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• W1999307515 abstract "Hepatocyte growth factor (HGF) plays a major role in the pathogenesis of a variety of human epithelial tumors including papillary carcinoma of the thyroid. Previous reports demonstrated that HGF, acting through the Met receptor, repressed thrombospondin-1 (TSP-1) expression. To study the mechanisms by which HGF down-regulated TSP-1 expression, we transiently transfected a panel of deleted human TSP-1 promoter reporter plasmids into papillary thyroid carcinoma cells. We identified a region between –1210 and –1123 bp relative to the transcription start site that is responsive to HGF treatment and harbors a cAMP-responsive element (CRE) at position –1199 (TGACGTCC). Overexpression of various members of the CRE-binding protein family identified activating transcription factor-1 (ATF-1) as the transcription factor responsible for HGF-induced repression of TSP-1 promoter activity. This inhibition was associated with a concomitant increase in the abundance of nuclear ATF-1 protein. Gel shift and antibody supershift studies indicated that ATF-1 was involved in DNA binding to the TSP-1-CRE site. Finally, we utilized small hairpin RNA to target ATF-1 and showed that these small interfering RNA constructs significantly inhibited ATF-1 expression at both the RNA and the protein level. ATF-1 knockdown prevented HGF-induced down-regulation of TSP-1 promoter activity and protein expression and also reduced HGF-dependent tumor cell invasion. Taken together, our results indicate that HGF-induced down-regulation of TSP-1 expression is mediated by the interaction of ATF-1 with the CRE binding site in the TSP-1 promoter and that this transcription factor plays a crucial role for tumor invasiveness in papillary carcinoma of the thyroid triggered by HGF. Hepatocyte growth factor (HGF) plays a major role in the pathogenesis of a variety of human epithelial tumors including papillary carcinoma of the thyroid. Previous reports demonstrated that HGF, acting through the Met receptor, repressed thrombospondin-1 (TSP-1) expression. To study the mechanisms by which HGF down-regulated TSP-1 expression, we transiently transfected a panel of deleted human TSP-1 promoter reporter plasmids into papillary thyroid carcinoma cells. We identified a region between –1210 and –1123 bp relative to the transcription start site that is responsive to HGF treatment and harbors a cAMP-responsive element (CRE) at position –1199 (TGACGTCC). Overexpression of various members of the CRE-binding protein family identified activating transcription factor-1 (ATF-1) as the transcription factor responsible for HGF-induced repression of TSP-1 promoter activity. This inhibition was associated with a concomitant increase in the abundance of nuclear ATF-1 protein. Gel shift and antibody supershift studies indicated that ATF-1 was involved in DNA binding to the TSP-1-CRE site. Finally, we utilized small hairpin RNA to target ATF-1 and showed that these small interfering RNA constructs significantly inhibited ATF-1 expression at both the RNA and the protein level. ATF-1 knockdown prevented HGF-induced down-regulation of TSP-1 promoter activity and protein expression and also reduced HGF-dependent tumor cell invasion. Taken together, our results indicate that HGF-induced down-regulation of TSP-1 expression is mediated by the interaction of ATF-1 with the CRE binding site in the TSP-1 promoter and that this transcription factor plays a crucial role for tumor invasiveness in papillary carcinoma of the thyroid triggered by HGF. Hepatocyte growth factor (HGF) 3The abbreviations used are: HGF, hepatocyte growth factor; TSP-1, thrombospondin-1; TPC-1, thyroid papillary carcinoma cells; ATF-1, activating transcription factor-1; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding; CREM, cAMP-response element modulator; ICER, inducible cAMP early repressor; USF, upstream stimulatory factor; LUC, luciferase; RT, reverse transcription; shRNA, small hairpin RNAs; shCTL, shRNA control vector; shATF-1, shRNA ATF-1 expression vector., also known as scatter factor-1, is a mesenchymal- or stromal-derived multifunctional cytokine/growth factor (1Stoker M. Perryman M. J. Cell Sci. 1985; 77: 209-223Crossref PubMed Google Scholar, 2Nakamura T. Nishizawa T. Hagiya M. Seki T. Shimonishi M. Sugimura A. Tashiro K. Shimizu S. Nature. 1989; 342: 440-443Crossref PubMed Scopus (1982) Google Scholar) that acts predominantly on cells of epithelial origin in an endocrine and/or paracrine fashion (3Stoker M. Gherardi E. Perryman M. Gray J. Nature. 1987; 327: 239-242Crossref PubMed Scopus (1131) Google Scholar, 4Sonnenberg E. Meyer D. Weidner K.M. Birchmeier C. J. Cell Biol. 1993; 123: 223-235Crossref PubMed Scopus (643) Google Scholar) through binding the high affinity c-Met tyrosine kinase receptor (5Bottaro D.P. Rubin J.S. Faletto D.L. Chan A.M. Kmiecik T.E. Vande Woude G.F. Aaronson S.A. Science. 1991; 251: 802-804Crossref PubMed Scopus (2090) Google Scholar). In vitro, HGF-Met interaction elicits a complex and specific genetic program leading to epithelial cell dissociation (“scattering”) (3Stoker M. Gherardi E. Perryman M. Gray J. Nature. 1987; 327: 239-242Crossref PubMed Scopus (1131) Google Scholar), migration and extracellular matrix invasion (3Stoker M. Gherardi E. Perryman M. Gray J. Nature. 1987; 327: 239-242Crossref PubMed Scopus (1131) Google Scholar, 6Scarpino S. Stoppacciaro A. Colarossi C. Cancellario F. Marzullo A. Marchesi M. Biffoni M. Comoglio P.M. Prat M. Ruco L.P. J. Pathol. 1999; 189: 570-575Crossref PubMed Scopus (31) Google Scholar), proliferation and protection from apoptosis (7Eccles N. Ivan M. Wynford-Thomas D. Mol. Cell. Endocrinol. 1996; 117: 247-251Crossref PubMed Scopus (32) Google Scholar, 8Fan S. Wang J.A. Yuan R.Q. Rockwell S. Andres J. Zlatapolskiy A. Goldberg I.D. Rosen E.M. Oncogene. 1998; 17: 131-141Crossref PubMed Scopus (131) Google Scholar), acquisition of polarity, and branching morphogenesis (9Montesano R. Matsumoto K. Nakamura T. Orci L. Cell. 1991; 67: 901-908Abstract Full Text PDF PubMed Scopus (1089) Google Scholar, 10Santos O.F. Moura L.A. Rosen E.M. Nigam S.K. Dev. Biol. 1993; 159: 535-548Crossref PubMed Scopus (89) Google Scholar, 11Nusrat A. Parkos C.A. Bacarra A.E. Godowski P.J. Delp-Archer C. Rosen E.M. Madara J.L. J. Clin. Investig. 1994; 93: 2056-2065Crossref PubMed Scopus (200) Google Scholar). In vivo, HGF-Met signaling clearly plays a role in normal cellular processes during embryonic development, and many of these normal activities have been implicated in tumor progression and metastasis. HGF is also a potent inducer of angiogenesis (12Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1204) Google Scholar) by regulating positively the pro-angiogenic factor vascular endothelial growth factor and negatively the anti-angiogenic protein thrombospondin-1 (TSP-1), an effect that was recently described in a breast cancer cell line (13Zhang Y.W. Su Y. Volpert O.V. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12718-12723Crossref PubMed Scopus (303) Google Scholar) and in thyroid carcinoma cells (14Scarpino S. Di Napoli A. Taraboletti G. Cancrini A. Ruco L.P. J. Pathol. 2005; 205: 50-56Crossref PubMed Scopus (15) Google Scholar). Papillary thyroid carcinoma represents the most common malignancy of the thyroid gland. Because Met is not expressed or only expressed at low level in the normal thyroid, aberrant expression and autocrine or mutational activation of c-Met receptor in papillary cancer suggest a possible role for the HGF-Met axis in tumor development and progression (15Prat M. Narsimhan R.P. Crepaldi T. Nicotra M.R. Natali P.G. Comoglio P.M. Int. J. Cancer. 1991; 49: 323-328Crossref PubMed Scopus (281) Google Scholar, 16Di Renzo M.F. Olivero M. Ferro S. Prat M. Bongarzone I. Pilotti S. Belfiore A. Costantino A. Vigneri R. Pierotti M.A. et al.Oncogene. 1992; 7: 2549-2553PubMed Google Scholar, 17Belfiore A. Gangemi P. Costantino A. Russo G. Santonocito G.M. Ippolito O. Di Renzo M.F. Comoglio P. Fiumara A. Vigneri R. J. Clin. Endocrinol. Metab. 1997; 82: 2322-2328Crossref PubMed Scopus (77) Google Scholar). TSP-1, the first protein to be recognized as a naturally occurring inhibitor of angiogenesis, is a large multifunctional extracellular matrix glycoprotein that affects tumor growth and metastases through modulation of angiogenesis and other stromal biological functions (18Hsu S.C. Volpert O.V. Steck P.A. Mikkelsen T. Polverini P.J. Rao S. Chou P. Bouck N.P. Cancer Res. 1996; 56: 5684-5691PubMed Google Scholar). However, the involvement of TSP-1 in tumor progression remains complex and controversial since both stimulatory (19Tuszynski G.P. Gasic T.B. Rothman V.L. Knudsen K. Gasic G.J. Cancer Res. 1987; 47: 4130-4133PubMed Google Scholar, 20Castle V. Varani J. Fligiel S. Prochownik E.V. Dixit V. J. Clin. Investig. 1991; 87: 1883-1888Crossref PubMed Scopus (71) Google Scholar) and inhibitory (21Zabrenetzky V. Harris C.C. Steeg P.S. Roberts D.D. Int. J. Cancer. 1994; 59: 191-195Crossref PubMed Scopus (202) Google Scholar, 22Castle V.P. Dixit V.M. Polverini P.J. Lab. Investig. 1997; 77: 51-61PubMed Google Scholar) effects in angiogenesis have been described and assigned to up-regulation of matrix-degrading enzymes and their inhibitors. TSP-1 gene expression is controlled by many factors including cytokines (23Morandi V. Cherradi S.E. Lambert S. Fauvel-Lafeve F. Legrand Y.J. Legrand C. J. Cell. Physiol. 1994; 160: 367-377Crossref PubMed Scopus (35) Google Scholar), growth factors (24Hugo C. Pichler R. Meek R. Gordon K. Kyriakides T. Floege J. Bornstein P. Couser W.G. Johnson R.J. Kidney Int. 1995; 48: 1846-1856Abstract Full Text PDF PubMed Scopus (80) Google Scholar), oncogenes (25Mettouchi A. Cabon F. Montreau N. Vernier P. Mercier G. Blangy D. Tricoire H. Vigier P. Binetruy B. EMBO J. 1994; 13: 5668-5678Crossref PubMed Scopus (100) Google Scholar, 26Slack J.L. Bornstein P. Cell Growth & Differ. 1994; 5: 1373-1380PubMed Google Scholar, 27Volpert O.V. Pili R. Sikder H.A. Nelius T. Zaichuk T. Morris C. Shiflett C.B. Devlin M.K. Conant K. Alani R.M. Cancer Cell. 2002; 2: 473-483Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 28Watnick R.S. Cheng Y.N. Rangarajan A. Ince T.A. Weinberg R.A. Cancer Cell. 2003; 3: 219-231Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), oncosuppressor genes (29Salnikow K. Cosentino S. Klein C. Costa M. Mol. Cell. Biol. 1994; 14: 851-858Crossref PubMed Scopus (63) Google Scholar, 30Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Science 1994. 1994; 265: 1582-1584Google Scholar, 31Dejong V. Degeorges A. Filleur S. Ait-Si-Ali S. Mettouchi A. Bornstein P. Binetruy B. Cabon F. Oncogene. 1999; 18: 3143-3151Crossref PubMed Scopus (57) Google Scholar, 32Schwarte-Waldhoff I. Volpert O.V. Bouck N.P. Sipos B. Hahn S.A. Klein-Scory S. Luttges J. Kloppel G. Graeven U. Eilert-Micus C. Hintelmann A. Schmiegel W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9624-9629Crossref PubMed Scopus (238) Google Scholar, 33Wen S. Stolarov J. Myers M.P. Su J.D. Wigler M.H. Tonks N.K. Durden D.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4622-4627Crossref PubMed Scopus (208) Google Scholar, 34Gutierrez L.S. Suckow M. Lawler J. Ploplis V.A. Castellino F.J. Carcinogenesis. 2003; 24: 199-207Crossref PubMed Scopus (56) Google Scholar, 35Soula-Rothhut M. Coissard C. Sartelet H. Boudot C. Bellon G. Martiny L. Rothhut B. Exp. Cell Res. 2005; 304: 187-201Crossref PubMed Scopus (36) Google Scholar), phorbol 12-myristate 13-acetate (36Kim S.A. Um S.J. Kang J.H. Hong K.J. Mol. Cell. Biochem. 2001; 216: 21-29Crossref PubMed Scopus (24) Google Scholar), nickel compounds (37Salnikow K. Wang S. Costa M. Cancer Res. 1997; 57: 5060-5066PubMed Google Scholar), glucose (38Wang S. Skorczewski J. Feng X. Mei L. Murphy-Ullrich J.E. J. Biol. Chem. 2004; 279: 34311-34322Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), and hypoxia (39Phelan M.W. Forman L.W. Perrine S.P. Faller D.V. J. Lab. Clin. Med. 1998; 132: 519-529Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Characterization and structural analysis of the human TSP-1 promoter region (40Donoviel D.B. Framson P. Eldridge C.F. Cooke M. Kobayashi S. Bornstein P. J. Biol. Chem. 1988; 263: 18590-18593Abstract Full Text PDF PubMed Google Scholar) has shown that control of TSP-1 gene expression relies on multiple cis-acting elements and transactivators that regulate the transcriptional rate of this gene (41Hennessy S.W. Frazier B.A. Kim D.D. Deckwerth T.L. Baumgartel D.M. Rotwein P. Frazier W.A. J. Cell Biol. 1989; 108: 729-736Crossref PubMed Scopus (54) Google Scholar, 42Framson P. Bornstein P. J. Biol. Chem. 1993; 268: 4989-4996Abstract Full Text PDF PubMed Google Scholar). Thus far, the transcriptional regulation of TSP-1 gene expression by HGF has not yet been investigated. The present study was undertaken to examine the molecular mechanisms by which HGF down-regulates TSP-1 expression in the human papillary thyroid carcinoma cell line TPC-1. We identified activating transcription factor 1 (ATF-1) binding to the ATF/CREB-responsive element as a negative regulator of TSP-1 expression. Functional analyses revealed that silencing of gene expression at the mRNA and protein level by small hairpin RNAs (shRNA) directed against ATF-1 reversed HGF-dependent down-regulation of TSP-1 expression and inhibited HGF-induced TPC-1 cell invasion. Cell Line and Materials—The human thyroid papillary carcinoma cell line TPC-1 was kindly provided by Dr. J. A. Fagin (University of Cincinnati, Cincinnati, OH). Cells were routinely grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mm glutamine, 5% fetal bovine serum, and 1% penicillin/streptomycin (Invitrogen) in a humidified CO2 incubator at 37 °C. Human recombinant HGF was from Calbiochem. Rabbit polyclonal anti-TSP-1 was used as described previously (35Soula-Rothhut M. Coissard C. Sartelet H. Boudot C. Bellon G. Martiny L. Rothhut B. Exp. Cell Res. 2005; 304: 187-201Crossref PubMed Scopus (36) Google Scholar). Mouse monoclonal antibody raised against recombinant human ATF-1 (clone C41-5.1), which does not cross-react with other members of the ATF/CREB family, was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-USF1 and anti-USF2 antibodies were obtained as described (43Viollet B. Lefrançois-Martinez A.M. Henrion A. Kahn A. Raymondjean M. Martinez A. J. Biol. Chem. 1996; 271: 1405-1415Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Mouse monoclonal anti-β-actin antibody was purchased from Sigma. Goat anti-mouse and goat anti-rabbit IgG-peroxidase conjugates were from Amersham Biosciences (Orsay, France). Expression Vectors, Transfections, and Luciferase Assays—Firefly luciferase (LUC) reporter plasmids pGL3-Basic containing various fragments of the human TSP-1 promoter (–1290/+750, –1210/+750, –1123/+750, –767/+750, –267/+750, –71/+750, and –43/+750) were from Dr. P. Bornstein (University of Washington, Seattle, WA) and from Dr. M. Okamoto (Kyushu University, Fukuoka, Japan). ATF-1 and ATF-1-S63A, with a serine to alanine mutation at position 63 subcloned into p3XFLAG-CMV, were a kind gift from Dr. R. Pryves (Columbia University, New York, NY). Plasmids pSV-CREB, pSV-CREMα, pSV-CREMβ, and pSV-ICER were obtained from Prof. P. Sassone-Corsi (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch-Strasbourg, France). Human USF1, USF2, and transdominant negative USF1 and USF2 mutants (ΔbTDU1 and ΔbTDU2, respectively), cloned into pCR3 vector, were a kind gift from Dr. B. Viollet (Institut Cochin, Paris, France) (44Lefrançois-Martinez A.M. Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The –96/+11 proximal bp fragment of the L-type pyruvate kinase promoter (–96PK-LUC) and a construct that comprises three copies of the L4 E-box binding USF1 and USF2 (i.e. (L4)3-96PK-LUC) were obtained from Dr. B. Viollet and used as described (45Vaulont S. Puzenat N. Levrat F. Cognet M. Kahn A. Raymondjean M. J. Mol. Biol. 1989; 209: 205-219Crossref PubMed Scopus (113) Google Scholar, 46Gourdon L. Lou D.Q. Raymondjean M. Vasseur-Cognet M. Kahn A. FEBS Lett. 1999; 459: 9-14Crossref PubMed Scopus (17) Google Scholar). TranSilent shRNA plasmid expression vectors for ATF-1 (shATF-1) and control (shCTL) were purchased from Panomics (Redwood City, CA). Cells grown to 70–80% confluency in 96-well microplates (PerkinElmer) were co-transfected with 0.2 μg of luciferase plasmid DNA, 50 ng of pRL-TK vector (Promega) together with the relevant expression vectors (i.e. ATF-1, ATF-1-S63A, CREB, cAMP-response element modulator α (CREMα), CREMβ, inducible cAMP early repressor (ICER), USF1, USF2, ΔbTDU1, ΔbTDU2, shATF-1, and shCTL) using Lipofectamine 2000 reagent following the protocol provided by the supplier (Invitrogen). At 24 h after transfection, cells were washed with phosphate-buffered saline and lysed with 100 μl of FireLite dual reporter lysis buffer (PerkinElmer Life Sciences, Courtaboeuf, France), and luciferase activities were measured with the PerkinElmer TopCount microplate counter. All firefly luciferase values were normalized to Renilla luciferase levels. In RNA interference studies using shRNA-based plasmid constructs, cells grown to 50% confluency in 10-cm dishes were transfected with shATF-1 or shCTL (4 μg) using Lipofectamine 2000 reagent. After 72 h of transfection, cells were used for immunoblot and RT-PCR analysis. Matrigel invasion assay and luciferase activity assays were performed after 48 h of transfection. RT-PCR—Semiquantitative RT-PCR was performed on total RNA prepared by an RNeasy Mini kit (Qiagen, Hilden, Germany). The purified RNA was suspended in diethylpyrocarbonate-treated water. One microgram of total RNA was reverse-transcribed using avian myeloblastosis virus reverse transcriptase and oligo(dT)15 primer from Promega. Amplification was performed using PCR Master Mix and TaqDNA polymerase from Promega. The optimal reaction conditions were 30 cycles of 45 s at 94 °C, 1 min at 55 °C for TSP-1 and ATF-1, at 60 °C for S26, and 1 min at 72 °C. Specific primer pairs were: for TSP-1, forward, 5′-CTC AGG AAC AAA GGC TGC TC-3′, reverse, 5′-ACT CCT GAA TGT GGC AGG TC-3′; for S26, forward, 5′-GTG CGT GCC CAA GGA TAA GG-3′, reverse, 5′-ATG GGC TTT GGT GGA GGT CG-3′; for ATF-1, forward, 5′-CAA CCT TCA GCA GTT CA-3′, reverse, 5′-TTT CTG CCC CGT GTA TCT TC-3′. Western Blot Analysis—Cultured cells were made quiescent by serum starvation for at least 24 h before incubation with human recombinant HGF. Conditioned media were collected and centrifuged at 5,000 × g for 10 min, and total cell protein was measured using bicinchoninic acid microassay from Pierce. Cells were then washed with ice-cold phosphate-buffered saline, lysed in buffer containing 25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mm EDTA, 1 mm sodium Na3VO4, 50 mm NaF, 1 mm 4-(2-aminoethyl)-benzenesulfonylfluoride, and 1 μg/ml each aprotinin, leupeptin, pepstatin, and antipain, placed on ice for 20 min, and then centrifuged at 14,000 × g for 15 min at 4 °C. Equal amounts of protein (whole cell extract, nuclear extracts, conditioned media) were resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions and then transferred to nitrocellulose membranes. The blot was stained with Ponceau S to confirm equal loading of proteins and then probed with the indicated antibodies. Immunoblots were developed using appropriate secondary horseradish peroxidase-coupled antibodies and an enhanced chemiluminescence (ECL) kit (Amersham Biosciences). To ensure equal loading of proteins from nuclear extracts, the membranes were stripped and reprobed with an anti-β-actin antibody under the same conditions as described above. Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared as described (47Guillemot L. Levy A. Raymondjean M. Rothhut B. J. Biol. Chem. 2001; 276: 39394-39403Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Nuclear pellets were resuspended in 20 mm HEPES, 25% glycerol, 0.5 m NaCl, 1.5 mm MgCl2, 0.5 mm EDTA. Protease inhibitors were introduced into each buffer. The amount of protein was determined using the BCA protein quantification kit (Interchim, Montluçon, France). Double-stranded oligonucleotide probes corresponding to a CRE consensus sequence (Promega, 5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′), to the sequence of the human TSP-1 CRE site (Invitrogen, 5′-GGAGCGAGGCGGCTGACGTCCCATCCCGAA-3′ (bp –1212 to –1183)), and to the sequence of the human TSP-1 CRE mutated site (Invitrogen, 5′-GCGGCTGACGGGCCATCCCGAA-3′ (bp –1204 to –1183)) were prepared by annealing sense and antisense specific sequences. Binding reactions for gel shift assays were performed as described (48Antonicelli F. Brown D. Parmentier M. Drost E.M. Hirani N. Rahman I. Donaldson K. MacNee W. Am. J. Physiol. 2004; 286: L1319-L1327Crossref PubMed Scopus (67) Google Scholar). Probes were prepared by labeling 3.5 pmol of the double-stranded oligonucleotides with [γ-32P]ATP and T4 polynucleotide kinase (Invitrogen). Five micrograms of each nuclear protein was diluted in 20 μl of gel retardation buffer (10 mm Tris/HCl, 100 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, 0.5 mm MgCl2, 10% glycerol). 32P-labeled double-stranded oligonucleotide probes (5,000 cpm) were incubated for 20 min with nuclear extracts in the presence of the nonspecific DNA sequence poly(dI-dC)·poly(dI-dC). In competition experiments, the nuclear extract was incubated with a 100-fold molar excess of the appropriate unlabeled specific and nonspecific competitor oligonucleotides. After incubation at room temperature, electrophoresis of the different samples was carried out on nondenaturating polyacrylamide gels for 3 h in 0.5× Tris borate/EDTA. Gels were dried under vacuum and exposed to Kodak XAR film overnight. In supershift studies, a mouse monoclonal anti-ATF-1 or a nonspecific IgG was preincubated with the crude nuclear extract for 3 h at 4°C before the addition of the labeled probe. Cell Invasion Assay—Invasion of TPC-1 cells in vitro was investigated using modified Boyden chambers (tissue culture-treated, 6.5-mm diameter, 8-μm pore size; Transwell Costar, Brumath, France). Cells were trypsinized, suspended (1 × 106 cells/ml) in serum-free Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin, and seeded onto membranes coated with 30 μg/cm2 Matrigel (extracellular matrix gel, Sigma). Purified TSP-1 or anti-TSP-1 antibody (clone 3F355, 30 μg/ml) were added to the upper chamber of the Transwell units. Complete medium containing HGF (50 ng/ml) was added to the lower compartment of the chamber. After 24 h at 37 °C, cells on the upper membrane surface were removed by careful wiping with a cotton swab, and the filters were fixed by treatment with methanol and stained with 0.5% crystal violet solution for 15 min. Invasive cells adhering to the undersurface of the filter were then counted (five high power fields/chamber) using an inverted microscope (Nikon Eclipse). Identification of Binding Sites That Mediate Down-regulation of the Human TSP-1 Promoter by HGF—HGF-induced down-regulation of TSP-1 expression has been reported in various cells including thyroid papillary carcinoma (13Zhang Y.W. Su Y. Volpert O.V. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12718-12723Crossref PubMed Scopus (303) Google Scholar, 14Scarpino S. Di Napoli A. Taraboletti G. Cancrini A. Ruco L.P. J. Pathol. 2005; 205: 50-56Crossref PubMed Scopus (15) Google Scholar). We performed RT-PCR analysis on total cellular RNA from HGF (50 ng/ml)-treated TPC-1 cells using specific primers for TSP-1 and S26. As shown in Fig. 1A, the PCR products of TSP-1 and S26 were amplified to the appropriate sizes of 604 and 250 bp, respectively, and HGF incubation induced a down-regulation in TSP-1 mRNA level. To determine the relationship of TSP-1 protein expression to TSP-1 mRNA expression after HGF stimulation, Western blot analysis was performed in culture supernatants (Fig. 1B). These results demonstrated that HGF caused a reduction in TSP-1 production at the protein level. To determine whether HGF was capable of down-regulating TSP-1 promoter activity, we transiently transfected TPC-1 cells with the –1290/+750-bp human TSP-1 promoter fused to the luciferase reporter gene. HGF treatment resulted in a repression of TSP-1 promoter activity in a dose- (Fig. 1C) and time-dependent (Fig. 1D) manner. The location of potential binding sites for transcription factors in the human TSP-1 promoter has been published (41Hennessy S.W. Frazier B.A. Kim D.D. Deckwerth T.L. Baumgartel D.M. Rotwein P. Frazier W.A. J. Cell Biol. 1989; 108: 729-736Crossref PubMed Scopus (54) Google Scholar, 42Framson P. Bornstein P. J. Biol. Chem. 1993; 268: 4989-4996Abstract Full Text PDF PubMed Google Scholar) and is shown in Fig. 1E. To identify sequences that mediate down-regulation of TSP-1 promoter activity by HGF, functional analysis was carried out using a series of luciferase reporter gene plasmids containing various lengths of the human TSP-1 5′-flanking sequences into TPC-1 cells. As shown in Fig. 1F, HGF decreased luciferase activity in the –1290/+750 and –1210/+750 plasmid constructs, respectively. When the sequence between nucleotides –1210/+750 and –1123/+750 was deleted, the inhibitory effect of HGF was lost, indicating that the region between nucleotides –1210/+750 to –1123/+750 contains a negative regulatory element required for transcriptional inactivation of TSP-1. A potential CRE binding site (TGACGTCC) for ATF/CREB transcription factors is located within this sequence. Fig. 1, E and F, also showed that further deletion of the TSP-1 promoter between nucleotides –1123/+750 and –767/+750 resulted in a supplementary increase in luciferase activity. This region harbors a putative E-box motif (CAGATG) able to bind USF proteins that might also participate in the negative regulation by HGF. ATF-1 Transcription Factor Is Involved in the Down-regulation of TSP-1 Expression by HGF—A number of transcription factors specifically bind to the CRE sequence as homo- and heterodimers via a carboxyl-terminal basic domain leucine zipper (bZip) motif (49Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2547) Google Scholar). To identify the proteins that could be involved in the regulation of TSP-1 promoter, TPC-1 cells were co-transfected with different plasmids encoding various members of the ATF/CREB family such as CREB, CREM isoforms, ATF-1, and ICER. Fig. 2A showed that only ATF-1 overexpression was able to inhibit basal TSP-1 promoter activity as well as to enhance the inhibitory effect elicited by HGF, and the observed effect was dose-dependent (data not shown). Expression of all the other members stimulated TSP-1 promoter activity. To test whether ATF-1 phosphorylation is required for HGF repression of the TSP-1 promoter, we used a dominant negative ATF-1 phosphorylation site mutant (S63A). Transfection with this construct had no effect on HGF-induced inhibition of the TSP-1 promoter reporter gene (data not shown). We than tested whether HGF had an effect on the abundance of ATF-1 protein. Western blot analysis with nuclear extracts from TPC-1 cells showed that HGF treatment caused accumulation of the ATF-1 transcription factor in a time-dependent manner (Fig. 2B). These findings suggest that ATF-1 plays a major role in HGF-mediated down-regulation of TSP-1 promoter activity in TPC-1 cells. Our results corroborate with earlier studies that demonstrated the involvement of ATF-1 in the regulation of TSP-1 in nickel-transformed cells (37Salnikow K. Wang S. Costa M. Cancer Res. 1997; 57: 5060-5066PubMed Google Scholar). USF1 and USF2 Do Not Participate in HGF-induced Down-regulation of TSP-1 Expression—USF1 and USF2, originally identified as activators of the adenovirus major late promoter (50Sawadogo M. Roeder R.G. Cell. 1985; 43: 165-175Abstract Full Text PDF PubMed Scopus (720) Google Scholar), are ubiquitously expressed basic helix-loop-helix/leucine zipper transcription factors (51Qyang Y. Luo X. Lu T. Ismail P.M. Krylov D. Vinson C. Sawadogo M. Mol. Cell. Biol. 1999; 19: 1508-1517Crossref PubMed Scopus (148) Google Scholar). They recognize and bind to DNA with an E-box motif as either homodimers or heterodimers. USF1 and USF2 regulate the expression of several genes (52Heckert L.L. Sawadogo M. Daggett M.A. Chen J.K. Mol. Endocrinol. 2000; 14: 1836-1848Crossref PubMed Google Scholar, 53Kennedy H.J. Viollet B. Rafiq I. Kahn A. Rutter G.A. J. Biol. Chem. 1997; 272: 20636-20640Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) including smooth muscle cell-expressed genes (54Johnson A.D. Owens G.K. Am. J. Physiol. 1999; 276: C1420-C1431Crossref PubMed Google Scholar). More recently, Wang et al. (38Wang S. Skorczewski J. Feng X. Mei L. Murphy-Ullrich J.E. J. Biol. Chem. 2004; 279: 34311-34322Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) showed that repression of glucose-induced TSP-1 gene expression by cGMP protein kinase involved down-regulation of USF2 protein levels, resulting in a decrease in USF2 binding to a single region (–932 to –915) in the human TSP-1 promoter. To check whether or not USF proteins are involved in the negative regulation of TSP-1 promoter by HGF, co-transfection assays were performed with either the –1290/+750-bp or the –1123/+750-bp human TSP-1-LUC promoters and plasmids encoding USF1, USF2, or the dominant negative ΔbTDU1 and ΔbTDU2 constructs. Results from Fig. 3, A and B, showed that both USF1 and USF2 stimulated basal and HGF-dependent TSP-1 promoter activity, whereas the dominant negative forms were either without effect (Fig. 3A) or inhibitory (Fig. 3B) when compared with control pCR3 plasmid. To prove the specificity of the USF plasmids, co-transfection experiments were also performed with a positive control construct that carries three copies of the L4 E-box ligated to the –96/+54 proximal bp of the L-type pyruvate kinase promoter. Fig. 3C showed that USF1 and USF2 can act as transactivators of the (L4)3-96PK-LUC promoter, whereas both USF1 and USF2 mutants repressed transcription. We then tested whether HGF had an effect on the amount of USF proteins. Western blot analysis with nuclear extracts from TPC-1 cells showed that HGF treatment had no impact on the amount of USF2 transcription factor level when compared with β-actin (Fig. 3D). Identical results were obtained when USF1 protein was analyzed (data not shown). In conclusion to our experimental data, we can rule out the possibility that USF1 and/or USF2 are involved in HGF-dependent repression of TSP-1 gene expression. Identification of ATF-1 Binding to the CRE—To determine whether the transcription factor ATF-1 was able to bind specifically to the CRE motif, we performed electrophoretic mobility shift assays with labeled double-stranded oligonucleotide probes encoding the CRE consensus, TSP-1-CRE, or TSP-1-CREmut (TSP-1 · CRE mutated) sequence and nuclear extracts prepared from control and HGF-stimulated TPC-1 cells. Results from Fig. 4A indicate that incubation of nuclear extracts from HGF-treated cells with the CRE consensus probe resulted in an increase in a DNA-protein complex (lane 2) when compared with control (lane 1, arrow). The complex was competed away by an excess of unlabeled CRE consensus oligonucleotide (lane 3) but not by excess of a nonspecific sequence (lane 4). A similar increase in DNA-protein complex was also observed when the TSP-1-CRE oligonucleotide was used as a probe (compare lanes 5 and 6, arrow). In contrast, when an oligonucleotide containing a mutated TSP-1-CRE site was used as a probe, the specific DNA-protein complex disappeared (lanes 7 and 8). These results indicate that in TPC-1 cells, one complex corresponds to a transcription factor that specifically binds to the CRE within the TSP-1 promoter. To identify whether ATF-1 is the transcription factor that binds the promoter, we performed electrophoretic mobility shift assays with nuclear extracts from HGF-stimulated cells, using either CRE consensus or TSP-1-CRE-labeled oligonucleotide probes and an antibody against human ATF-1, which does not cross-react with other members of the ATF/CREB family. Two supershifts were observed with the anti-ATF-1 antibody (Fig. 4B, lane 2, asterisks) but not with an irrelevant antibody (lane 3). Similar results were also obtained with the TSP-1-CRE probe (Fig. 4B, lanes 5 and 6), although the migration of the lower supershifted band differed (lanes 2 and 5, compare the asterisks). These data clearly identify ATF-1 as the binding protein for the TSP-1-CRE site in TPC-1 cells. However, we cannot rule out the possibility that heterodimerization occurs with other members of the ATF-CREB family as it has been described by others (55Ellis M.J. Lindon A.C. Flint K.J. Jones N.C. Goodbourn S. Mol. Endocrinol. 1995; 9: 255-265PubMed Google Scholar). This can be emphasized by the fact that two bands are supershifted with a specific antibody (Fig. 4B, lanes 2 and 5). One plausible explanation could be that ATF-1 may be bound to DNA as a homodimer or a complex with additional proteins in the extract and, owing to a difference in size or charge, each band is supershifted to a different position. Silencing of the ATF-1 Gene by shRNA Significantly Reduced HGF-induced Down-regulation of TSP-1—To further confirm the involvement of ATF-1 during HGF-induced TSP-1 down-regulation, a shRNA plasmid targeted at the human ATF-1 mRNA sequence was used in this study. Luciferase reporter assays using the shRNA construct and the –1290/+750 TSP-1-LUC reporter plasmid were performed. As shown in Fig. 5A, gene silencing of ATF-1 prevented HGF-induced inhibition of TSP-1 promoter activity when compared with control (shCTL) vector. In addition, shATF-1 down-regulated ATF-1 expression at the mRNA (Fig. 5B) and protein level (Fig. 5C). This effect was correlated with the up-regulation of TSP-1 protein expression/secretion independently of HGF treatment. These findings clearly indicate that ATF-1 is an essential transcription factor that regulates TSP-1 expression. ATF-1-dependent TSP-1 Down-regulation Promotes Tumor Cell Invasion—The effect of HGF on in vitro Matrigel invasion of TPC-1 cells was examined. As shown in Fig. 6A, HGF stimulated Matrigel invasion of TPC-1 cells. To confirm the involvement of TSP-1 in HGF-mediated promotion of tumor invasion, cells were allowed to invade into the Matrigel matrix in the presence or absence of TSP-1 protein. Incubation of cells with TSP-1 protein reduced basal as well as HGF-stimulated Matrigel cell invasion. To further determine the involvement of TSP-1 in TPC-1 cell invasion, we performed assays in the presence of a neutralizing antibody against TSP-1. Anti-TSP-1 antibody was able to significantly enhance basal TPC-1 cell invasion. Taken together, these results demonstrate that HGF-induced cell invasion is mediated at least in part by the regulation of TSP-1 expression. To address the involvement of ATF-1, invasion assays were performed using control and ATF-1 shRNA-expressing cells. Fig. 6B showed that knockdown of ATF-1 inhibited HGF-induced TPC-1 cell invasion. Collectively, our data demonstrate that ATF-1 transcription factor is a major intermediary in the signaling pathway that is responsible for HGF-induced TSP-1 down-regulation, leading to thyroid tumor cell invasion. As reported previously, disruption of ATF-1 activity in human metastatic melanoma cells by using an inhibitory anti-ATF-1 antibody fragment suppressed their tumorigenicity and metastatic potential in nude mice (56Jean D. Tellez C. Huang S. Davis D.W. Bruns C.J. McConkey D.J. Hinrichs S.H. Bar-Eli M. Oncogene. 2000; 19: 2721-2730Crossref PubMed Scopus (59) Google Scholar). Therefore ATF-1 may represent a target of choice to develop novel strategies for thyroid cancer therapy. We are grateful to Dr. James A. Fagin, University of Cincinnati, OH, who provided us the TPC-1 cells. We thank Prof. Paolo Sassone-Corsi, University of California, Irvine, CA and Dr. Benoit Viollet, Institut Cochin, Paris, France for valuable reagents." @default.
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• W1999307515 title "Activating Transcription Factor-1-mediated Hepatocyte Growth Factor-induced Down-regulation of Thrombospondin-1 Expression Leads to Thyroid Cancer Cell Invasion" @default.
• W1999307515 cites W146861865 @default.
• W1999307515 cites W1555964835 @default.
• W1999307515 cites W1564139422 @default.
• W1999307515 cites W1576971163 @default.
• W1999307515 cites W1803277453 @default.
• W1999307515 cites W1965641790 @default.
• W1999307515 cites W1968004681 @default.
• W1999307515 cites W1969065141 @default.
• W1999307515 cites W1974188350 @default.
• W1999307515 cites W1984294894 @default.
• W1999307515 cites W1992177486 @default.
• W1999307515 cites W1993253159 @default.
• W1999307515 cites W1995605351 @default.
• W1999307515 cites W1997134767 @default.
• W1999307515 cites W1999288105 @default.
• W1999307515 cites W2003114121 @default.
• W1999307515 cites W2005393972 @default.
• W1999307515 cites W2005782838 @default.
• W1999307515 cites W2007904495 @default.
• W1999307515 cites W2015353751 @default.
• W1999307515 cites W2016302051 @default.
• W1999307515 cites W2028886206 @default.
• W1999307515 cites W2029362226 @default.
• W1999307515 cites W2035174231 @default.
• W1999307515 cites W2040193953 @default.
• W1999307515 cites W2045115099 @default.
• W1999307515 cites W2054042846 @default.
• W1999307515 cites W2060444151 @default.
• W1999307515 cites W2060671555 @default.
• W1999307515 cites W2069761967 @default.
• W1999307515 cites W2069978422 @default.
• W1999307515 cites W2072346277 @default.
• W1999307515 cites W2074970072 @default.
• W1999307515 cites W2077817593 @default.
• W1999307515 cites W2080356314 @default.
• W1999307515 cites W2085149005 @default.
• W1999307515 cites W2087447167 @default.
• W1999307515 cites W2101173615 @default.
• W1999307515 cites W2104856326 @default.
• W1999307515 cites W2129886211 @default.
• W1999307515 cites W2132482143 @default.
• W1999307515 cites W2139102988 @default.
• W1999307515 cites W2151005088 @default.
• W1999307515 cites W2186242595 @default.
• W1999307515 cites W36200721 @default.
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