Transactivation of the Urokinase-type Plasminogen Activator Receptor Gene through a Novel Promoter Motif Bound with an Activator Protein-2α-related Factor
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.4702
ISSN1083-351X
AutoresHeike Allgayer, Heng Wang, Yao Wang, M. M. Heiss, Reinhard Bauer, Okot Nyormoi, Douglas Boyd,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe urokinase receptor overexpressed in invasive cancers promotes laminin degradation. The current study was undertaken to identify cis elements and trans-acting factors activating urokinase receptor expression through a footprinted (−148/−124) region of the promoter containing putative activator protein-2- and Sp1-binding motifs. Mobility shifting experiments using nuclear extract from a high urokinase receptor-expressing cell line (RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct from, activator protein-2α bound to this region. Mutations preventing the binding of the activator protein 2α-related factor diminished urokinase receptor promoter activity. In RKO cells, the expression of a negative regulator of activator protein-2 function diminished urokinase receptor promoter activity, protein, and laminin degradation. Conversely, urokinase receptor promoter activity in low urokinase receptor-expressing GEO cells was increased by activator protein-2αA expression. Although using GEO nuclear extract, little activator protein-2α-related factor bound to the footprinted region, phorbol 12-myristate 13-acetate treatment, which induces urokinase receptor expression, increased complex formation. Mutations preventing the activator protein-2α-related factor and Sp1/Sp3 binding reduced urokinase receptor promoter stimulation by this agent. Thus, the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the urokinase receptor is mediated partly throughtrans-activation of the promoter via a sequence (−152/−135) bound with an activator protein-2α-related factor. The urokinase receptor overexpressed in invasive cancers promotes laminin degradation. The current study was undertaken to identify cis elements and trans-acting factors activating urokinase receptor expression through a footprinted (−148/−124) region of the promoter containing putative activator protein-2- and Sp1-binding motifs. Mobility shifting experiments using nuclear extract from a high urokinase receptor-expressing cell line (RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct from, activator protein-2α bound to this region. Mutations preventing the binding of the activator protein 2α-related factor diminished urokinase receptor promoter activity. In RKO cells, the expression of a negative regulator of activator protein-2 function diminished urokinase receptor promoter activity, protein, and laminin degradation. Conversely, urokinase receptor promoter activity in low urokinase receptor-expressing GEO cells was increased by activator protein-2αA expression. Although using GEO nuclear extract, little activator protein-2α-related factor bound to the footprinted region, phorbol 12-myristate 13-acetate treatment, which induces urokinase receptor expression, increased complex formation. Mutations preventing the activator protein-2α-related factor and Sp1/Sp3 binding reduced urokinase receptor promoter stimulation by this agent. Thus, the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the urokinase receptor is mediated partly throughtrans-activation of the promoter via a sequence (−152/−135) bound with an activator protein-2α-related factor. The urokinase-type plasminogen activator (urokinase) is a serine protease that converts the inert zymogen plasminogen into plasmin, a protease with broad substrate specificity leading to extracellular matrix degradation and tumor invasion (1Nielsen L. Hansen J. Skriver L. Wilson E. Kaltoft K. Zenthen J. Dano K. 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Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (591) Google Scholar) composed of three similar repeats approximately 90 residues each (6Behrendt N. Ploug M. Patthy L. Houen G. Blasi F. Dano K. J. Biol. Chem. 1991; 266: 7842-7847Abstract Full Text PDF PubMed Google Scholar,7Riittinen L. Limongi P. Crippa M.P. Conese M. Hernandez-Marrero L. Fazioli F. Blasi F. FEBS Lett. 1996; 381: 1-6Crossref PubMed Scopus (19) Google Scholar). The amino-terminal domain binds the plasminogen activator with the carboxyl terminus domain serving to anchor the binding protein to the cell surface via a glycosyl-phosphatidylinositol chain (6Behrendt N. Ploug M. Patthy L. Houen G. Blasi F. Dano K. J. Biol. Chem. 1991; 266: 7842-7847Abstract Full Text PDF PubMed Google Scholar, 7Riittinen L. Limongi P. Crippa M.P. Conese M. Hernandez-Marrero L. Fazioli F. Blasi F. FEBS Lett. 1996; 381: 1-6Crossref PubMed Scopus (19) Google Scholar). urokinase-type plasminogen activator receptor activator protein-1 and -2, respectively chloramphenicol acetyltransferase electrophoretic mobility shift assay phorbol 12-myristate 13-acetate Rous sarcoma virus The u-PAR has multiple functions. First, urokinase bound to the u-PAR activates plasminogen at a much faster rate than fluid phase plasminogen activator (8Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 9Higazi A. Cohen R. Henkin J. Kniss D. Schwartz B.S. Cines D. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and this contributes to type IV collagenase activation (10Ginestra A. Monea S. Seghezzi G. Dolo V. Nagase H. Mignatti P. Vittorelli M. J. Biol. Chem. 1997; 272: 17216-17222Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Second, the binding site clears urokinase-inhibitor complexes from the extracellular space (11Cubellis M. Wun T. Blasi F. EMBO J. 1990; 9: 1079-1085Crossref PubMed Scopus (332) Google Scholar) via a α2-macroglobulin receptor-dependent mechanism (12Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar). Third, the u-PAR interacts with the extracellular domain of integrins to connect to the cytoskeleton, thereby mediating cell adhesion and migration (13Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar, 14Yebra M. Parry G.C.N. Stromblad S. Mackman N. Rosenberg S. Mueller B.M. Cheresh D.A. J. Biol. Chem. 1996; 271: 29393-29399Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 15Bohuslav J. Horejsi V. Hansmann C. Stockl J. Weidle U.H. Majdic O. Bartke I. Knapp W. Stockinger H. J. Exp. Med. 1995; 181: 1381-1390Crossref PubMed Scopus (355) Google Scholar). Fourth, the u-PAR is chemotactic for human monocytes and mast cells, and this may require the cleavage of the binding site between domains 1 and 2 (16Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar, 17Sillaber C. Baghestanian M. Hofbauer R. Virgolini I. Bankl H.C. Fureder W. Agis H. Willheim M. Leimer M. Scheiner O. Binder B.R. Kiener H.P. Bevec D. Fritsch G. Majdic O. Kress H.G. Gadner H. Lechner K. Valent P. J. Biol. Chem. 1997; 272: 7824-7832Crossref PubMed Scopus (75) Google Scholar). The u-PAR gene is 7 exons long and is located on chromosome 19q13 (18Vagnarelli P. Raimondi E. Mazzieri R. De Carli L. Mignatti P. Cytogenet. Cell Genet. 1992; 60: 197-199Crossref PubMed Scopus (10) Google Scholar,19Casey J.R. Petranka J.G. Kottra J. Fleenor D.E. Rosse W.F. Blood. 1994; 84: 1151-1156Crossref PubMed Google Scholar). Transcription of the u-PAR gene yields a 1.4-kilobase mRNA or an alternatively spliced variant lacking the membrane attachment peptide sequence (20Roldan A. Cubellis M. Masucci M. Behrendt N. Lund L. Dano K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (542) Google Scholar, 21Pyke C. Eriksen J. Solberg H. Schnack B. Nielsen S. Kristensen P. Lund L.R. Dano K. FEBS Lett. 1993; 326: 69-74Crossref PubMed Scopus (79) Google Scholar). The amounts of u-PAR are controlled mainly at the transcriptional level, but altered message stability and receptor recycling may represent other means of controlling the amount of this gene product at the cell surface (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 23Shetty S. Kumar A. Idell S. Mol. Cell. Biol. 1997; 17: 1075-1083Crossref PubMed Scopus (107) Google Scholar, 24Lund L.R. Ellis V. Ronne E. Pyke C. Dano K. Biochem. J. 1995; 310: 345-352Crossref PubMed Scopus (114) Google Scholar, 25Nykjaer A. Conese M. Cremona O. Gliemann J. Blasi F. EMBO J. 1997; 16: 2610-2620Crossref PubMed Scopus (295) Google Scholar). The transcriptional regulation of the u-PAR gene is still poorly understood. Soravia et al. (26Soravia E. Grebe A. De Luca P. Helin K. Suh T.T. Degen J.L. Blasi F. Blood. 1995; 86: 624-635Crossref PubMed Google Scholar) reported that the basal expression of the gene was regulated via Sp1 motifs proximal and upstream of the transcriptional start site. Our laboratory showed that both the constitutive and PMA-inducible expression of the gene required a footprinted region (−190/−171) of the promoter containing an AP-1 motif (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). We also observed a second footprinted region of the promoter (−148/−124), and deletion of this region caused a dramatic reduction in the constitutive u-PAR promoter activity in a colon cancer cell line characterized by its high expression of this gene. Interestingly, this region of the promoter contained noncanonical AP-2 (−142/−134) and Sp1 (−147/−138) motifs overlapping with each other as well as nonconsensus polyomavirus activator 3 (−133/−127) motifs. The u-PAR has been implicated in a number of physiological and pathological processes involving tissue remodeling, although it is not critical for mouse development (27Carmeliet P. Moons L. Dewerchin M. Rosenberg S. Herbert J. Lupu F. Collen D. J. Cell Biol. 1998; 140: 233-245Crossref PubMed Scopus (117) Google Scholar). In cancer, several experimental and clinical findings support the view that the u-PAR plays a prominent role in tumor cell invasion and metastasis. For example, the u-PAR mRNA is expressed in the tumor cells of invasive colon cancers (28Pyke C. Salo S. Ralfkiaer E. Romer J. Dano K. Tryggvason K. Cancer Res. 1995; 55: 4132-4139PubMed Google Scholar,29Pyke C. Kristensen P. Ralfkiaer E. Grondahl-Hansen J. Eirksen J. Blasi F. Dano K. Am. J. Pathol. 1991; 138: 1059-1067PubMed Google Scholar), and a high u-PAR protein level is predictive of short survival times for patients with this disease (30Ganesh S. Sier C.F.M. Heerding M.M. Griffioen G. Lamers C.B.H. Verspaget H.W. Lancet. 1994; 344: 401-402Abstract PubMed Scopus (173) Google Scholar). Further, earlier studies have shown that the overexpression of a human u-PAR cDNA increased the ability of human osteosarcoma cells to invade into an extracellular matrix-coated porous filter (31Kariko K. Kuo A. Boyd D. Okada S. Cines D. Barnathan E. Cancer Res. 1993; 53: 3109-3117PubMed Google Scholar). Conversely, down-regulating u-PAR levels using antisense expression constructs, oligonucleotides, or synthetic compounds reduced the ability of divergent invasive cancers to invade in vitro and in vivo (32Kook Y.H. Adamski J. Zelent A. Ossowski L. EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar, 33Xing R.H. Mazar A. Henkin J. Rabbani S.A. Cancer Res. 1997; 57: 3585-3593PubMed Google Scholar, 34Quattrone A. Fibbi G. Anichini E. Pucci M. Zamperini A. Capaccioli S. Del Rosso M. Cancer Res. 1995; 55: 90-95PubMed Google Scholar, 35Min H.Y. Doyle L.V. Vitt C.R. Zandonella C.L. Stratton-Thomas J.R. Shuman M.A. Rosenberg S. Cancer Res. 1996; 56: 2428-2433PubMed Google Scholar, 36Wilhelm O. Weilde U. Rettenberger S. Schmitt M. Graeff H. FEBS Lett. 1994; 337: 131-134Crossref PubMed Scopus (105) Google Scholar). Since the u-PAR is a key factor in promoting tumor-associated proteolysis, down-regulation of its expression could be a promising strategy for inhibiting cancer invasion and metastasis. We therefore undertook a study with two objectives: (a) to identifycis-elements and trans-acting factors regulating constitutive and PMA-inducible u-PAR gene expression via the footprinted region spanning nucleotides −148/−124 and (b) to determine the effect of interfering with transcription factors binding to this region on u-PAR-directed laminin degradation. The u-PAR CAT reporter consisted of 449 base pairs of sequence (37Wang Y. Dang J. Johnson L.K. Selhamer J.J. Doe W.F. Eur. J. Biochem. 1995; 227: 116-122Crossref PubMed Scopus (69) Google Scholar) stretching from −398 to +51 (relative to the transcription start site) cloned into the XbaI site of the pCAT-Basic vector (Promega, Madison, WI). Reporter constructs regulated by truncated u-PAR promoter fragments were as described previously (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The urokinase CAT reporter consisted of 2345 base pairs of 5′-flanking region fused directly to the reporter (38Verde P. Boast S. Franze A. Robbiati F. Blasi F. Nucleic Acids Res. 1988; 16: 10699-10715Crossref PubMed Scopus (103) Google Scholar). Antibodies to Sp1, Sp2, Sp3, and AP-2 isoforms were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Jun-D expression construct was described elsewhere (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Oligonucleotides were purchased from Genosys Biotechnologies (The Woodlands, TX). Recombinant AP-2αA and Sp1 (full-length human proteins) were obtained from Promega (Madison, WI). Expression vectors for AP-2αA, AP-2αB, and AP-2 antisense (39Buettner R. Kannan P. Imhof A. Bauer R. Yim S.O. Glockshuber R. Van Dyke M.W. Tainsky M.A. Mol. Cell. Biol. 1993; 13: 4174-4185Crossref PubMed Scopus (123) Google Scholar) consisted of the cloned sequences inserted into theEcoRI site of pSG5 (Stratagene, La Jolla, CA) and were kindly provided by Dr. Michael Tainsky. The AP-2 pBLCAT2 reporter construct contained three consensus AP-2 motifs 5′ of the pBLCAT2 reporter (40Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar). For the generation of the R2 CAT reporter construct, an oligonucleotide spanning nucleotides −154/−128 was cloned into theXbaI site of pCATbasic (Promega). Nuclear extracts and EMSA were carried out as described elsewhere (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). EMSA was carried out using nuclear extract (8 μg), 0.6 μg of poly(dI/dC), and 2 × 104 cpm of a T4 polynucleotide kinase-labeled [γ-32P]ATP oligonucleotide. The sequences of the AP-2 and Sp1 consensus oligonucleotides were: 5′-GAT CGA ACT GAC CGC CCG CGG CCC GT-3′ (Santa Cruz Biotechnology catalog no. sc-2513) and 5′-ATT CGA TCG GGG CGG GGC GAG C-3′ (Santa Cruz Biotechnology catalog no. sc-2502), respectively. The sequence of the mutated (underlined nucleotides) AP-2 consensus-containing oligonucleotide was 5′-GAT CGA ACT GAC CGC TTG CGG CCC GT-3′ (Santa Cruz Biotechnology catalog no. sc-2516). This was performed according to the protocol of the Site-Directed Mutagenesis Kit (5Z701) ofCLONTECH (Palo Alto, CA). For generation of the Sp1/Sp3mt u-PAR CAT, pCATbasic (Promega) regulated by 398 base pairs of the u-PAR promoter (37Wang Y. Dang J. Johnson L.K. Selhamer J.J. Doe W.F. Eur. J. Biochem. 1995; 227: 116-122Crossref PubMed Scopus (69) Google Scholar) served as a template. A mutation primer substituting T for G at positions −148, −147, −144, and −142 and its corresponding selection primer (5′-CTTATCATGTCTGGTACCCCCGGAATTC-3′) converting the BamHI site of pCATbasic to a KpnI site were annealed to the denatured template plasmid, and the plasmid was amplified according to the protocol. Remaining wild-type plasmid was eliminated by two BamHI digestions for 4 h, each of them followed by transformation of nondigested DNA into BMH 71–18mutS cells (CLONTECH, Palo Alto, CA). DNA of selected clones was isolated and sequenced using the Amersham Pharmacia Biotech T7-Sequenase 2.0 Kit. For generation of the AP-2/Sp1/Sp3mt u-PAR CAT construct, the Sp1/Sp3mt u-PAR CAT plasmid served as a template. The second mutation primer substituted A for C at positions −146, −145, −142, and −141 of the u-PAR promoter, and the selection primer (5′-CTTATCATGTCTGGATCCCCCGGAATTC-3′) changed the KpnI site generated above back to BamHI. Selection for AP-2/Sp1/3-mutated plasmids was done by KpnI digestion. The procedure was continued as described above. Cells were transfected at 60% confluency using poly-l-ornithine as described previously (41Nead M.A. McCance D.J. J. Invest. Dermatol. 1995; 105: 668-671Abstract Full Text PDF PubMed Scopus (31) Google Scholar). All transient transfections were performed in the presence of a luciferase expression vector (4 μg), and transfection efficiencies were determined by assaying for luciferase activity. CAT activity was measured as described previously (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The amount of acetylated [14C]chloramphenicol was determined using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software. Student's t test analysis was performed two-sided using the SPSS for Windows statistical software (release 6.1.3) (SPSS Inc., Chicago, IL). Statistical significance was defined asp ≤ 0.05. Transfected cells were enriched by the MACS-Select method of Miltenyi Biotech (Auburn, CA). RKO cells were co-transfected with the AP-2αB expression construct and a plasmid encoding a mutated CD4 molecule (pMACS 4) in a 3:1 ratio. Cells were harvested after 42 h in 320 μl of PBE buffer (phosphate-buffered saline, 0.5% bovine serum albumin, 5 mm EDTA) and incubated for 15 min with 80 μl of a magnetic bead-conjugated antibody directed against the mutated CD4 molecule. The cell suspension was then run through VS+ separation columns using the VarioMACS magnet according to the manufacturer's protocol. RKO cells were extracted into a buffer (10 mmTris, pH 7.4, 0.15 m NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 20 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 1 mm EGTA, 1 mm EDTA) for 10 min at 4 °C. Insoluble material was removed by centrifugation, and 750 μg protein of cell extract was immunoprecipitated at 4 °C for 16 h with 0.25 μg of a polyclonal anti-u-PAR antibody and protein A-agarose beads. The polyclonal antibody (kindly provided by Dr. Andrew Mazar, Angstrom Pharmaceuticals, San Diego, CA) was raised in rabbits against amino acids 1–281 of the human u-PAR and purified on a Sepharose-immobilized u-PAR column. The immunoprecipitated material was subjected to standard Western blotting (42Burnette W. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5936) Google Scholar), and the blot was probed with 5 μg/ml of an anti-u-PAR monoclonal antibody (catalog no. 3931, American Diagnostica, Greenwich, CT) and a horseradish peroxidase-conjugated goat anti-mouse IgG. Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). For the determination of u-PAR by enzyme-linked immunosorbent assay, resected tissue was prepared as described by the manufacturer (American Diagnostica). These were carried out as described previously (43Schlechte W. Murano G. Boyd D.D. Cancer Res. 1989; 49: 6064-6069PubMed Google Scholar). RKO cells were harvested with 3 mm EDTA/phosphate-buffered saline, washed twice, and seeded (500,000 cells) on radioactive laminin-coated (2 μg/dish) dishes. The cells were allowed to attach overnight. Subsequently, cell surface u-PARs were saturated by incubating the cells at 37 °C for 30 min with 5 nm urokinase, and unbound plasminogen activator was removed by washing. The cells were then replenished with serum-free medium with or without 10 μg/ml plasminogen (final concentration). After varying times at 37 °C, aliquots of the culture medium were withdrawn and counted for radioactivity. Solubilized laminin represents the degraded glycoprotein (43Schlechte W. Murano G. Boyd D.D. Cancer Res. 1989; 49: 6064-6069PubMed Google Scholar). Region II of the u-PAR Promoter Footprinted by Nuclear Extract from a High u-PAR-expressing Cell Line Is Bound with Sp1, Sp3, and an AP-2α-related Factor—We previously reported (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) that nuclear extract from a high (3 × 105 binding sites/cell) u-PAR-expressing colon cancer cell line (RKO) footprinted a region (referred to as region II) of the u-PAR promoter (nucleotides −148/−124). As a first step to identifying transcription factor(s) bound to this region, EMSA was carried out using an oligonucleotide spanning nucleotides −154/−128 (Fig.1 A). The oligonucleotide was not extended to the 3′-end (−124) of the footprinted region II, since preliminary EMSA utilizing a probe that included sequences 3′ of −128 had failed to reveal any specific binding complexes. Employing the −154/−128 oligonucleotide, three slower migrating bands (indicated by a brace, arrow, and asterisk) were apparent (Fig. 1 B, lanes 2 and8) with a 100-fold excess of the nonradioactive oligonucleotide eliminating (lane 3) all of these bands. Computer analysis of this region of the u-PAR promoter revealed the presence of putative AP-2- (−142/−134) and Sp1/Sp3- (−147/−138) binding motifs all bearing one mismatch with the corresponding canonical (AP-2, GCCNNNGGC; Sp1, RYYCCGCCCM) sequences. The addition of a 100-fold excess of a consensus AP-2-containing oligonucleotide (Fig.1 B, lane 7 and 11) from the human metallothionein IIa promoter sequence (44Williams T. Admon A. Luscher B. Tjian R. Genes Dev. 1988; 2: 1557-1569Crossref PubMed Scopus (450) Google Scholar) eliminated one of the shifted bands (*). In contrast, substitution of this oligonucleotide at the AP-2 motif (CC to TT) prevented it from competing for the shifted bands (Fig. 1 B, lane 10). Increasing amounts of a nonradioactive oligonucleotide bearing a consensus Sp1 motif caused a dose-dependent decrease in the intensity of two of the shifted bands (indicated with a brace andarrow) (Fig. 1 C) while having little effect on the band (*) competed with the AP-2 motif-containing oligonucleotide. The ability of the consensus Sp1 motif to compete for the binding of nuclear-extracted proteins to the u-PAR promoter footprinted region II oligonucleotide suggested that transcription factors recognized by these motifs were bound to the u-PAR promoter. To examine this possibility, "supershifting" experiments were carried out. The addition of an anti-Sp1-specific antibody to the RKO nuclear extract resulted (Fig. 1 B, lane 4) in a slower migrating band (indicated with a line) with a concomitant decreased intensity of the complex (indicated by a brace) competed with the Sp1 consensus sequence. On the other hand, the addition of an antibody against Sp2 had no effect (lane 5) on the migration pattern, while an antibody directed at the DNA-binding domain of Sp3 completely abolished (Fig. 1 B,lane 6) the shifted band with the intermediate mobility (arrow). These data suggested that the region of the u-PAR promoter footprinted with nuclear extract from a high u-PAR-expressing cell line (22Lengyel E. Wang H. Stepp E. Juarez J. Wang Y. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) is bound with Sp1 and Sp3. Since the fastest migrating complex evident in the EMSA (indicated by an asterisk) was competed with an AP-2 consensus motif (Fig.1 B), we hypothesized that the bound protein was an AP-2 isoform. To test this hypothesis, two sets of experiments were carried out. First, RKO nuclear extract was mixed with 2 μg of an anti-AP-2α antibody or an equal amount of IgG and subsequently with protein A-agarose beads. The supernatant (depleted of AP-2α-immunoreactive proteins) was then used in band shifting experiments. The fastest migrating band (*), which was competed with an excess of the AP-2 consensus, was practically abolished (Fig.1 D) by treatment of the nuclear extract with the anti-AP-2α antibody, whereas the bands recognized by the Sp1 (brace) and Sp3 (arrow) antibodies were unaffected. Further, the IgG control had no effect on the intensity or mobility of any of the shifted bands and antibodies specific for AP-2β, or AP-2γ failed to deplete the RKO nuclear extract of the binding factor (Fig. 1 E). Second, we determined if authentic AP-2α could bind to the u-PAR promoter region II. In EMSA using an oligonucleotide spanning −154/−128, authentic AP-2α (Promega) gave rise to a shifted band (line) (Fig.2), which could be competed with an excess of either the u-PAR region II oligonucleotide (−154/−128) or a consensus AP-2 motif. The authentic AP-2α bound to the −154/−128 probe had a different mobility from the retarded band (which was removed by immunoprecipitation with the anti-AP-2α antibody; Fig.1 D) observed using RKO nuclear extract (*). To delineate the minimal sequence of footprinted region II required for the binding of Sp1/Sp3 and the factor recognized by the anti-AP-2α antibody, band-shifting experiments were carried out with the u-PAR promoter oligonucleotide truncated from either the 5′- or 3′-end (Fig.3 A). The removal of two base pairs at the 5′ terminus (generating oligonucleotide −152/−128) had little effect on the intensities of the Sp1- (brace) and Sp3- (arrow) bound complexes when compared with probe −154/−128. However, a severe attenuation in the binding of these factors was apparent with further truncation from the 5′-end as evident with probe −150/−128. These data suggested that nucleotides 5′ of the nonconsensus Sp1 motif (−147/−138) are required for the optimal binding of Sp1 and Sp3 to region II of the u-PAR promoter. On the other hand, the fastest migrating band (*), which is recognized by the anti-AP-2α antibody, was unaffected by the removal of 5′ nucleotides with binding maintained with probe −143/−128. These data rule out the possibility that an AP-2-like motif located further upstream (−151/−143) is mediating the binding of this factor to region II of the u-PAR promoter. While the binding of Sp1/Sp3 and the AP-2α antibody-reactive factor demonstrated different 5′ requirements, the binding of these transcription factors showed identical requirements for 3′ sequences. Thus, the removal of up to 5 base pairs from the 3′ terminus of oligonucleotide −154/−130 (generating oligonucleotide −154/−135) had little effect on transcription factor binding. However, the deletion of an additional 3 base pairs from the 3′-end (oligonucleotide −154/−138) completely abolished the binding of these three transcription factors. Thus, sequences in the u-PAR promoter extending 3′ to −135 are required for the optimal binding of Sp1, Sp3, and the factor recognized by the anti-AP-2α antibody.Figure 3Identification of minimal sequences of footprinted region II required for binding of nucleus-derived transcription factors from RKO cells. A, RKO nuclear extract (8 μg) was incubated at 21 °C for 20 min with end-labeled oligonucleotides with (+), or without (−) a 100-fold excess of the indicated competitor or an oligonucleotide containing a consensus AP-2 motif (AP-2 consensus). Binding complexes were resolved by EMSA as described in the legend to Fig. 1. The experiments were carried out three times. B, the nucleotide sequence of the u-PAR promoter, including the footprinted region II is shown. Underlined and overlined nucleotides indicate putative motifs identified by the Genetics Computer Group program (Madison, WI). C, RKO nuclear extract (8 μg) or AP-2α (10–20 ng) was incubated at 21 °C for 20 min with a radioactive oligonucleotide corresponding to nucleotides −147/−128 of the u-PAR promoter in the presence or absence of a 100-fold excess of the unlabeled competitor sequences. After this time, 2 μg of the indicated antibody was added to the reaction mixture where indicated. Binding complexes were subsequently analyzed by EMSA. The data are typical of duplicate experiments.View Large Image Figure ViewerDownload (PPT) While the anti-AP-2α antibody "supershifted" (arrow) authentic AP-2α bound to the u-PAR promoter oligonucleotide −154/−128 (Fig. 2), in contrast, we were unable to detect a "supershift" of the fastest migrating band (using RKO nuclear extract) with this antibody (data not shown). However, this could be
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