bcl-2 Induction of Urokinase Plasminogen Activator Receptor Expression in Human Cancer Cells through Sp1 Activation
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m308938200
ISSN1083-351X
AutoresDaniela Trisciuoglio, Angela Iervolino, Antonio Candiloro, Gabriella Fibbi, Maurizio Fanciulli, Uwe Zangemeister‐Wittke, Gabriella Zupi, Donatella Del Bufalo,
Tópico(s)Cancer-related molecular mechanisms research
ResumoWe have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway. We have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway. Angiogenesis is a fundamental process required for tumor growth, invasion, and metastasis and is strongly induced by hypoxia. In fact, several cell types including tumor cells, macrophages, and endothelial cells respond to hypoxia by producing angiogenic factors, fibrinolytic factors, and adhesion molecules involved in pathologic angiogenesis (1Desbaillets I. Diserens A.C. de Tribolet N. Hamou M.F. Van Meir E.G. Oncogene. 1999; 8: 1447-1456Crossref Scopus (100) Google Scholar, 2Harmey J.H. Dimitriadis E. Kay E. Redmond H.P. Bouchier-Hayes D. Ann. Surg. Oncol. 1998; 5: 271-278Crossref PubMed Scopus (172) Google Scholar, 3Karakurum M. Shreeniwas R. Chen J. Pinsky D. Yan S.D. Anderson M. Sunouchi K. Major J. Hamilton T. Kuwabara K. J. Clin. Invest. 1994; 93: 1564-1570Crossref PubMed Scopus (322) Google Scholar, 4Suzuma K. Takagi H. Otani A. Honda Y. Invest. Ophthalmol. Vis. Sci. 1998; 39: 1028-1035PubMed Google Scholar, 5Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4163) Google Scholar). Four sequential steps can be distinguished during angiogenesis: the degradation of the basement membrane and interstitial matrix, endothelial cell migration, endothelial cell proliferation, and the formation of tubular structures with a lumen and a new basement membrane (6Folkman J. Cancer Res. 1986; 46: 467-473PubMed Google Scholar). Three of these steps critically depend on proteolytic activity generated by the matrix metalloproteinases and the plasminogen activator/plasmin system. In particular, the role of urokinase plasminogen activator receptor (uPAR) 1The abbreviations used are: uPAR, urokinase plasminogen activator receptor; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor; ADR, adriamycin; MTR, mitramycin A; CHX, cycloheximide; ERK, extracellular signal-regulated kinase; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay. in tumor cell invasion and migration and in the formation of new microvascular structures has been largely demonstrated (7Min 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, 8Evans C.P. Elfman F. Parangi S. Conn M. Cunha G. Shuman M.A. Cancer Res. 1997; 57: 3594-3599PubMed Google Scholar, 9Kroon M.E. Koolwijjk P. van Goor H. Weidle U.H. Collen A. van der Pluijm G. van Hinsbergh V.W. Am. J. Pathol. 1999; 154: 1731-1742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Angiogenesis is also controlled by alterations in oncogene and tumor suppressor gene expression (10Kieser A. Welch H.A. Brandner G. Marme D. Kolch W. Oncogene. 1994; 9: 963-969PubMed Google Scholar, 11Ueba T. Nosaka T. Takahashi J.A. Shibata F. Florkiewicz R.Z. Vogelstein B. Oda Y. Kikuchi H. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9009-9013Crossref PubMed Scopus (138) Google Scholar, 12Mukhopadhyay D. Tsiokas L. Zhou X.M. Foster D. Brugge J.S. Sukhatme V.P. Nature. 1995; 375: 577-581Crossref PubMed Scopus (540) Google Scholar, 13Rak J. Yu J.L. Klement G. Kerbel R.S. J. Invest. Dermatol. Symp. Proc. 2000; 5: 24-33Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 14Krieg M. Haas R. Brauch H. Acker T. Flamme I. Plate K.H. Oncogene. 2000; 19: 5435-5443Crossref PubMed Scopus (324) Google Scholar). In this context, we previously demonstrated that the bcl-2 oncogene increases in vitro and in vivo angiogenesis in two different tumor histotypes (15Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (119) Google Scholar, 16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar). In particular, we found an increase in the level of vascular endothelial growth factor (VEGF) when breast carcinoma (15Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (119) Google Scholar) and melanoma (16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar) cells overexpressing Bcl-2 were exposed to hypoxic conditions. We also demonstrated that Bcl-2 overexpression in human melanoma cells enhances hypoxia-induced VEGF mRNA stability and promoter activation (16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar) and that treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity (17Del Bufalo D. Trisciuoglio D. Scarsella M. Zangemeister-Wittke U. Zupi G. Oncogene. 2003; 22: 8441-8447Crossref PubMed Scopus (53) Google Scholar). The involvement of bcl-2 in angiogenesis of prostate carcinoma (18Fernandez A. Udagawa T. Schwesinger C. Beecken W. Achilles-Gerte E. McDonnell T. D'Amato R. J. Natl. Cancer Inst. 2001; 93: 208-213Crossref PubMed Scopus (87) Google Scholar) and microvascular endothelial cells has also been described (19Nor J.E. Christensen J. Liu J. Peters M. Mooney D.J. Strieter R.M. Polverini P.J. Cancer Res. 2001; 61: 2183-2188PubMed Google Scholar). Thus, the leading property of bcl-2 to inhibit apoptosis is associated with its ability to induce angiogenesis. The aim of this study was to evaluate the role of Bcl-2 overexpression in the regulation of uPAR expression in human tumors. Since a minimal promoter region required for the basal transcription of the human uPAR gene has been demonstrated to contain GC-rich proximal sequences that are specifically bound by the transcription factor Sp1 (20Soravia E. Grebe A. De Luca P. Helin K. Suh T.T. Degen J.L. Blasi F. Blood. 1995; 86: 624-635Crossref PubMed Google Scholar, 21Allgayer H. Wang H. Gallick G.E. Crabtree A. Mazar A. Jones T. Kraker A.J. Boyd D.D. J. Biol. Chem. 1999; 274: 18428-18437Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we also investigated whether the regulation of uPAR by Bcl-2 overexpression is due to the effect of Bcl-2 on Sp1 expression, phosphorylation, and DNA binding activity. To address the molecular mechanism that mediates the effect of Bcl-2, the role of extracellular signal-regulated kinase (ERK) signaling in Sp1 transcriptional activity was investigated. Our results demonstrate that Bcl-2 overexpression in hypoxia increases Sp1 expression and activity through ERK signaling with the result of enhanced uPAR transcription and expression. Cell Lines and Cell Cultures—M14 human melanoma cells, MCF7 ADR human breast carcinoma cells resistant to adriamycin (ADR), and Bcl-2-overexpressing clones, previously obtained after transfection (16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar, 22Del Bufalo D. Biroccio A. Leonetti C. Zupi G. FASEB J. 1997; 11: 947-953Crossref PubMed Scopus (120) Google Scholar), were maintained in RPMI medium (Invitrogen) containing 10% fetal calf serum, 2 mm l-glutamine, and antibiotics. Hypoxic Treatment—Parental cells and bcl-2 transfectants were seeded and grown for 24 h in complete medium. Then cells were incubated for 24 h in serum-free medium under normoxic or hypoxic conditions as reported previously (15Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (119) Google Scholar). Cells were harvested, counted, and used for protein preparation and total RNA extraction. Antisense Treatment—These studies were performed with antisense 2009, a 20-mer phosphorothioate oligonucleotide directed against the coding region (codons 141-147) of the bcl-2 messenger RNA. The antisense 2009 and the scrambled control sequence were previously described (23Ziegler A. Luedke G.H. Fabbro D. Altmann K.H. Stahel R.A. Zangemeister-Wittke U. J. Natl. Cancer Inst. 1997; 89: 1027-1036Crossref PubMed Scopus (215) Google Scholar). Oligonucleotides were delivered into cells in the form of complexes with the transfection reagent Lipofectin (Invitrogen). Lipofectin at 100 μg/ml was allowed to complex with oligonucleotides, and this solution was further diluted to the desired concentration in serum and antibiotic-free medium prior to addition to the cells. M14 parental cells and bcl-2 transfectants were incubated for 24 h in the presence of 300 nm oligonucleotides and Lipofectin and then exposed to hypoxia for 24 h in serum-free medium. ELISA and Western blot analyses were performed at the end of treatment. ELISA Analysis—To determine the amount of uPAR in the cell lysates and in tumor xenografts, an ELISA kit was used. The sensitivity of the uPAR assays was 0.1 ng/ml (American Diagnostica inc., Greenwich, CT). To evaluate in vitro uPAR expression, cells were exposed to normoxia or hypoxia for 24 h in the absence or presence of 100 or 200 nm mitramycin A (MTR; Sigma) or were treated with bcl-2 antisense oligonucleotide as reported above. Then cells were harvested, washed twice in PBS, resuspended, and counted. Cells were then centrifuged, and the pellet was lysed in lysis buffer (partial cell lysate). The combined trypsin/EDTA treatment leaves a complex material, referred to as surface-adherent material (24Del Rosso M. Pedersen N. Fibbi G. Pucci M. Dini G. Anichini E. Blasi F. Exp. Cell Res. 1992; 203: 427-434Crossref PubMed Scopus (23) Google Scholar), on the culture dish, which primarily represents the cell focal contacts and is enriched with uPAR. Thus, the partial cell lysate was replaced in the culture dish for 1 h at 4 °C to lyse the surface-adherent material-associated uPAR (total cell lysate) as well. The total cell lysate was stirred for 12 h at 4 °C to allow an optimal solubilization of the cell extract and centrifuged at 10,000 × g for 60 min at 4 °C to separate cell debris. The material was frozen and maintained at -80 °C. For in vivo analysis of uPAR expression, tumor xenografts (22Del Bufalo D. Biroccio A. Leonetti C. Zupi G. FASEB J. 1997; 11: 947-953Crossref PubMed Scopus (120) Google Scholar) (100-200 mg) was homogenized in 500 μl of lysis buffer. Homogenates were centrifuged (20 min at 16,000 × g), and the supernatant was used for uPAR protein analysis. Immunoprecipitation Assay—Nuclear extracts were prepared as described previously (16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar). Extracts from ∼3 × 106 cells (100 μl containing 200 μg of nuclear proteins) were diluted to 1 ml in whole-cell extraction buffer (25 mm HEPES, pH 7.4, 1 mm MgCl2, 100 mm KCl, 0.1 mm EDTA, 10% glycerol, 1 mm dithiothreitol, and inhibitor proteases and phosphatases) and incubated with 3 μl of polyclonal rabbit antibody to Sp1 (Pep2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 °C with gentle shaking. After the addition of 50 μl of protein A-agarose beads (Santa Cruz Biotechnology), the suspension was incubated for another 2 h at 4 °C. The beads were pelleted by centrifugation, washed three times with 1.5 ml of cold extraction buffer, and resuspended in 50 μl of 2× SDS sample buffer. After the suspension was heated to 95 °C for 10 min, 20-μl samples were resolved on denaturing SDS-polyacrylamide gels and transferred to membranes. Anti-human anti-phosphoserine and anti-phosphothreonine antibodies (Chemicon International, Temacula, CA) were used at dilutions of 1:500 and 1:100, respectively. Western Blot Analysis—For analysis of Sp1 protein expression, equal amounts of nuclear extracts (35 μg of protein) were fractionated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Monoclonal antibody against Sp1 (clone 1C6; BD Pharmigen Technical, Franklin Lakes, NJ) was used at a dilution of 1:200. For the Sp1 phosphorylation study, nuclear extracts obtained after 24 h of hypoxic treatment were treated for 60 min at 37 °C with 10 units of calf intestinal phosphatase (New England Biolabs) before Western blot analysis. For the Sp1 stability study, cycloheximide (CHX; Calbiochem) at a dose of 10 μg/ml was added after 24 h of hypoxic treatment, and the cells were further incubated under hypoxia for varying times ranging from 30 min to 4 h. To evaluate uPAR and Bcl-2 expression and ERK1/ERK2 expression and phosphorylation, whole-cell protein extracts were electrophoresed on an 11% SDS-polyacrylamide gel. Anti-human uPAR mouse monoclonal antibody (American Diagnostica Inc.) was used at a 1:400 dilution. Anti-human Bcl-2 mouse monoclonal antibody (clone 124; Dako s.p.a., Milan, Italy) was used at a 1:200 dilution. To detect phospho-ERKs, the phospho-p44/42 mitogen-activated protein kinase (Thr202/Tyr204) antibody (Cell Signaling Technology Inc.) was used at a 1:1000 dilution; to probe total ERK1 and ERK2, the p44/42 mitogen-activated protein kinase polyclonal antibody (Cell Signaling Technology Inc.) was used at a 1:1000 dilution. Immunoreactive bands were visualized using horseradish peroxidase-coupled goat anti-rabbit immunoglobulin and the ECL detection system (Amersham Biosciences). To check the amount of proteins transferred to the nitrocellulose membranes, β-actin or heat shock protein (Hsp70/72) was used as control and detected by an anti-β-actin polyclonal antibody (Santa Cruz Biotechnology) or anti-Hsp70/72 monoclonal antibody (Calbiochem) at a 1:1000 dilution. Densitometric analysis was performed after Western blot analysis. Western blot analysis of ERK1/ERK2 and Sp1 expression and phosphorylation was performed in the absence or presence of ERK inhibitor UO126 (10 μm; Calbiochem). Northern Blot Analysis—Total RNA was prepared, and Northern blot analysis was performed as described previously (15Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (119) Google Scholar). A 585-bp fragment of the plasmid specific for human uPAR kindly provided by Dr. F. Blasi (25Roldan A.L. Cubellis M.V. Masucci M.T. Behrendt N. Lund L.R. Dano K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (541) Google Scholar), a 717-bp fragment of human Sp1 kindly provided by Dr. R. C. Simmen (26Simmen R.C. Zhang X.L. Zhang D. Wang Y. Michel F.J. Simmen F.A Mol. Cell. Endocrinol. 2000; 159: 159-170Crossref PubMed Scopus (12) Google Scholar), and a probe for glyceraldehyde-3-phosphate dehydrogenase (27Scotlandi K. Serra M. Manara M.C. Lollini P.L. Maurici D. Del Bufalo D. Baldini N. Int. J. Cancer. 1994; 58: 95-101Crossref PubMed Scopus (14) Google Scholar) were used. Promoter Activity—For transient transfection, 3 × 105 cells were seeded into 60-mm dishes, and 24 h later each dish was transfected with 6 μg of pGL3-mouse uPAR promoter vector kindly provided by Dr. A. Maity (28Maity A. Solomon D. Exp. Cell Res. 2000; 255: 250-257Crossref PubMed Scopus (33) Google Scholar). Cells were cotransfected in triplicates with an internal control PEQ-176 plasmid (1.5 μg) using a calcium-phosphate method (Promega Italia, Milan, Italy). Twenty-four h later, half of the dishes were subjected to hypoxia, and the other half were kept under normoxic conditions. Samples were collected 24 h after the induction of hypoxia and analyzed for luciferase and β-galactosidase activity. Relative luciferase expression was determined as a ratio of β-galactosidase activity. The mean of five independent experiments was calculated for each condition. Electrophoretic Mobility Shift Assay (EMSA)—Cells were exposed for 24 h to hypoxia in the presence or absence of the Sp1 inhibitor MTR (100 or 200 nm) or ERK inhibitor UO126 (10 μm). Then nuclear proteins were prepared as described for immunoprecipitation, and EMSA was performed as previously described (29Ricca A. Biroccio A. Del Bufalo D. Mackay A.R. Santoni A. Cippitelli M. Int. J. Cancer. 2000; 86: 188-196Crossref PubMed Scopus (95) Google Scholar). Oligonucleotides were purchased from Invitrogen. The following double-stranded oligomer-containing Sp1 consensus sequence was used as labeled probes (104 cpm) or cold competitor (100 ng): 5′-ATTCGATCGGGGCGGGGCGAGC-3′ (30Zannetti A. Del Vecchio S. Carriero M.V. Fonti R. Franco P. Botti G. D'Aiuto G. Stoppelli M.P. Salvatore M. Cancer Res. 2000; 60: 1546-1551PubMed Google Scholar). To analyze the specificity of DNA-binding complexes, supershift with Sp1 (Pep2; Santa Cruz Biotechnology) and Sp3 (D-20; Santa Cruz Biotechnology) antibodies was performed by preincubating nuclear extracts with the antibody (2 μg) for 30 min prior to the addition of labeled DNA probe. Statistical Analysis—Multiple comparisons were performed by the Student-Newman-Keuls test after demonstration of significant differences among medians by nonparametric variance analysis according to Kruskal-Wallis. Bcl-2 Overexpression Increases uPAR Protein Expression under Hypoxic Conditions—We previously demonstrated the ability of Bcl-2 to synergize with hypoxia to increase angiogenesis in melanoma and breast carcinoma cells (15Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (119) Google Scholar, 16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar). Since uPAR plays an important role in angiogenesis (7Min 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, 8Evans C.P. Elfman F. Parangi S. Conn M. Cunha G. Shuman M.A. Cancer Res. 1997; 57: 3594-3599PubMed Google Scholar, 9Kroon M.E. Koolwijjk P. van Goor H. Weidle U.H. Collen A. van der Pluijm G. van Hinsbergh V.W. Am. J. Pathol. 1999; 154: 1731-1742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), we evaluated whether uPAR was also involved in the Bcl-2-induced angiogenesis. For this purpose, two Bcl-2-overexpressing clones, previously obtained after transfection of the M14 parental melanoma line (16Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar), were used. We first determined the levels of cell-associated uPAR protein in the M14 cell line, the MN8 control clone, and two bcl-2 transfectants (MB5 and MB6) grown under normoxic (Fig. 1A, white columns) and hypoxic (black columns) conditions for 24 h. As shown in Fig. 1A, the difference in uPAR amounts measured in the control lines and bcl-2-transfected clones were not statistically significant when cells were grown under normoxic conditions. In hypoxia, the M14 parental line and the MN8 control clone did not show significant differences, neither when compared with normoxic conditions nor between each other. On the contrary, the two bcl-2 transfectants grown in hypoxia showed significantly higher levels of uPAR protein than in normoxia (∼2-fold increase). To confirm the role of bcl-2 in the regulation of uPAR expression, Bcl-2 protein expression was down-regulated both in the M14 parental cells and the MB5 and MB6 bcl-2 transfectants using 2009 antisense oligonucleotide (23Ziegler A. Luedke G.H. Fabbro D. Altmann K.H. Stahel R.A. Zangemeister-Wittke U. J. Natl. Cancer Inst. 1997; 89: 1027-1036Crossref PubMed Scopus (215) Google Scholar) before cells were exposed to hypoxia for 24 h. Western blot analysis of Bcl-2 expression (Fig. 1B) showed that bcl-2 antisense oligonucleotide treatment decreased Bcl-2 protein levels by about 45 and 60% in parental cells and in the two transfectants, respectively. Performing ELISA assays, also a decrease of the level of uPAR protein in parental cells by about 40% was observed. The reduction in the two bcl-2 transfectants was about 60-70% (Fig. 1C). Down-regulation of uPAR expression in bcl-2 transfectants by bcl-2 antisense oligonucleotide treatment was also confirmed by Western blot. As revealed in Fig. 1D, antisense treatment induced about 60% inhibition of uPAR expression in both clones. The scrambled sequence oligonucleotide did not affect Bcl-2 or uPAR expression. These results confirm a link between Bcl-2 and uPAR expression. Bcl-2 Overexpression Increases uPAR mRNA Expression and Promoter Activity under Hypoxic Conditions—Because uPAR expression can be modulated by transcriptional regulation (28Maity A. Solomon D. Exp. Cell Res. 2000; 255: 250-257Crossref PubMed Scopus (33) Google Scholar), we were interested to know the mechanism through which Bcl-2 synergizes with hypoxia to induce uPAR expression. To this end, we determined whether transactivation of the uPAR promoter differently occurred in control cells and the bcl-2 transfectants (Fig. 2A). Transient transfections were performed using the uPAR promoter coupled to the luciferase-reporter gene (28Maity A. Solomon D. Exp. Cell Res. 2000; 255: 250-257Crossref PubMed Scopus (33) Google Scholar), and control and Bcl-2-overexpressing cells were exposed to normoxia or hypoxia for 24 h. As shown in Fig. 2A, under normoxic conditions, uPAR promoter activity was low and similar in all cells tested, regardless of the level of Bcl-2 protein. Exposure to hypoxia induced a slight increase in promoter activity in control cells, whereas an increase of about 4-fold was observed in Bcl-2-overexpressing cells. In the MN8 control clone, the uPAR promoter response was comparable with that of M14 both in normoxia and hypoxia (data not shown). Since the steady-state level of mRNA also represents a control point for uPAR expression (28Maity A. Solomon D. Exp. Cell Res. 2000; 255: 250-257Crossref PubMed Scopus (33) Google Scholar), we examined the effect of Bcl-2 on uPAR at the transcriptional level. Northern blot analysis of uPAR mRNA was performed after exposure of M14 parental line, MN8 control clone, and two bcl-2 transfectants (MB5 and MB6) to hypoxic conditions for 24 h (Fig. 2B). An increase of about 2-fold of uPAR mRNA expression was observed in both bcl-2 transfectants, compared with the M14 parental line and MN8 control clone exposed to hypoxia for 24 h. Sp1 Protein Stability and Phosphorylation Is Increased in bcl-2 Transfectants Exposed to Hypoxia—A minimal promoter region, containing GC-rich proximal sequences that are specifically bound by the transcription factor Sp1, has been identified for the basal transcriptional activity of the human uPAR gene (20Soravia E. Grebe A. De Luca P. Helin K. Suh T.T. Degen J.L. Blasi F. Blood. 1995; 86: 624-635Crossref PubMed Google Scholar, 21Allgayer H. Wang H. Gallick G.E. Crabtree A. Mazar A. Jones T. Kraker A.J. Boyd D.D. J. Biol. Chem. 1999; 274: 18428-18437Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The regulation of Sp1-dependent transcription can be affected by changes in the overall amount of Sp1 or in transactivation activity due to biochemical modifications such as phosphorylation (31Black A.R. Black J.D. Azizkhan-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (898) Google Scholar, 32Black A.R. Jensen D. Lin S.Y. Azizkhan J.C. J. Biol. Chem. 1999; 274: 1207-1215Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 33Rohlff C. Ahmad S. Borellini F. Lei J. Glazer R.I. J. Biol. Chem. 1997; 272: 21137-21141Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 34Reisinger K. Kaufmann R. Gille J. J. Cell Sci. 2003; 116: 225-238Crossref PubMed Scopus (97) Google Scholar, 35Fojas de Borja P. Collins N.K. Du P. Azizkhan-Clifford J. Mudryj M. EMBO J. 2001; 20: 5737-5747Crossref PubMed Scopus (110) Google Scholar, 36Alroy I. Soussan L. Seger R. Yarden Y. Mol. Cell. Biol. 1999; 19: 1961-1972Crossref PubMed Scopus (82) Google Scholar). To address this issue, we analyzed Sp1 protein expression, stability, and phosphorylation in M14 parental cells, the MN8 control clone, and the MB5 and MB6 bcl-2 transfectants grown in normoxia or exposed to hypoxia for 24 h. To evaluate the effect of Bcl-2 on Sp1 mRNA expression, Northern blot analysis was performed 24 h after exposure to hypoxic conditions (Fig. 3A). Similar levels of Sp1 were observed between the control lines and the bcl-2 transfectants grown in hypoxia. The expression of Sp1 was also analyzed at the protein level. A representative Western blot analysis of Sp1 protein in nuclear extracts is shown in Fig. 3B. As expected, Sp1 protein migrates as two bands with molecular masses of 95 and 105 kDa. The two species are the result of differential post-translational modification of the Sp1 polypeptide, corresponding to the unphosphorylated (lower band) and phosphorylated (upper band) protein (37Schewe D.M. Leupold J.H. Boyd D.D. Lengyel E.R. Wang H. Gruetzner K.U. Schildberg F.W. Jauch K.W. Allgayer H. Clin. Cancer Res. 2003; 9: 2267-2276PubMed Google Scholar). Whereas no differences in Sp1 expression between control and Bcl-2-overexpressing cells were observed under normoxic conditions (data not shown), after 24 h of hypoxia, an increase in both bands was observed in the bcl-2 transfectants compared with parental cells. In particular, densitometric analysis revealed an increase of about 2- and 3-fold in the lower and the phosphorylated higher molecular weight forms of Sp1, respectively. To assess whether Bcl-2 overexpression stabilizes the Sp1 protein, we monitored the levels of Sp1 protein after blocking de novo protein synthesis by CHX treatment. To this end, the M14 parental line and a representative bcl-2 transfectant (MB5) were exposed to hypoxia for 24 h and then treated, under hypoxia, with 10 μg/ml CHX for varying times ranging from 30 min to 4 h (Fig. 3C). Western blot analysis showed a rapid decay of Sp1 protein in M14 parental cells within 30 min and Sp1 was hardly detectable 2 h after the start of CHX treatment. In contrast, Sp1 protein levels in Bcl-2-overexpressing cells remained stable during the first 2 h of CHX treatment and then gradually but slowly decreased about 40% within 4 h. The results obtained with the MB6 clone
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