The ERK/MAPK Pathway Regulates the Activity of the Human Tissue Factor Pathway Inhibitor-2 Promoter
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m210935200
ISSN1083-351X
AutoresChristina Kast, Minglun Wang, Malcolm Whiteway,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoHuman tissue factor pathway inhibitor-2 (hTFPI-2) is a 32-kDa serine protease inhibitor that is associated with the extracellular matrix. hTFPI-2 inhibits several extracellular matrix-degrading serine proteases and may play a role in tumor invasion and metastasis. To study the signal transduction pathway that leads to the activation of the hTFPI-2, we cloned the potential promoter region of this gene adjacent to a heterologous luciferase reporter gene. Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells, and the cervical HeLa cells. This PMA induction was blocked with the MEK inhibitor UO126, suggesting that the PMA-induced activation of the hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal growth factor induced the luciferase reporter gene in HeLa cells. Cotransfection of the luciferase construct with constitutively active components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that regulation of hTFPI-2 is mediated through this pathway. A series of luciferase reporter gene constructs with progressive deletions of the 5′-flanking region suggested that the minimal basal promoter activity is located between nucleotide positions −89 and −384, whereas the minimal inducible promoter activity is between −89 and −222. We have used the computer program TFSEARCH and mutagenesis to analyze potential transcription factor binding sites. We identified an AP-1 binding site at nucleotide position −156 (inducible activity) and a Sp1 site at position −134 (basal activity) as potential cis-acting elements in the promoter region of the hTFPI-2. Human tissue factor pathway inhibitor-2 (hTFPI-2) is a 32-kDa serine protease inhibitor that is associated with the extracellular matrix. hTFPI-2 inhibits several extracellular matrix-degrading serine proteases and may play a role in tumor invasion and metastasis. To study the signal transduction pathway that leads to the activation of the hTFPI-2, we cloned the potential promoter region of this gene adjacent to a heterologous luciferase reporter gene. Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells, and the cervical HeLa cells. This PMA induction was blocked with the MEK inhibitor UO126, suggesting that the PMA-induced activation of the hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal growth factor induced the luciferase reporter gene in HeLa cells. Cotransfection of the luciferase construct with constitutively active components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that regulation of hTFPI-2 is mediated through this pathway. A series of luciferase reporter gene constructs with progressive deletions of the 5′-flanking region suggested that the minimal basal promoter activity is located between nucleotide positions −89 and −384, whereas the minimal inducible promoter activity is between −89 and −222. We have used the computer program TFSEARCH and mutagenesis to analyze potential transcription factor binding sites. We identified an AP-1 binding site at nucleotide position −156 (inducible activity) and a Sp1 site at position −134 (basal activity) as potential cis-acting elements in the promoter region of the hTFPI-2. phorbol 12-myristate 13-acetate activating protein-1 Dulbecco's modified Eagle's medium epidermal growth factor extracellular signal regulated kinase human tissue factor inhibitor-2 mitogen-activated protein kinase MAPK/ERK kinase MEK kinase phosphate-buffered saline protein kinase C c-Jun amino-terminal kinase stress-activated protein kinase cytomegalovirus green fluorescence protein matrix metalloprotease Growth hormones and the tumor promoting agent PMA1 initiate diverse intracellular signaling pathways that lead to the phosphorylation of transcription factors and ultimately to the regulation of target genes. Among the pathways often used to transduce signals are the mitogen-activated protein kinase (MAPK) cascades. These cascades consist of a three-kinase module that includes an MEK kinase (MEKK), which activates an MAPK/ERK kinase (MEK), which in turn activates a MAPK (1Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar). Three well characterized MAPKs have been described in mammalian cells: the mitogen-responsive ERK, the stress-responsive JNK/SAPK, and the p38 MAPK. The Ras/Raf/MEK/ERK signaling cascade regulates cell proliferation and differentiation (2Khosravi-Far R. Campbell S. Rossman K.L. Der C.J. Adv. Cancer Res. 1998; 72: 57-107Crossref PubMed Google Scholar). Components of this pathway are often activated in human tumors and oncogenic Ras, and constitutively activated ERKs have been found in a large variety of malignancies (3Shapiro P. Crit. Rev. Clin. Lab. Sci. 2002; 39: 285-330Crossref PubMed Scopus (86) Google Scholar, 4Sivaraman V.S. Wang H. Nuovo G.J. Malbon C.C. J. Clin. Invest. 1997; 99: 1478-1483Crossref PubMed Scopus (419) Google Scholar, 5Oka H. Chatani Y. Hoshino R. Ogawa O. Kakehi Y. Terachi T. Okada Y. Kawaichi M. Kohno M. Yoshida O. Cancer Res. 1995; 55: 4182-4187PubMed Google Scholar). We have used transcript profiling to identify genes that are differentially regulated by this pathway. Among the many activated genes, we have identified the human tissue factor pathway inhibitor-2 (hTFPI-2) as a gene that is highly up-regulated by the ERK/MAPK pathway. hTFPI-2 is a 32-kDa serine proteinase inhibitor with three tandem Kunitz-type domains (6Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Crossref PubMed Scopus (185) Google Scholar, 7Miyagi Y. Koshikawa N. Yasumitsu H. Miyagi E. Hirahara F. Aoki I. Misugi K. Umeda M. Miyazaki K. J. Biochem. (Tokyo). 1994; 116: 939-942Crossref PubMed Scopus (95) Google Scholar) and has high homology to hTFPI-1, a regulator of the extrinsic blood coagulation pathway. The second Kunitz-type domain of hTFPI-1 binds to factor Xa, and this complex inhibits the activity of the factor VIIa-tissue factor complex through interaction of the first Kunitz-type domain in hTFPI-1 and the active site of VIIa/TF (8Girard T.J. Warren L.A. Novotny W.F. Likert K.M. Brown S.G. Miletich J.P. Broze Jr., G.J. Nature. 1989; 338: 518-520Crossref PubMed Scopus (429) Google Scholar). Despite the high homology of hTFPI-2 to hTFPI-1, hTFPI-2 is a weak inhibitor of the activation of factor X (9Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Crossref PubMed Scopus (139) Google Scholar) and hTFPI-2 poorly inhibits tissue factor. However, hTFPI-2 inhibits the tissue factor-factor VIIa complex and a variety of serine proteases, including trypsin, plasmin, plasma kallikrein, chymotrypsin, and cathepsin G, but it does not inhibit thrombin, urokinase-type plasminogen activator, and tissue-type plasminogen activator (6Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Crossref PubMed Scopus (185) Google Scholar, 9Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Crossref PubMed Scopus (139) Google Scholar). Most of the hTFPI-2 expressed in dermal fibroblasts and endothelial cells localizes within the extracellular matrix, probably bound to heparan sulfate (10Rao C.N. Liu Y.Y. Peavey C.L. Woodley D.T. Arch. Biochem. Biophys. 1995; 317: 311-314Crossref PubMed Scopus (65) Google Scholar, 11Rao C.N. Reddy P. Liu Y. O'Toole E. Reeder D. Foster D.C. Kisiel W. Woodley D.T. Arch. Biochem. Biophys. 1996; 335: 82-92Crossref PubMed Scopus (88) Google Scholar, 12Iino M. Foster D.C. Kisiel W. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 40-46Crossref PubMed Scopus (93) Google Scholar). hTFPI-2 can prevent the conversion of Pro-MMP-1 (matrix metalloprotease 1, interstitial collagenase) and Pro-MMP-3 (matrix metalloprotease 3, stromelysin-1/transin-1) into MMP-1 and MMP-3 by plasmin and trypsin (13Rao C.N. Mohanam S. Puppala A. Rao J.S. Biochem. Biophys. Res. Commun. 1999; 255: 94-98Crossref PubMed Scopus (87) Google Scholar) and therefore might indirectly regulate matrix proteolysis and connective tissue turnover. The role of hTFPI-2 in cancer progression is not completely elucidated. On one hand, hTFPI-2 has an anti-invasive effect that might be mediated via inhibition of plasmin that activates proteases promoting degradation of the extracellular matrix and tumor invasion. Several tumor cell lines were less invasive when they were stably transfected with hTFPI-2 cDNA (14Jin M. Udagawa K. Miyagi E. Nakazawa T. Hirahara F. Yasumitsu H. Miyazaki K. Nagashima Y. Aoki I. Miyagi Y. Gynecol. Oncol. 2001; 83: 325-333Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 15Konduri S.D. Tasiou A. Chandrasekar N. Nicolson G.L. Rao J.S. Clin. Exp. Metastasis. 2000; 18: 303-308Crossref PubMed Scopus (41) Google Scholar, 16Konduri S.D. Rao C.N. Chandrasekar N. Tasiou A. Mohanam S. Kin Y. Lakka S.S. Dinh D. Olivero W.C. Gujrati M. Foster D.C. Kisiel W. Rao J.S. Oncogene. 2001; 20: 6938-6945Crossref PubMed Scopus (72) Google Scholar, 17Konduri S.D. Tasiou A. Chandrasekar N. Rao J.S. Int. J. Oncol. 2001; 18: 127-131PubMed Google Scholar). On the other hand, hTFPI-2 has been shown to have a pro-invasive effect in hepatocellular carcinoma cells (18Neaud V. Hisaka T. Monvoisin A. Bedin C. Balabaud C. Foster D.C. Desmouliere A. Kisiel W. Rosenbaum J. J. Biol. Chem. 2000; 275: 35565-35569Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this study, we investigated the signaling pathway and transcriptional elements that regulate the expression of the hTFPI-2 gene in epithelial cells. Although it has been shown that PMA can stimulate hTFPI-2 gene expression in glioma cells and that the promoter region −312 to +1 is critical for minimal and inducible promoter activity of hTFPI-2 (19Konduri S.D. Osman F.A. Rao C.N. Srinivas H. Yanamandra N. Tasiou A. Dinh D.H. Olivero W.C. Gujrati M. Foster D.C. Kisiel W. Kouraklis G. Rao J.S. Oncogene. 2002; 21: 921-928Crossref PubMed Scopus (14) Google Scholar), the signal transduction pathway by which PMA induces gene expression of hTFPI-2 and the promoter elements involved in hTFPI-2 regulation have not been studied in detail. Here we show that the hTFPI-2 gene expression is regulated by the ERK/MAPK signaling pathway and that the activity of this pathway is directed to an AP-1 site in the promoter of the hTFPI-2 gene. The Phorbol 12-myristate 13-acetate was ordered from Sigma-Aldrich Chemicals Co. (St. Louis, MO) and recombinant human epidermal growth factor from Austral Biologicals (San Raman CA). The LipofectAMINE Plus reagent as well as the Ecdysone-Inducible Mammalian Expression system, including EcR-293 cells, zeocin, and pronasteron A, and the expression vector pIND were purchased from Invitrogen Corp. (Carlsbad, CA). The MEK inhibitor UO126 and the Luciferase assay system were obtained from Promega Corp. (Madison, WI). The Phospho-p44/42 MAPK (Thr202/Tyr204) antibody was purchased fromCell Signaling Technology (Beverly, MA). The c-Myc (9E10) monoclonal antibody and the MEK-1 (C-18) and Raf-1 (C-12) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The QuikChangeTM site-directed mutagenesis kit was from Stratagene (La Jolla, CA). The FuGENE6 transfection reagent was obtained from Roche Diagnostics Corp. The HEK293, HeLa, A549, and MCF7 cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). All the cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Wisent Inc., St. Bruno, Quebec, Canada) with 10% fetal bovine serum (HyClone Laboratories, Mississauga, Ontario, Canada), except for the A549 cell line, which was cultured in DMEM supplemented with 5% fetal bovine serum. The plasmid pRK5 containing the myc-RasV12 gene with a glycine to valine mutation at amino acid position 12 and a sequence (EQKLISEEDLGS) containing a myc epitope inserted between a methionine and a threonine (amino acid positions one and two) was a generous gift from Nathalie Lamarche-Vane, McGill University, Montreal, Canada. The plasmid was digested with the restriction enzyme ClaI and the 5′-overhang filled in with Klenow. The myc-RasV12 fragment was subsequently released by ApaI and cloned into the EcoRV/ApaI site of pIND. pAN130-containing Raf-1 (20Nantel A. Mohammad-Ali K. Sherk J. Posner B.I. Thomas D.Y. J. Biol. Chem. 1998; 273: 10475-10484Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) served as a template to amplify the carboxyl-terminal, catalytic domain of Raf-1 (RafCT) using the oligonucleotides O-1 and O-2 listed in TableI. At the same time an EcoRI site (printed in boldface) was created to facilitate the cloning of the amplified 962-bp fragment into theEcoRI/XhoI sites of pcDNA3. A Kozak sequence (printed in italic) was inserted in-frame by cloning the phosphorylated, annealed oligonucleotides O-3 and O-4 (Table I) into the EcoRI site of RafCT, creating an AflII site (printed in boldface). The nucleotide sequence was verified by sequencing. The Kozak-RafCT fragment was released byAflII/XhoI and cloned in the corresponding sites of pIND.Table IOligonucleotidesO-15′-GCGAATTCAGGCCTCGTGGACAGAGAGATTCAAGC-3′O-25′-GTAACTCGAGTCAACTAGAAGACAGGCAGCC-3′O-35′-AATTCCTTAAG ACCATGG-3′O-45′-AATTCCATGGT CTTAAGG-3′O-55′-GCAGCTCATCGACGACATGGCGAACGACTTCGTGGGCACAAGG-3′O-75′-CGGGGTACCAGCTTCATACATGCTTGGTTGG-3′O-85′-GGAAGATCTGGTGCAGGGGGTCGGGCG-3′O-95′-AGCTTGCAGCGCGGGGGCAACGGGGTGACAGTCCCCGTGCATGAATCAGCCACCCCTCAGGCTCCGCCCCGGCGGGGGTC-3′O-105′-GGCCGACCCCCGCCGGGGCGGAGCCTGAGGGGTGGCTGATTCATGCACGGGGACTGTCACCCCGTTGCCCCCGCGCTGCA-3′O-115′-AGCTTGCAGCGCGGGGGCGGCGGGGTGACAGTCCCCGTGCAGCTAGCAGCCACCCCTCAGGCTCCGCCCCGGCGGGGGTC-3′O-125′-GGCCGACCCCCGCCGGGGCGGAGCCTGAGGGGTGGCTGCTAGCTGCACGGGGACTGTCACCCCGCCGCCCCCGCGCTGCA-3′O-135′-AGCTTGCAGCGCGGGGGCGGCGGGGTGACAGTCCCCGTGCATGAATCAGCCACCCCTCAGGCTTTGCCCCAACGGGGGTC-3′O-145′-GGCCGACCCCCGTTGGGGCAAAGCCTGAGGGGTGGCTGATTCATGCACGGGGACTGTCACCCCGCCGCCCCCGCGCTGCA-3′O-155′-AACTTGGAAGCTTATTCCTCTCCCTCTTACAC-3′O-165′-AATATCCGGCCGACCCCCGCCGGGGCAAAGCCTGAGGGGTGGCTGCTAGCTGCACGGGGA-3′O-175′-AATATCCCGGCCGACCCTTGCCGGGGCGGAGCCTGAGGGGTGGCTGCTAGCTGCACGGGGA-3′ Open table in a new tab MEK-1 was released from pAN104 (20Nantel A. Mohammad-Ali K. Sherk J. Posner B.I. Thomas D.Y. J. Biol. Chem. 1998; 273: 10475-10484Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) by BamHI/XhoI restriction digest and cloned into the corresponding sites of Bluescript KS. MEK-1SD was created by site-directed mutagenesis (QuikChangeTM site-directed mutagenesis kit, Stratagene) using two complementary oligonucleotides. The nucleotides that were changed to obtain a serine to aspartic acid mutation in MEK-1SD are underlined in O-5 in Table I. The nucleotide sequence was verified by sequencing, and the MEK-1SD was released from Bluescript withBamHI/XhoI and cloned into the corresponding sites of pIND. The plasmid mycCMV5-ERK2-MEK1-LA was kindly provided by Melanie H. Cobb, University of Texas Southwestern, Dallas, TX. The plasmid was digested with HindIII, the 5′-overhand was filled in with Klenow fragment, and ERK2-MEK1-LA was subsequently released byKpnI and cloned into the KpnI/EcoRV sites of pIND. Genomic DNA was isolated from 293 cells. The cells were washed in PBS, lysed in a buffer containing 10 mm NaCl, 10 mm EDTA, 0.5% Sarkosyl, and 10 mm Tris, pH 8, and incubated with proteinase K (10 mg/ml) at 50 °C overnight. After two phenol and two chloroform extractions, the genomic DNA was ethanol-precipitated and dissolved in TE, pH 8. A 1.5-kb fragment of the 5′-flanking region of the hTFPI-2 was amplified with an Expand High Fidelity PCR system (Roche Molecular Biochemicals) using the oligonucleotides O-7 and O-8 (Table I) to create a KpnI and a BglII restriction site (printed in boldface). ThisKpnI/BglII fragment was cloned into the plasmid pXP2, which contains the firefly luciferase reporter (21Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar) and was a generous gift from Mark Featherstone, McGill University, Montreal, Canada. The resulting construct was named p-1511-luc. The sequence was found identical to the one published by Kamei (22Kamei S. Kazama Y. Kuijper J.L. Foster D.C. Kisiel W. Biochim. Biophys. Acta. 2001; 1517: 430-435Crossref PubMed Scopus (24) Google Scholar) except for a C to A change at nucleotide position −47 relative to the translation start site. The hTFP-2promoter/luciferase reporter plasmids p-1293-luc, p-1055-luc, p-881-luc, p-733-luc, p-384-luc, p-222-luc, and p-89-luc were created by restriction digest on the original p-1511-luc plasmid. The p-1511-luc plasmid was digested with DraI, EarI,NarI, MscI, EcoNI, EcoRI, and SmaI, respectively, and the DNA fragments were released by BglII digest. The 5′-overhang created by EarI,NarI, EcoNI, and EcoRI restriction digest was filled in with Klenow fragment, and the restriction fragments were cloned into the SmaI/BglII site of pXP2. The putative Sp1 transcription factor binding site GGGGCGG between nucleotide positions −190 and −184 was changed to GGGGCAA, the putative AP-1 siteTGAATCA between nucleotide positions −162 and −156 was altered to GCTAGCA, and the overlapping Sp1/AP-2 and GC box GGCTCCGCCCCGGCGGGGG between nucleotide positions −144 and −126 was modified to GGCTTTGCCCCAACGGGGG. Double-stranded oligonucleotides containing the corresponding nucleotide changes were phosphorylated, annealed, and cloned asHindIII/EagI fragments into the corresponding sites of p-1055-luc. Each mutation was confirmed by DNA sequencing. The oligonucleotides used for p-198MSP1A-luc were O-9 and O-10, for p-198MAP1-luc O-11 and O-12, and for p-198MSP1B/MAP2-luc O-13 and O-14 (Table I). p-222MAP1/MSP1B-luc and p-222MAP1/MAP2-luc were created by PCR using the Expand High Fidelity PCR system using the oligonucleotides O-15 and O-16 or O-17, respectively. In both constructs the putative AP-1 site TGAATCA between nucleotide positions −162 and −156 was altered toGCTAGCA, and either the putative Sp1 site between nucleotide positions −140 and −134 CCGCCCC altered to TTGCCCC or the putative AP-2 site between nucleotide positions −136 and −126 CCCGGCGGGGG was altered to CCCGGCAAGGG. The sequence for theHindIII and EagI restriction sites are printed in boldface, and the nucleotide changes are underlined (Table I). The PCR products were verified by sequencing and subcloned asHindIII/EagI fragments into the corresponding sites of p-1055-luc. For inhibitor studies, the HEK293 cells (150,000 cells/well) were seeded the day prior to transfection. The cells were cotransfected with 1 μg of p-1511-luc and 70 ng of a GFP-spectrin control plasmid (23Kalejta R.F. Shenk T. Beavis A.J. Cytometry. 1997; 29: 286-291Crossref PubMed Scopus (111) Google Scholar). 24 h after transfection, the cells were serum-starved for 24 h. Where indicated, the MEK1/2-specific inhibitor UO126 was added 15 min prior to PMA induction. The cells were harvested in phosphate-buffered saline (PBS) and divided into two tubes. Cells in one tube were lysed using cell culture lysis reagent (Promega) and centrifuged at 12,000 × g for 5 min, and the cell extract was assayed for firefly luciferase activity using the luciferase reporter assay system (Promega). Light intensity was measured by using a microtiter plate luminometer (DYNEX Technologies, Inc., Chantilly, VA). The cells in the second tube were trypsinized and washed, and the percentage of cells that express GFP was determined by cytofluorimetry (EPICS XL-MCL) to control for transfection efficiency. Determination of protein by the Bradford assay (Bio-Rad) was carried out, controlling for harvesting efficiencies. Luciferase activity was expressed as firefly light units/μg of protein and normalized for transfection efficiency. HEK293, HeLa, A549, and MCF7 cells were seeded into six-well plates at 150,000, 250,000, 250,000, and 400,000 cells/well, respectively, 1 day prior to transfection. All cells were transfected with 1 μg of the reporter gene constructs. HEK293 and HeLa cells were transfected with FuGENE6 at a 3 μl of FuGENE/1 μg of DNA ratio, and A549 and MCF7 cells were transfected with LipofectAMINE Plus according to the manufacturer's protocol. 24 h after transfection, the cells of each well were split into six wells of a 24-well plate. 7 h later, the cells were serum-starved overnight and subsequently treated with PMA (250 nm) or EGF (50 ng/ml) as indicated. Cell extracts were lysed and analyzed as described above. The -fold stimulation of luciferase was calculated as firefly light units/μg of protein of PMA- or EGF-treated cells divided by the firefly light units/μg of protein of nontreated cells. Two days prior to transfection, EcR-293 cells cultivated in DMEM supplemented with 10% fetal bovine serum and Zeocin (400 μg/ml) were seeded into six-well plates at 200,000 cells/well. EcR-293 cells stably express the modified ecdysone receptor. A gene of interest, cloned into a pIND-based inducible expression vector, can be induced with the ecdysone analog pronasteron A that binds to the ecdysone receptor. Cells were transfected with 6 μl of FuGENE6 and 1.07 μg of total DNA (0.9 μg of RasV12, RafCT, MEK-1SD, ERK2-MEK1-LA, or pIND control vector, 0.1 μg of reporter plasmid, and 70 ng of GFP control plasmid). The medium containing fetal bovine serum was replaced 24 h after transfection with DMEM without serum, pronasteron A was added 4 h later to the medium to induce protein expression at a concentration of 6 μm unless indicated differently, and cells were harvested 20 h later. Luciferase activity was expressed as firefly light units/μg of protein and normalized for transfection efficiency. Cells were harvested and washed with PBS. The cells were lysed in PBS containing 0.1 mm sodium vanadate, 10 mm sodium pyrophosphate, 1.5% Triton X-100, and the proteinase inhibitors aprotinin (10 μg/ml), leupeptin (10 μg/ml), and phenylmethylsulfonyl fluoride (1 mm). 15 μg of total protein was separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 1% bovine serum albumin in TBST (150 mm NaCl, 0.05% Tween 20, 20 mm TrisCl, pH 7.5) and subsequently incubated with the anti-myc (9E10) at a dilution of 1:250 at 4 °C overnight, or with anti-Raf-1 (C12), or anti-MEK1 (C18) at dilutions of 1:1000 for 1 h at room temperature. The Phospho-p44/42 MAPK (Thr202/Tyr204) antibody was diluted 1:1000 in TBST containing 4% skin milk powder and incubated for 1 h. After incubation with the secondary antibodies conjugated to horseradish peroxidase (1:1000) for 1 h, the bands were visualized by ECL (Roche Diagnostics Corp., Indianapolis, IN). We have observed that the hTFPI-2 gene was highly up-regulated on microarrays that were probed with cDNAs originating from HEK293 cells treated with 250 nm PMA for 4 h (data not shown). Recently, PMA induction of a hTFPI-2-luciferase reporter gene has also been shown in glioma cells (19Konduri S.D. Osman F.A. Rao C.N. Srinivas H. Yanamandra N. Tasiou A. Dinh D.H. Olivero W.C. Gujrati M. Foster D.C. Kisiel W. Kouraklis G. Rao J.S. Oncogene. 2002; 21: 921-928Crossref PubMed Scopus (14) Google Scholar), and mRNA of hTFPI-2 is up-regulated in BeWo and JEG-3 trophoblast cells treated with PMA (24Iochmann S. Reverdiau-Moalic P. Hube F. Bardos P. Gruel Y. Thromb. Res. 2002; 105: 217-223Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). To study the signaling pathway that leads to the induction of hTFPI-2 gene expression by PMA and to investigate important promoter elements involved in its transcriptional regulation, we PCR-amplified a 1511-bp fragment of the 5′-flanking region of the hTFPI-2 gene from genomic DNA isolated from HEK293 cells and cloned the 1511-bp fragment adjacent to the firefly luciferase reporter gene (p-1511-luc). To assess whether this potential promoter region allows transcription of luciferase, we transiently transfected HEK293 cells with the reporter plasmid p-1511-luc and monitored changes in luciferase activity of PMA-treated and untreated cells. Cells incubated with 250 nm PMA showed a 10-fold stimulation of luciferase compared with cells without PMA treatment (Fig.1 A). Induction of the luciferase reporter gene was decreased by 90% if cells were preincubated with the MEK1 inhibitor UO126 at a concentration of 10 μm. UO126 completely inhibited PMA induction at a concentration of 100 μm, suggesting that PMA activates the hTFPI-2 promoter through the MEK signaling pathway (Fig.1 A). As shown in the Western blot in Fig. 1 B, PMA activated p44/p42 MAPK in HEK293 cells and the phosphorylation of p44/p42 could be inhibited by the MEK1-inhibitor UO126 in a dose-dependent manner. This result indicates that MEK activation is necessary for the induction hTFPI-2 promoter activity by PMA. HEK293 cells have very recently been reported as atypical epithelial cells and may originate from neuronal cells (25Shaw G. Morse S. Ararat M. Graham F.L. FASEB J. 2002; 16: 869-871Crossref PubMed Scopus (578) Google Scholar). To determine whether the PMA induction of the hTFPI-2 gene observed in HEK293 cells was unique to this cell line or whether PMA promotes up-regulation of the hTFPI-2 gene in other epithelial cells lines as well, we transiently transfected human lung carcinoma A549 cells, breast carcinoma MCF7 cells and cervical carcinoma HeLa cells with the luciferase reporter plasmid p-1511-luc. A 3.7-fold PMA-dependent induction of the hTFPI-2 promoter activity was observed in A549 cells, whereas PMA induced the hTFPI-2 promoter activity in MCF7 and HeLa cells 30- or 20-fold, respectively (Fig.2). Because PMA has been reported to transactivate the epidermal growth factor receptor (26Chen N. Ma W.Y. She Q.B. Wu E. Liu G. Bode A.M. Dong Z. J. Biol. Chem. 2001; 276: 46722-46728Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we tested whether the hTFPI-2 gene could be induced by the growth factor EGF. Although EGF up-regulated the hTFPI-2 promoter activity in HeLa cells 10-fold, no substantial stimulation of the luciferase reporter gene was obtained in A549 and MCF7 cells (Fig. 2). To assess the importance of the components of the ERK/MAPK signaling pathway in the regulation the hTFPI-2 promoter activity, we cotransfected EcR-293 cells with the plasmid p-1511-luc and vectors containing constitutively activated signaling components, such as RasV12, RafCT, MEK-1SD, ERK2-MEK1-LA, or the empty control vector (pIND). The -fold stimulation of luciferase was calculated as normalized luciferase activity obtained in cells expressing active signaling components divided by the luciferase activity of samples originating from vector-transfected control cells (Fig.3 A). Protein expression of RasV12, RafCT, MEK-1SD, and ERK2-MEK1-LA in transiently transfected EcR-293 cells is shown on Western blots in Fig. 3 B. All the constitutively activated signaling components were well expressed. Expression of RasV12 stimulated the luciferase reporter gene 37-fold compared with the control vector. Expression of constitutively active signaling MAPK components further downstream of Ras, such as RafCT, MEK-1SD, and ERK2-MEK1-LA, induced the luciferase reporter the gene 7-, 92-, or 39-fold, respectively (Fig. 3 A), indicating that the hTFPI-2 gene expression can be regulated by the Ras/Raf/MEK/ERK pathway in EcR-293 cells. The highest activation of the hTFPI-2 promoter was obtained by MEK-1SD containing aspartic acids at amino acid positions 218/222 (27Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar), whereas the RasV12 and ERK2-MEK1-LA fusion protein (28Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar) activated the hTFPI-2 promoter to a similar extent. In conclusion, these results demonstrate that the Ras/Raf/MEK/ERK signaling pathway mediates regulation of the hTFPI-2 gene. We transiently cotransfected EcR-293 cells with a series of luciferase reporter gene constructs containing progressive deletions of the 5′-flanking region with either a vector containing RasV12 or the empty control vector pIND. The -fold stimulations of the Rasversus control vector are shown in Fig.4 A. 35- to 51-fold stimulations of luciferase were obtained with constructs p-1511-luc through p-222-luc, whereas no inducible luciferase activity was obtained with construct p-89-luc, suggesting that the minimal inducible promoter activity is located between nucleotide positions −222 and −89 (Fig. 4 A). Similar results were obtained in PMA-treated HEK293 cells expressing the deletion constructs. PMA stimulated p-1511-luc through p-222-luc 5- to 8-fold, whereas p-89-luc was not induced by PMA (Fig. 4 B). Therefore, we decided to investigate the promoter elements in the −222/−89 region that are responsible for the 51-fold Ras and 8-fold PMA stimulations as compared with control cells. The minimal basal promoter activity includes the −89/−384-bp region, because p-384-luc showed similar basal luciferase activity as longer constructs, whereas no basal activity was obtained with construct p-89-luc (Table II). As shown in Table II, the luciferase activity dropped by 74% in vector (pIND)-transfected cells expressing p-222-luc compared with cells expressing p-384-luc, indicating that the region,
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