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Published ahead of print on March 23, 2004, doi:10.1165/rcmb.2003-0296OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 209-215, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0296OC

The PAI-1 Gene as a Direct Target of Endothelial PAS Domain Protein-1 in Adenocarcinoma A549 Cells

Mahito Sato, Toru Tanaka, Koji Maemura, Tsuyoshi Uchiyama, Hiroko Sato, Toshitaka Maeno, Tatsuo Suga, Tatsuya Iso, Yoshio Ohyama, Masashi Arai, Junichi Tamura, Hironosuke Sakamoto, Ryozo Nagai and Masahiko Kurabayashi

Second Department of Internal Medicine, and Department of General Medicine, Gunma University School of Medicine, Gunma; and Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan

Address correspondence to: Masahiko Kurabayashi, M.D., Ph.D., Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, 371-8511, Japan. E-mail: mkuraba{at}med.gunma-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelial PAS domain protein-1 (EPAS1) regulates transcription of the genes encoding erythropoietin and vascular endothelial growth factor, which are important for maintaining oxygen homeostasis. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) gene expression is induced by hypoxia. In this study, we sought to determine whether PAI-1 gene expression is directly regulated by EPAS1 in cancer cells because activities of proteases and their inhibitors are tightly regulated for tumor invasion. Hypoxia increased the PAI-1 mRNA levels in human adenocarcinoma A549 cells. Overexpression of EPAS1 significantly increased the PAI-1 mRNA and protein levels. Transient transfection assays revealed that EPAS1 increased PAI-1 gene transcription through a sequence containing 5'-CACGTACA-3' located at –194 (we refer to it as site HREPAI-1) and GT-box located at –78. Electrophoretic gel mobility shift assays revealed that HREPAI-1 serves as a binding site for EPAS1, and Sp1 constitutively binds to GT-box. In conclusion, PAI-1 expression is induced by EPAS1 through HREPAI-1 and through an Sp1-binding site. These results indicate that the PAI-1 gene is a direct target of EPAS1 and suggest the role of EPAS1 and Sp1 in the hypoxic response of cancer cells.

Abbreviations: cAMP response element, CRE • CRE binding protein, CREB • electrophoretic mobility shift assay, EMSA • endothelial PAS domain protein-1, EPAS1 • erythropoietin, EPO • hypoxia-inducible factor-1{alpha}, HIF-1{alpha} • HIF-1{alpha}–like factor, HLF • hypoxia response element, HRE • kilobases, kb • messenger RNA, mRNA • multiplicity of infection, MOI • plasminogen activator inhibitor-1, PAI-1 • phosphate-buffered saline, PBS • vascular endothelial growth factor, VEGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxygen tension is an important regulator of mammalian gene expression. Physiologic functions of hypoxia-inducible genes are diverse, but modulation of gene expression in response to hypoxia is considered to be metabolic adaptation to compensate for an inadequate oxygen supply. In the case of systemic hypoxia due to anemia or decreased environmental oxygen concentration, erythropoiesis is stimulated by inducing erythropoietin (EPO) gene expression (1). In the case of local hypoxia due to ischemia, angiogenesis is stimulated by the production of vascular endothelial growth factor (VEGF) (2). In addition, severe hypoxia induces the transition from oxidative phosphorylation to glycolysis as the principal means of generating ATP by inducing glycolytic enzymes aldolase A, phosphoglycerate kinase 1, enolase 1, and lactate dehydrogenase A (3, 4).

There has been substantial progress in understanding the molecular basis of oxygen sensing and transcriptional regulation of hypoxia-inducible genes described above. Notably, transcription of these diverse genes is under the control of a crucial transcription factor: hypoxia-inducible factor (HIF)-1{alpha}. Functional binding sites for HIF-1{alpha} have been identified in the 3'-flanking region of the EPO gene and 5'-flanking region of the VEGF gene and the genes encoding glycolytic enzymes (5). The HIF-1{alpha} binding sequences in the human EPO and VEGF genes are 5'-TACGTGCT-3' and 5'-TACGTGGG-3', respectively. Endothelial PAS domain protein-1 (EPAS1), also known as hypoxia-inducible factor-like factor (HLF) (6), HIF-related factor, HIF-2{alpha} (7) is one of the transcription factors which belong to basic helix-loop-helix PAS family. EPAS1 has been identified as a molecule that has close sequence similarity to HIF-1{alpha}8. Analogously to HIF-1{alpha}, EPAS1 regulates transcription of the VEGF, EPO, and Tie-2 genes (6, 8, 9).

Hypoxia has a significant effect on the growth of tumors, not only through the induction of VEGF but also through the induction of proteolytic enzyme (10). Because cell migration is a central event in tumor invasion, changes in proteolytic and fibrinolytic activity should be tightly regulated. Plasminogen activator inhibitor-1 (PAI-1) is a single-chain polypeptide with an Mr of 50 kD that inhibits urokinase plasminogen activator as well as single- and double-chain forms of tissue plasminogen activator by rapidly forming a 1:1 stoichiometric complex (11). Although the mechanism of the role of PAI-1 in tumor invasion and angiogenesis is incompletely understood, there is strong experimental evidence both in vivo and in vitro suggesting that balance between the protease and their respective inhibitors plays a central role for tumor invasion to occur. Local invasion and vascularization of transplanted malignant keratinocytes do not occur in PAI-1–deficient (PAI-1–/–) mice (12). Furthermore, recent study has revealed that PAI-1 related to the migration of cancer cells and cancer invasiveness (13).

In this study, we investigated the role of EPAS1 in the hypoxia-induced expression of the PAI-1 gene, identified an EPAS1-binding site within the human PAI-1 promoter, correlated EPAS1 binding with transcriptional activation, and demonstrated transcriptional activation by forced expression of EPAS1. Furthermore, we showed that GT-box, which acts as an Sp1-binding site, mediates the inducible expression by EPAS1. These results indicate that PAI-1 gene is the direct target of EPAS1, and that Sp1 plays a role in the response of tumor cells to hypoxic stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
O2 absorbent, Anaeropack, was purchased from Mitsubishi Gas Chemistry, Co. Ltd (Tokyo, Japan). The human PAI-1 exon 2 cDNA has previously been described (14). Polyclonal antibodies to Sp1, AP1, CREB, and Egr-1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibody for HIF-2{alpha} (EPAS1) was purchased from Novus Biologicals, Inc (Littleton, CO). Anti-myc monoclonal antibody was purchased from Invitrogen (Groningen, The Netherlands). Expression plasmid encoding EPAS1/pcDNA3 and adenoviral vector expressing EPAS1 cDNA was prepared as previously described (15). Adenoviral vector expressing dominant negative EPAS1 (Ad-phEP-1-[1–485]) has been previously described (10).

Cell Culture
The human lung adenocarcinoma cell line A549 was purchased from Riken cell bank (Tsukuba, Japan). Approximately 106 trypsinized cells were seeded into 100-mm dishes and grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Before being used for experiments, cells were starved under serum-free conditions for at least 24 h. Then cells were cultured in hypoxic chamber with Anaeropack for several hours as previously described (15). Oxygen tension within the culture medium was dropped to 53 mm Hg after 4 h and stayed at the same level even after 12 h as previously described (14).

Total RNA Isolation and Northern Blot Analysis
Total RNA was isolated from the cells by using Isogen (Nippon Gene, Tokyo, Japan). Total RNA (20 µg) from cells cultured in hypoxic condition for various time periods were electrophoresed in 1% agarose gels in the presence of 2.2 M formaldehyde, and blotted to Byodine membranes (Buckinghamshire, UK). The EPAS1 and PAI-1 cDNAs were labeled by [{alpha}-32P] dCTP using Random Primer Labeling Kit (Takara Biochemicals, Osaka, Japan). Hybridization was performed in a solution containing 5 x SSPE, 1% SDS, 5 x Denhalt's solution and 10 µg/ml salmon sperm DNA at 42°C for 16 h. The filters were washed at room temperature with 2 x SSC, 1% SDS for 10 min twice, at 42°C with 0.2 x SSC, 1% SDS for 10 min twice. The signals were detected by autoradiography.

Enzyme-Linked Immunosorbent Assay for PAI-1
The concentrations of PAI-1 produced were measured as previously described (16). The culture supernatants were collected after infection of Ad-EPAS1 or Ad-LacZ for 48 h and the absorbance was measured at 450 nm. PAI-1 production was normalized to the volume of the medium and cell number.

Plasmids
Human PAI-1 promoter-luciferase reporter genes were constructed by PCR methods using a plasmid containing an approximate 3.4-kilobase (kb) DNA insert (courtesy of Dr. D. E. Vaughan, Vanderbilt University Medical Center, Nashville, TN) as a template. The following forward primers with a KpnI site preceded by several nucleotides were used in a PCR reaction (KpnI site and additional bases are indicated by lowercase letters): PAI–262-LUC forward primer, 5'-cccggtaccAAAGGTCAAGGGAGGTTC-3'; PAI-202WT-LUC forward primer, 5'-cccggtaccCTCTTACACACGTACACACAC-3'; PAI-202HREµ-LUC forward primer, 5'-cccggtaccCTCTTACACAATTAATGACAC-3'; PAI-84-LUC forward primer, 5'-cccggtaccCAGTGAGTGGGTGGGGCTGGA-3'; PAI-84Sp1 µ-LUC forward primer, 5'-cccggtaccCAGTGAGTAAATAAGGCTGGA-3'; PAI-61-Luc forward primer, 5'-cccggtaccATGAGTTCATCTATTTCCTGC-3'; PAI-51-Luc forward primer, 5'-cccggtaccCTATTTCCTGCCCACATCTGG-3'.

The 3' primer with an XhoI site was 5'-gggctcgagCTGCAGGAATTCAGCTGCTGG-3' (XhoI site and additional bases are indicated by lowercase letters). The products were sequenced, and confirmed as human PAI-1 promoter.

Transient Transfection, Luciferase Assay, and Preparation of Cell Lysates
For transient transfection, cells were seeded at 5 x 105 cells per dish. Cells were transfected with 1 µg of reporter plasmid or if indicated 1 µg of expression plasmid by calcium phosphate precipitation method. After 24 h, transfected cells were washed twice with phosphate-buffered saline (PBS) and incubated in the presence or absence of hypoxia. After 4 h incubation, cells were harvested for luciferase assays. Cells were washed with PBS two times and lysated in 120 µl of Cell Cultured Lysate Regent (CCLR; Promega Corporation, Madison, WI). The cell lysate was scraped and centrifuged to remove cell debris. Luciferase activity was measured immediately with a Berthold Lumat LB9501 luminometer (Berthold Technologies, Bad Wildbag, Germany). Luciferase activities were expressed relative to those of pcDNA3 and promoterless plasmid PGL3BV, which set at 1.0.

Nuclear Extracts
Nuclear extracts were prepared from A549 control cells or cells exposed to hypoxia after 24 h of serum starvation. Briefly, confluent cells were washed two times with PBS and scraped and collected with PBS. After that, 5 ml of ice cold Buffer A (50 mM Hepes-KOH [pH 7.8], 420 mM KCl, 0.1 mM EDTA [pH 8.0], 5 mM MgCl2, 20% of glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM DTT, 2 µg/ml of aprotinin, leupeptin, and pepstatin, 1 mM sodium orthovanadate) was added and incubated on ice for 5 min. After centrifugation at 1,500 rpm for 5 min nuclei were Dounce homogenized. Nuclei were pelleted by centrifuging at 1,500 rpm for 5 min again and resuspended in 100–200 µl of nuclear extraction buffer C (50 mM Hepes-KOH [pH 7.8], 420 mM KCl, 0.1 mM EDTA [pH 8.0], 5 mM MgCl2, 20% of glycerol, 1 mM PMSF, 1 mM DTT, 2 µg/ml of aprotinin, leupeptin, and pepstatin, 1 mM sodium orthovanadate). After incubation and rocking at 4°C, the lysates were cleared of debris by centrifugation.

Oligonucleotide and Electrophoretic Mobility Shift Assays
The sequence of the oligonucleotides used as probes or competitors in electrophoretic mobility shift assays (EMSAs) were as follows with the consensus motif in boldface and mutations of the wild-type sequence underlined: PAI-1(–201/–181): 5'-TCTTACACACGTACACACACA-3'; Mutated PAI-1(–201/–181): 5'-TCTTACACAATTAATGACACA-3'; PAI-1(–84/–64): 5'-CAGTGAGTGGGTGGGGCTGGA-3'; Mutated PAI-1(–84/–64): 5'-CAGTGAGTAAATAAGGCTGGA-3'; Sp1: 5'-ATTCGATCGGGGCGGGGCGAGC-3'; VEGF-HRE: 5'-CCACAGTGCATACGTGGGCTCCAACAGGTCCTGTT-3'; CRE: 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; AP1: 5'-GGCTTGATGACTCAGCCGGAA-3'.

All probes were 5' end labeled with T4 polynucleotide kinase and [{gamma}-32P]dATP. Binding reactions were performed for 15 min on ice with 10–15 µg of nuclear extracts in 10 µL of Buffer C, and ~ 10,000 cpm of 32P-labeled oligonucleotides (17). Six percent or 5% polyacrylamide gels were used for EMSA. For the competition experiments, 0.5 ng of the labeled oligonucleotides were mixed with 50 ng of unlabeled competitor oligonucleotides.

Statistical Analysis
Statistical analyses were performed using Student's t test with significant difference determined as P < 0.05. Data are presented as means ± SE for at least three separate experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of PAI-1 mRNA Expression by Hypoxia in A549 Cells
To examine the effects of hypoxia on PAI-1 gene expression in cancer cells, A549 cells were exposed to hypoxia by incubating them in the chamber containing oxygen absorbents, Anaeropack, for 12 h. Northern blot showed that PAI-1 mRNA was markedly induced within 2 h and up to for 12 h by hypoxia (Figure 1A). Both of two distinct PAI-1 mRNAs (3.2 kb and 2.4 kb), which are generated from the single gene in humans by alternative splicing mechanisms (18), were induced by hypoxia. As we have previously reported, such an increase in PAI-1 mRNA levels was accompanied by an induction of the EPAS1 gene expression (15). To test directly whether EPAS1 induces the PAI-1 gene expression, we have constructed adenoviral vector expressing EPAS1 under the control of viral promoter, to which we refer as Ad-EPAS1. Infection of the A549 cells with Ad-EPAS1 but not with the control Ad-LacZ virus, which expresses ß-galactosidase, markedly induced the PAI-1 gene expression (Figure 1B). In addition, infection with Ad-EPAS1 at 10 MOI (multiplicity of infection) increased PAI-1 protein levels in the medium by 8-fold compared with infection with Ad-LacZ (Figure 1C). The same results were obtained by infecting Ad-EPAS1 at 50 MOI. These results suggest that hypoxia increases PAI-1 expression and that EPAS1 may play a role in inducing PAI-1 gene expression in response to hypoxia.



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  		Figure 1. Effects of hypoxia or EPAS1 overexpression on PAI-1 expression. (A) Induction of PAI-1 mRNA expression by hypoxia in A549 cells. Confluent cultures of A549 cells were exposed to hypoxia for indicated hours, and total cellular RNA (20 µg) prepared at the indicated times was analyzed by Northern blot analysis for PAI-1 mRNA. (B) Effect of EPAS1 overexpression on PAI-1 mRNA expression. A549 cells were infected with adenoviral vector expressing either EPAS1 or LacZ. At 48 h after infection, total cellular RNA (20 µg) was analyzed by Northern blot analysis for PAI-1 and ß-actin mRNAs. (C) Effect of EPAS1 overexpression on PAI-1 protein expression. A549 cells were infected with adenoviral vector expressing either EPAS1 or LacZ. At 48 h after infection, each culture medium was analyzed by ELISA for PAI-1. PAI-1 protein levels were expressed relative to those of Ad-LacZ 10 MOI, which set at 1.0. *P < 0.05 relative to control.

 
Induction of PAI-1 Promoter Activity by EPAS1
The promoter region of the human PAI-1 gene contains several potential consensus-binding sites for AP1, AP2, Egr-1, and Sp1 transcription factors. To define the regions that confer EPAS1-induced promoter activity, a series of 5'-deletion mutants was constructed (Figure 2). The various constructs having 5'-ends between –262 and –61, and a common 3'-end at +82 (relative to the transcriptional start site), were inserted into the luciferase reporter plasmid pGL3BV and transfected into A549 cells. PAI-262-Luc, which covers the promoter region –262 to +82, showed 8-fold induction of luciferase activity by cotransfection of EPAS1 expression vector as compared with pcDNA3-transfected cells. The basal activity of PAI-262-Luc was comparable in the presence or absence of co-transfected pcDNA3. Deletion of the promoter sequence to –84 decreased the levels of EPAS1 responsiveness to 3.5-fold. PAI-61-Luc only minimally responded. Basal promoter activity of PAI-61-Luc was significantly higher compared with that of promoterless pGL3BV, suggesting that unresponsiveness of this construct to EPAS1 does not seem to be due to the deletion of the essential sequence for basal transcription (Figure 2A).




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  		Figure 2. Upregulation of the PAI-1 promoter activity by EPAS1 expression vector. (A) A549 cells were transiently transfected with indicated 5'-deletion constructs of the PAI-1 luciferase reporter gene along with either EPAS1/pcDNA3 (solid bars) or control empty plasmid pcDNA3 (open bars). (B) Site-specific mutational analysis of the HRE sequence in PAI-1 promoter. A549 cells were transfected with indicated expression and reporter plasmids. (C) Site-specific mutational analysis of the GT-box in the PAI-1 promoter. A549 cells were transfected with indicated expression and reporter plasmids. All results represent means ± SE at least three experiments in duplicate. Luciferase activities were expressed relative to those of pcDNA3 and promoterless plasmid PGL3BV, which set at 1.0.

 
Identification of Sequence Elements Required for Transactivation by EPAS1
Based on the results of 5'-deletion analysis of PAI-1 promoter/luciferase constructs, we expected cis-acting elements within the promoter region between –262 and –84, and the region between –84 and –61 (Figure 2A). The sequence 5'-CACGTACA-3', which corresponds to a consensus hypoxia response element (HRE) (19–21), was identified at position –194. To test the contribution of this element to EPAS1 inducibility, we generated a PAI-1 promoter reporter construct carrying a mutation in the HRE within the context of PAI-202WT-Luc, which contains PAI-1 promoter sequence between –202 and +82 (Figure 2B). Results showed that induction of promoter activity by EPAS1 was significantly reduced in mutated PAI-1 promoter construct PAI-202HREµ-Luc compared with that of wild-type promoter (Figure 2B). Indeed, recent studies have revealed that this element can serve as hypoxia response element in HepG2 cells (19). In consistent with their data, we observed the hypoxic induction of PAI-202WT-Luc and mutation of HRE significantly attenuated this response (data not shown). Thus, we refer to this element as HREPAI-1. It is of importance to note that PAI-202HREµ-Luc still has a substantial ability to respond to EPAS1 expression vector (~ 3-fold induction). This result was consistent with 5'-deletion analysis and strongly supports our initial hypothesis that sequence between –84 and –61 contains sequence element which is required for responsiveness to EPAS1.

Identification of GT-Box as an EPAS1-Responsive Element
Because the region between –84 and –61 contains GT-box, which can serve as Sp1-binding sequence, we tested whether this sequence mediates the transactivation of PAI-1 promoter by EPAS1. PAI-84Sp1 µ-Luc reporter plasmid, which contains mutations known to abolish Sp1 binding, was constructed. Transfection assays in A549 cells revealed that basal transcription from mutated promoter was reduced ~ 4-fold compared with that of wild-type promoter and more importantly, transactivation by EPAS1 was completely eliminated. It is noteworthy that basal promoter activity of PAI-84Sp1 µ-Luc was significantly higher compared with that of PAI-61-Luc and PAI-51-Luc, suggesting that PAI-84Sp1 µ-Luc retains sequence required for basal transcription (Figure 2C). Although It seems that PAI-51-Luc and PAI-61-Luc constructs have some inducibility, it is unlikely that these constructs contain sequence responsive to hypoxia because PAI-84Sp1 µ-Luc has no inducibility and the EPAS1-induced luciferase activity of PAI-51-Luc and PAI-61-Luc constructs are much less than that of PAI-84-Luc construct. The effects of EPAS1 on promoter activity appear to be gene-specific because ß-actin promoter was only minimally affected by EPAS1 expression vector (data not shown).

Binding of Nuclear Factors to HREPAI-1 by Hypoxia
To examine the binding of nuclear factors to HREPAI-1 site, we performed EMSAs using the nuclear extracts prepared from A549 cells, which were cultured under either normoxic or hypoxic condition, and the 32P-labeled double-stranded oligonucleotide probe containing sequences between –201 and –181, which is designated as PAI(–201/–181). As shown in lanes 1 and 2 in Figure 3A, hypoxia-induced (B1) and constitutive (B2) DNA binding activities were detected. Both B1 and B2 binding activities were sequence-specific because wild-type sequence, but not mutated sequence (which contains mutation within HREPAI-1 site), effectively abolished B1 and B2 binding activities. Whereas unlabeled oligonucleotide containing Sp1 binding site did not compete with the probe for binding of both B1 and B2 activities, VEGF-HRE, containing HRE sequence within VEGF promoter, competed with the probe for B1 activity and B2 activity. Oligonucleotide CRE, which contains consensus CRE sequence, competed with the probe for B2 activity, and to a lesser extent for the B1 activity. As shown in Figure 3B, B1 DNA binding activity was supershifted by anti-EPAS1 antibodies. Antibodies against Sp1 had no effects. These results indicate that hypoxia-induced endogenous EPAS1 or immunologically related proteins constitute B1 complex.




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  		Figure 3. Identification of nuclear proteins binding to the HREPAI-1 in A549 cells. (A) EMSA was performed using a 32P-labeled probe PAI-1 (–201/–181), which contains the 5'-CACGTCA-3' sequence and nuclear extracts from A549 cells exposed to normoxia or hypoxia for 12 h. Various nonlabeled oligonucleotides were added as competitors including probe sequence and mutated sequences. (B) Nuclear extracts from A549 cells exposed to normoxia or hypoxia were prepared and incubated with antibodies against EPAS1 ({alpha}-EPAS1), Sp1({alpha}-Sp1), 30 min before incubation with radiolabeled PAI-1(–201/–181) probe. (C) Nuclear extracts from A549 cells infected with adenoviral vector expressing either EPAS1 or LacZ were prepared and incubated with various nonlabeled oligonucleotides before incubation with radiolabeled probe. (D) Nuclear extracts from A549 cells infected with adenoviral vector expressing either EPAS1 or LacZ were prepared and incubated with antibodies against myc ({alpha}-myc), Sp1({alpha}-Sp1), Egr-1 ({alpha}-Egr-1), and CREB ({alpha}-CREB) 30 min before incubation with radiolabeled PAI-1(–201/–181) probe.

 
Binding of EPAS1 to HREPAI-1
To ascertain that HREPAI-1 can serve as a binding site for EPAS1, we performed EMSA using the nuclear extracts from A549 transduced with either Ad-LacZ or Ad-EPAS1, and the 32P-labeled double-stranded oligonucleotide probe, PAI(–201/–181). Analogous to the experiments shown in Figure 3A, the probe again detected two distinct DNA binding activities, B1 and B2 (Figure 3C, lanes 1 and 2). DNA binding activity B2 was detected in nuclear extracts prepared from either Ad-LacZ–transduced or Ad-EPAS1–transduced cells. B1, on the other hand, was detected in nuclear extracts from Ad-EPAS1–transduced cells but not from Ad-LacZ–transduced cells. Binding activity B2 was competed by wild-type sequence but not by mutated sequence or Sp1 sequence. VEGF-HRE competed with probe for binding of both B1 and B2 activities.

Ad-EPAS1 expresses EPAS1 fused to c-myc, which allowed us to detect the expression of EPAS1 by using anti-myc antibody instead of anti-EPAS1 antibody. As shown in Figure 3D, when anti-myc antibody was included in the binding reaction, B1 but not B2 was supershifted. Neither anti-Sp1 nor anti–Egr-1 antibodies had effects on B1 binding activity. Interestingly, anti-CREB antibody interfered with B2 binding activity (Figure 3D). These results indicate that HREPAI-1 serves as a binding site for EPAS1, and CREB is the major nuclear factor that constitutes B2 binding activity.

Binding of Sp1 and Sp3 to the G+T-Motif
Because co-transfection experiments described above showed that EPAS1 transactivates PAI-1 promoter partly through the region between –84 and –61, we next asked the nuclear factors binding to GT-box between –84 and –61 within the PAI-1 promoter. EMSAs showed that two prominent DNA binding activities C1 and C2 are detected in the nuclear extracts prepared from either normoxic or hypoxic cells (Figure 4A). Both C1 and C2 binding activities were sequence-specific because wild-type PAI(–84/–64) but not mutated sequence competed with the probe for the binding. Whereas irrelevant oligonucleotide sequences CRE or AP1 had no effect on C1 and C2 complexes, Sp1 binding sequence effectively abolished the complex formation. Intensity of both C1 and C2 was comparable between normoxic and hypoxic nuclear extracts. As shown in Figure 4B, three distinct complexes (C1-a, C1-b, and C2) were detected in EMSAs using 5% PAGE instead of 6% PAGE as shown in Figure 4A. C1-a DNA binding activity was supershifted by anti-Sp1 antibodies, PEP2 and 1C6, but not by anti-Sp3 antibody. Conversely, C1-b and C2 complexes were supershifted by anti-Sp3 antibody but not by anti-Sp1 antibodies. Antibodies against CREB or Egr-1 had no effects. These results indicate that Sp1 or immunologically related proteins constitute C1-a complex and Sp3 or immunologically related proteins constitute the C1-b and C2 complexes.



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  		Figure 4. Identification of nuclear proteins binding to GT-box in A549 cells. (A) EMSA was performed using a 32P-labeled probe containing the GT-box and nuclear extracts from A549 cells exposed to normoxia or hypoxia for 12 h. Various nonlabeled oligonucleotides were added as a competitors including wild-type sequence and mutated sequences. (B) Nuclear extract from A549 cells exposed to normoxia or hypoxia were prepared and incubated with antibodies against Sp1({alpha}-Sp1), CREB ({alpha}-CREB), Egr-1 ({alpha}-Egr-1), and Sp3 ({alpha}-Sp3) 30 min before incubation with radiolabeled PAI-1 probe.

 
Inhibition of Hypoxia-Mediated PAI-1 Induction by Dominant-Negative EPAS1
It has been reported that phEP-1-(1–485), which expresses truncated form of EPAS1 protein which contains amino acid 1–485, acts as a dominant-negative mutant of wild-type EPAS1 (10). To determine whether EPAS1 is required for hypoxia-mediated increase in PAI-1 mRNA levels, we performed Northern blot analysis of the RNA from either Ad-LacZ– or Ad-phEP-1-(1–485)–transduced cells. In cells transduced with 50 MOI of Ad-phEP-1-(1–485), hypoxic induction of PAI-1 mRNA expression was almost completely inhibited, whereas 1.9-fold increases was observed in Ad-LacZ–transduced cells (P < 0.05). These results indicate that EPAS1 plays a significant role in mediating the induction of PAI-1 mRNA expression under hypoxic conditions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to understand the molecular mechanisms of the hypoxia-induced PAI-1 gene expression. The present study provides several lines of evidence indicating that the PAI-1 gene is the direct target of transcription factor EPAS1 in human adenocarcinoma A549 cells. Data supporting this conclusion are as follows. First, adenovirus expressing EPAS1 dramatically induced the endogenous PAI-1 mRNA expression. Second, EPAS1 transactivated PAI-1 promoter through the sequence containing HRE and G+T-rich sequence, which act as EPAS1-binding site and Sp1/Sp3-binding site, respectively. Third, overexpression of dominant-negative form of EPAS1, phEP-1-(1–485), significantly attenuated the hypoxia-induced PAI-1 mRNA expression.

To date, EPAS1 has been described to regulate the expression of the genes encoding Epo (6), VEGF (10), Tie-2 (9), and KDR (8) in hypoxia. Because these genes are relevant to the erythropoiesis and vascular development, EPAS1-inducible genes contribute to the maintenance of oxygen homeostasis. In this line, the increased expression of PAI-1 in hypoxic cells suggested to us a role for PAI-1 in the hypoxic response of the cells. It is well established that besides its role in thrombus formation, PAI-1 plays a crucial role in the control of matrix degradation by interfering with the conversion of plasminogen to plasmin. In addition, PAI-1 has been demonstrated to induce the progression of tumor tissue. Thus, an induction of PAI-1 by hypoxia is considered to be a mechanism underlying the hypoxia-mediated tumor progression.

Two points revealed by our study are particularly remarkable. The first is that not only EPAS1 but also CREB bind to the HREPAI-1. Recently, Kvietikova and coworkers have reported that CREB and its homologous protein ATF-1 bind constitutively to the HIF-1 DNA recognition site (22). By extensive characterization of the binding sequence of ATF-1/CREB, they demonstrated that ATF-1/CREB binding site does indeed match the HIF-1 DNA recognition site; the core CRE motif overlaps the core HIF-1 motif ACGT, which is in conserved in all HIF-1 binding sites found so far. The HREPAI-1 sequence 5'-CACGTACA-3' identified in our study also contains this motif. Beitner-Johnson and colleagues revealed that hypoxia strongly induced phosphorylation of CREB and EPAS1 in PC12 cells (23). Kvietikova and coworkers have raised the possibility that cAMP-dependent phosphorylation of ATF-1/CREB may be a critical component of HIF-1–mediated hypoxia-inducible gene expression (24). In fact, our recent study has shown that VEGF expression is induced by cAMP-dependent protein kinase (PKA) activator under hypoxic condition. However, we also found that PKA activator inhibited the hypoxia-mediated increase in PAI-1 expression (data not shown). Thus, PKA signaling and phosphorylation of ATF-1/CREB may have a different effect on HIF-1/EPAS1–mediated transcription depend upon the context of the motif. The physiologic role of the overlapping CRE and HIF-1 binding site deserves further study.

A second noteworthy finding of our experiment is that EPAS1 induces PAI-1 promoter activity partly through GT-box, which is constitutively bound by Sp1/Sp3. Functional reporter gene studies demonstrated that disruption of the Sp1/Sp3 binding sequence within the PAI-84-Luc context completely abolished the inducible expression by EPAS1. This finding obviously leaves open the question of how EPAS1 increases the activity of transcription through Sp1-binding site. Although the ubiquitous transcription factor Sp1 has long been considered to exclusively play a role in basal transcription, the increasing amount of evidence indicates that Sp1 regulates the tissue-specific gene expression, and also Sp1 mediates the inducible expression of a variety of genes in response to extracellular stimuli such as TGF-ß1 (25), phorbol 12-myristate 13-acetate (PMA) (26), and inflammatory cytokines (17). Particularly, recent studies have demonstrated that nuclear hormone receptors, including retinoic acid (27) and androgen receptors (28), increase the gene expression by modulating the Sp1 activity. Shimada and colleagues have reported that physical interaction between RAR/RXR and Sp1 enhances Sp1 binding activity to some G+C-motifs possibly through the conformation change of Sp1 (29). We are currently testing whether analogous mechanisms account for the Sp1-mediated transactivation of PAI-1 gene in response to hypoxia. In the present study, we performed transient transfection assays of the PAI-1 promoter construct to identify the EPAS1 response element. We did not use the reference plasmid such as ß-galactosidase reporter gene to normalize for transfection efficiency. Thus, we cannot formally rule out the possibility that differences in transfection efficiency might contribute to our findings.

Notably, we showed that endogenous EPAS1 binds to the HREPAI-1 site in response to hypoxia, as determined by supershift assays using anti-EPAS1 antibody (Figure 3B). This finding is somewhat contradictory to the report by Park and coworkers (30), who demonstrated that endogenous EPAS1, which has been referred to as HIF-2{alpha} in their study, does not translocate into the nucleus and remains transcriptionally inactive under hypoxic conditions in mouse embryonic fibroblasts. Although the reason for these discrepant results remains to be determined, it is most likely that the role of EPAS1 in a hypoxic response varies depending upon the cell types, because EPAS1 is not upregulated in response to hypoxia in mouse embryonic fibroblasts (30), whereas we have observed robust induction of EPAS1 in A549 cells (15).

In conclusion, the data presented in this study document an effect of EPAS1 on the gene encoding PAI-1 which regulates matrix degradation and thrombus formation, and provide a new insight into the pathophysiologic function of hypoxia-induced, tissue-restricted transcription factor EPAS1. Identification of the PAI-1 gene as a target of EPAS1 in cancer cells could be of great importance in understanding the molecular basis for tumor-associated coagulation activity as well as for tumor angiogenesis.



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  		Figure 5. Dominant-negative EPAS1 attenuated the induction of PAI-1 mRNA expression by hypoxia. (A) A549 cells were transduced with either Ad-phEP-1-(1–485) or Ad-LacZ at 50 MOI. At 48 h after transfection, cells were exposed to hypoxia for 12 h and total cellular RNA (20 µg) was analyzed by Northern blot analysis for PAI-1 mRNA. (B) Bar graphs show PAI-1 mRNA levels normalized by intensity of 28S RNA. PAI-1 mRNA levels were expressed relative to those of Ad-LacZ-transduced cells under normoxic condition. *P < 0.05 relative to normoxia.

 

   Acknowledgments
 
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sport, and Culture of Japan, and by a grant from Japan Cardiovascular Foundation (to M.K.).

Received in original form August 9, 2003

Received in final form March 13, 2004


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gidding, S. S., and J. A. Stockman, III. 1988. Erythropoietin in cyanotic heart disease. Am. Heart J. 116:128–132.[CrossRef][Medline]
  2. Shweiki, D., A. Itin, D. Soffer, and E. Keshet. 1992. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359:843–845.[CrossRef][Medline]
  3. Helfman, T., and V. Falanga. 1993. Gene expression in low oxygen tension. Am. J. Med. Sci. 306:37–41.[Medline]
  4. Wang, G. L., B. H. Jiang, E. A. Rue, and G. L. Semenza. 1995. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92:5510–5514.[Abstract/Free Full Text]
  5. Forsythe, J. A., B. H. Jiang, N. V. Iyer, F. Agani, S. W. Leung, R. D. Koos, and G. L. Semenza. 1996. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16:4604–4613.[Abstract]
  6. Ema, M., S. Taya, N. Yokotani, K. Sogawa, Y. Matsuda, and K. Y. Fujii. 1997. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc. Natl. Acad. Sci. USA 94:4273–4278.[Abstract/Free Full Text]
  7. Elvert, G., S. Lanz, A. Kappel, I. Flamme. 1999. mRNA cloning and expression studies of the quail homologue of HIF-2alpha. Mech. Dev. 87:193–197.[CrossRef][Medline]
  8. Tian, H., S. L. McKnight, and D. W. Russell. 1997. Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev. 11:72–82.[Abstract/Free Full Text]
  9. Maemura, K., C. M. Hsieh, M. K. Jain, S. Fukumoto, M. D. Layne, Y. Liu, S. Kourembanas, S. F. Yet, M. A. Perrella, and M. E. Lee. 1999. Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain. J. Biol. Chem. 274:31565–31570.[Abstract/Free Full Text]
  10. Graham, C. H., J. Forsdike, C. J. Fitzgerald, and S. Macdonald-Goodfellow. 1999. Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression. Int. J. Cancer 80:617–623.[CrossRef][Medline]
  11. Morton, P. A., D. A. Owensby, B. E. Sobel, and A. L. Schwartz. 1989. Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2: modulation by plasminogen activator inhibitor type 1. J. Biol. Chem. 264:7228–7235.[Abstract/Free Full Text]
  12. Bajou, K., A. Noel, R. D. Gerard, V. Masson, N. Brunner, C. Holst-Hansen, M. Skobe, N. E. Fusenig, P. Carmeliet, D. Collen, and J. M. Foidart. 1998. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat. Med. 4:923–928.[CrossRef][Medline]
  13. Chazaud, B., R. Ricoux, C. Christov, A. Plonquet, R. K. Gherardi, and G. Barlovatz-Meimon. 2002. Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations. Am. J. Pathol. 160:237–246.[Abstract/Free Full Text]
  14. Uchiyama, T., M. Kurabayashi, Y. Ohyama, T. Utsugi, N. Akuzawa, M. Sato, S. Tomono, S. Kawazu, and R. Nagai. 2000. Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 20:1155–1161.[Abstract/Free Full Text]
  15. Sato, M., T. Tanaka, T. Maeno, Y. Sando, T. Suga, Y. Maeno, H. Sato, R. Nagai, and M. Kurabayashi. 2002. Inducible expression of endothelial PAS domain protein-1 by hypoxia in human lung adenocarcinoma A549 cells: role of Src family kinases-dependent pathway. Am. J. Respir. Cell Mol. Biol. 26:127–134.[Abstract/Free Full Text]
  16. Watanabe, A., M. Kurabayashi, M. Arai, K. Sekiguchi, and R. Nagai. 2001. Combined effect of retinoic acid and basic FGF on PAI-1 gene expression in vascular smooth muscle cells. Cardiovasc. Res. 51:151–159.[Abstract/Free Full Text]
  17. Tanaka, T., H. Kanai, K. Sekiguchi, Y. Aihara, T. Yokoyama, M. Arai, T. Kanda, R. Nagai, and M. Kurabayashi. 2000. Induction of VEGF gene transcription by IL-1 beta is mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes. J. Mol. Cell. Cardiol. 32:1955–1967.[CrossRef][Medline]
  18. Fattal, P. G., and J. J. Billadello. 1993. Species-specific differential cleavage and polyadenylation of plasminogen activator inhibitor type 1 hnRNA. Nucleic Acids Res. 21:1463–1466.[Abstract/Free Full Text]
  19. Fink, T., A. Kazlauskas, L. Poellinger, P. Ebbesen, and V. Zachar. 2002. Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor-1. Blood 99:2077–2083.[Abstract/Free Full Text]
  20. Kietzmann, T., U. Roth, and K. Jungermann. 1999. Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes. Blood 94:4177–4185.[Abstract/Free Full Text]
  21. Kietzmann, T., A. Samoylenko, U. Roth, and K. Jungermann. 2003. Hypoxia-inducible factor-1 and hypoxia response elements mediate the induction of plasminogen activator inhibitor-1 gene expression by insulin in primary rat hepatocytes. Blood 101:907–914.[Abstract/Free Full Text]
  22. Kvietikova, I., R. H. Wenger, H. H. Marti, and M. Gassmann. 1997. The hypoxia-inducible factor-1 DNA recognition site is cAMP-responsive. Kidney Int. 51:564–566.[Medline]
  23. Beitner-Johnson, D., R. T. Rust, T. C. Hsieh, and D. E. Millhorn. 2001. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells. Cell. Signal. 13:23–27.[CrossRef][Medline]
  24. Kvietikova, I., R. H. Wenger, H. H. Marti, and M. Gassmann. 1995. The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site. Nucleic Acids Res. 23:4542–4550.[Abstract/Free Full Text]
  25. Poncelet, A. C., and H. W. Schnaper. 2001. Sp1 and Smad proteins cooperate to mediate transforming growth factor-beta 1-induced alpha 2(I) collagen expression in human glomerular mesangial cells. J. Biol. Chem. 276:6983–6992.[Abstract/Free Full Text]
  26. Noe, V., C. Alemany, M. Nicolas, and C. Ciudad. 2001. Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells. Eur. J. Biochem. 268:3163–3173.[Medline]
  27. Akiyama, H., T. Tanaka, T. Maeno, H. Kanai, Y. Kimura, S. Kishi, and M. Kurabayashi. 2002. Induction of VEGF gene expression by retinoic acid through Sp1-binding sites in retinoblastoma Y79 cells. Invest. Ophthalmol. Vis. Sci. 43:1367–1374.[Abstract/Free Full Text]
  28. Lu, S., G. Jenster, and D. E. Epner. 2000. Androgen induction of cyclin-dependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. Mol. Endocrinol. 14:753–760.[Abstract/Free Full Text]
  29. Shimada, J., Y. Suzuki, S. J. Kim, P. C. Wang, M. Matsumura, and S. Kojima. 2001. Transactivation via RAR/RXR-Sp1 interaction: characterization of binding between Sp1 and GC box motif. Mol. Endocrinol. 15:1677–1692.[Abstract/Free Full Text]
  30. Park, S., A. M. Dadak, V. H. Haase, L. Fontana, A. J. Giaccia, and R. S. Johnson. 2003. Hypoxia-induced gene expression occurs solely through the action of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}): role of cytoplasmic trapping of HIF-2a. Mol. Cell. Biol. 23:4959–4971.[Abstract/Free Full Text]



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