the mutant pBK2(−1184) constructs, up to 2-fold further increase in CAT activity was observed. This finding suggests that the P2 site has a negative modulation effect on p53-mediated activation of the BK2promoter. The bradykinin type 2 receptor, BK2, is a heptahelical G protein-coupled receptor. Upon ligand activation, BK2 engages numerous second messenger systems such as phospholipase C and D, phosphoinositol 3-kinase, protein kinase C, and tyrosine kinases. Among the nuclear factors activated by BK are AP-1 (9.El-Dahr S.S. Dipp S. Baricos W.H. Am. J. Physiol. 1998; 275: F343-F352Crossref PubMed Google Scholar) and NF-κB (46.Pan Z.K. Zuraw B.L. Lung C-C. Prossnitz E.R. Browning D.D. Ye R.D. J. Clin. Invest. 1996; 98: 2042-2049Crossref PubMed Scopus (131) Google Scholar) which are believed to mediate the effects of BK on growth, differentiation, and gene expression of inflammatory cytokines and growth factors. Activation of BK2 on endothelial cells mediates nitric oxide release and vasorelaxation, consistent with the hypertensive phenotype ofBK2-deficient mice (47.Alfie M.E. Sigmon D.H. Pomposiello S.I. Carretero O.A. Hypertension. 1997; 29: 483-487Crossref PubMed Google Scholar, 48.Madeddu P. Varoni M.V. Palomba D. Emanueli C. Demontis M.P. Glorioso N Dessi-Fulgheri P. Sarzani R. Anania V. Circulation. 1997; 96: 3570-3578Crossref PubMed Scopus (148) Google Scholar, 49.Cervenka L. Harrison-Bernard L.M. Dipp S. Primrose G. Imig J.D. El-Dahr S.S. Hypertension. 1999; 34: 176-180Crossref PubMed Scopus (63) Google Scholar). In the present study, transient transfections into HeLa cells of promoter-reporter constructs containing serial deletions of theBK2 5′-flanking region localized the p53 activation effect to a 56-bp region extending from −38 to −94 of the promoter. This region contains a p53-like binding motif (P1 site) at −70 to −50. This finding together with the sequence-specific binding of p53 to the P1 site, the inhibition of p53-mediated activation by co-expression of a mutant p53, and the species conservation of this site strongly implicate this DNA element in p53-mediated activation of theBK2 promoter. Cyclic AMP response element-binding protein and p300 (CBP/p300) are structurally related activators of numerous transcription factors including p53. CBP/p300 possess intrinsic histone acetyltransferase activity (18.Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 19.Gu W. Shi X.-L. Roeder R.G. Nature. 1997; 387: 296-299Crossref PubMed Scopus (522) Google Scholar, 20.Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (596) Google Scholar, 21.Liu L. Scolnick M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (654) Google Scholar). Acetylation of lysine residues in the N-terminal domains of histones facilitates gene activation, perhaps by reducing histone tail affinity for DNA, leading to a more favorable transcription factor binding to nucleosomal DNA. CBP/p300 acetylate components of the basal transcription machinery, such as TFIIEβ and TFIIF in vitro (50.Truant R. Xiao H. Ingles C.J. Greenblatt J. J. Biol. Chem. 1993; 268: 2284-2287Abstract Full Text PDF PubMed Google Scholar, 51.Thut C.J. Chen J.L. Klemm R. Tjian R. Science. 1995; 267 (104): 110Crossref Scopus (407) Google Scholar, 52.Lu H. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5154-5158Crossref PubMed Scopus (282) Google Scholar). Furthermore, p300 acetylates p53 on its C terminus thereby enhancing its DNA binding activity in vitro (21.Liu L. Scolnick M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (654) Google Scholar). In our model, co-expression of CBP/p300 with p53 augmented the transactivation function of p53. Based on these results, we propose that the interaction of p53 with the P1 site acts as a recruiting vehicle for CBP/p300 to the vicinity of the proximal promoter thereby activating the basal transcription machinery. We also identified a second p53-like binding site (P2) at position −707 of the rat BK2 gene. This site differs from P1 in many aspects. First, unlike P1, P2 is not conserved in the mouse or humanBK2 genes. Second, removal of P2 by truncation or deletion amplified p53-mediated transactivation. It is not clear how the P2 site down-modulates the positive transcriptional activity of p53 that is exerted at the level of the proximal promoter. One possibility may involve the displacement of an activator protein from binding to the same regulatory element, as recently reported in the α-fetoprotein gene (40.Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (151) Google Scholar). Indeed, the P2 sequence overlaps with a putative binding site for the transcription factor, NF-κB. Another may be that p53, when bound to the P2 site, interacts with an early stage of the assembling complex of general transcription factors, blocking further assembly. It is also conceivable that HeLa cells have a limited pool of CBP/p300 (53.Wadgaonkar R. Phelps K.M. Haque Z. Williams A.J. Silverman E.S. Collins T. J. Biol. Chem. 1999; 274: 1879-1882Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and that binding of p53 to P2 “sequesters” the available pool of p53 and its co-activators. Ongoing studies in our laboratory are testing these various hypotheses. The biological significance of p53-mediated regulation ofBK2 gene expression remains unclear. We speculate that endogenous p53 may be important in the regulation of BK2 expression under at least two conditions. Both p53 and BK2 are highly expressed in certain tissues during development (3.El-Dahr S.S. Figueroa C.D. Gonzalez C.B. Müller-Esterl W. Kidney Int. 1997; 51: 739-749Abstract Full Text PDF PubMed Scopus (63) Google Scholar). Our preliminary studies have shown a remarkable overlap in the cellular expression of p53 and BK2 in the developing kidney (54.Saifudeen Z. El-Dahr S.S. J. Am. Soc. Nephrol. 1998; 9 (abstr.): 367AGoogle Scholar). Thus, it is conceivable that p53 may have a role in the developmental regulation of the BK2 gene, as has recently been proposed for the α-fetoprotein gene (40.Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (151) Google Scholar). In addition, p53 is induced and activated by cellular injury and chronic inflammation (55.Arai N. Mitomi H. Ohtani Y. Igarashi M. Kakita A. Okayasu I. Mod Pathol. 1999; 12: 604-611PubMed Google Scholar) and this may represent a new mechanism to activateBK2 gene expression in response to inflammation (5.Schmidlin F. Scherrer D. Daeffler L. Bertrand C. Landry I. Gies J-P Mol. Pharmacol. 1998; 53: 1009-1015PubMed Google Scholar,6.Lung, C-C., Jagels, M. A., Daffern, P. J., Tan, E. M., and Zuraw, B. L. (1998) 39, 243–253Google Scholar). We conclude that the rat bradykinin B2 receptor gene is a target for the tumor suppressor p53. This is the first report of a G-protein-coupled receptor gene regulated by p53. Future challenges will be to determine the biological relevance of p53-mediated regulation of BK2 in vivo as well as whether p53 regulates other G protein-coupled receptor genes. We thank Dr. Michelle Barton for generously providing activated purified p53 and for helpful discussions and critical reading of the manuscript. We also thank Drs. R. Kwok for the CBP expression vector, and G. Morris for the wild-type and mutant p53 expression plasmids." @default.
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