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Expression of NAG-1, a Transforming Growth Factor-β Superfamily Member, by Troglitazone Requires the Early Growth Response Gene EGR-1

2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês

10.1074/jbc.m305295200

1083-351X

Seung Joon Baek, Jong‐Sik Kim, Jennifer B. Nixon, Richard P. DiAugustine, Thomas E. Eling,

Macrophage Migration Inhibitory Factor

Troglitazone (TGZ) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) are peroxisome proliferator-activated receptor-γ (PPARγ) ligands that have been shown to possess pro-apoptotic activity in human colon cancer. Although these compounds bind to PPARγ transcription factors as agonists, emerging evidence suggests that TGZ acts independently of PPARγ in many functions, including apoptosis. We previously reported that TGZ induces an early growth response transcription factor (EGR-1) by the ERK phosphorylation pathway rather than by the PPARγ pathway (Baek, S. J., Wilson, L. C., Hsi, L. C., and Eling, T. E. (2003) J. Biol. Chem. 278, 5845-5853). In this report, we show that the expression of the antitumorigenic and/or pro-apoptotic gene NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) is induced by TGZ and correlates with EGR-1 induction. In cotransfection and gel shift assays, we show that EGR-1-binding sites are located within region -73 to -51 of the NAG-1 promoter and have an important role in the transactivation of TGZ-induced NAG-1 expression. In contrast, PGJ2 induced NAG-1 protein expression, but PJG2 may not affect the same region that TGZ does in the NAG-1 promoter. The effect of PGJ2 is probably PPARγ-dependent because a PPARγ antagonist inhibited the PGJ2-induced expression of NAG-1. TGZ-induced NAG-1 expression was not inhibited by the PPARγ antagonist. The fact that TGZ-induced NAG-1 expression was accompanied by the biosynthesis of EGR-1 also suggests that EGR-1 plays a pivotal role in TGZ-induced NAG-1 expression. Our results suggest that EGR-1 induction is a unique property of TGZ, but is independent of PPARγ activation. The up-regulation of NAG-1 may provide a novel explanation for the antitumorigenic property of TGZ. Troglitazone (TGZ) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) are peroxisome proliferator-activated receptor-γ (PPARγ) ligands that have been shown to possess pro-apoptotic activity in human colon cancer. Although these compounds bind to PPARγ transcription factors as agonists, emerging evidence suggests that TGZ acts independently of PPARγ in many functions, including apoptosis. We previously reported that TGZ induces an early growth response transcription factor (EGR-1) by the ERK phosphorylation pathway rather than by the PPARγ pathway (Baek, S. J., Wilson, L. C., Hsi, L. C., and Eling, T. E. (2003) J. Biol. Chem. 278, 5845-5853). In this report, we show that the expression of the antitumorigenic and/or pro-apoptotic gene NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) is induced by TGZ and correlates with EGR-1 induction. In cotransfection and gel shift assays, we show that EGR-1-binding sites are located within region -73 to -51 of the NAG-1 promoter and have an important role in the transactivation of TGZ-induced NAG-1 expression. In contrast, PGJ2 induced NAG-1 protein expression, but PJG2 may not affect the same region that TGZ does in the NAG-1 promoter. The effect of PGJ2 is probably PPARγ-dependent because a PPARγ antagonist inhibited the PGJ2-induced expression of NAG-1. TGZ-induced NAG-1 expression was not inhibited by the PPARγ antagonist. The fact that TGZ-induced NAG-1 expression was accompanied by the biosynthesis of EGR-1 also suggests that EGR-1 plays a pivotal role in TGZ-induced NAG-1 expression. Our results suggest that EGR-1 induction is a unique property of TGZ, but is independent of PPARγ activation. The up-regulation of NAG-1 may provide a novel explanation for the antitumorigenic property of TGZ. The peroxisome proliferator-activated receptors (PPARs) 1The abbreviations used are: PPARs, peroxisome proliferator-activated receptors; PPRE, peroxisome proliferator-activated receptor response element; PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; TGZ, troglitazone; ERK, extracellular signal-regulated kinase; TGF-β, transforming growth factor-β; RA, retinoic acid; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase. are nuclear hormone receptors that can be activated by a specific ligand (1Schoonjans K. Martin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-166Crossref PubMed Scopus (468) Google Scholar). Three isoforms (α, β/δ, and γ) have been identified and are encoded by separate genes. PPARγ has been further characterized into three subtypes, γ1, γ2, and γ3 (2Fajas L. Auboeuf D. Raspe E. Schoonjans K. Lefebvre A.M. Saladin R. Najib J. Laville M. Fruchart J.C. Deeb S. Vidal-Puig A. Flier J. Briggs M.R. Staels B. 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Carcinogenesis. 2001; 22: 1379-1383Crossref PubMed Scopus (190) Google Scholar, 13Wakino S. Kintscher U. Liu Z. Kim S. Yin F. Ohba M. Kuroki T. Schonthal A.H. Hsueh W.A. Law R.E. J. Biol. Chem. 2001; 276: 47650-47657Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Masamune A. Satoh K. Sakai Y. Yoshida M. Satoh A. Shimosegawa T. Pancreas. 2002; 24: 130-138Crossref PubMed Scopus (43) Google Scholar). Among the PPARγ ligands, the antitumorigenic activity of troglitazone (TGZ) and PGJ2 has been well established (15Kubota T. Koshizuka K. Williamson E.A. Asou H. Said J.W. Holden S. Miyoshi I. Koeffler H.P. Cancer Res. 1998; 58: 3344-3352PubMed Google Scholar, 16Sarraf P. Mueller E. Jones D. King F.J. DeAngelo D.J. Partridge J.B. Holden S.A. Chen L.B. Singer S. Fletcher C. Spiegelman B.M. Nat. Med. 1998; 4: 1046-1052Crossref PubMed Scopus (929) Google Scholar). For example, TGZ and PGJ2 significantly inhibit tumor growth of human colorectal cancer cells (HCT-116), human breast cancer cells (MCF-7), and human prostate cancer cells (PC-3) in immunodeficient mice (15Kubota T. Koshizuka K. Williamson E.A. Asou H. Said J.W. Holden S. Miyoshi I. Koeffler H.P. Cancer Res. 1998; 58: 3344-3352PubMed Google Scholar, 16Sarraf P. Mueller E. Jones D. King F.J. DeAngelo D.J. Partridge J.B. Holden S.A. Chen L.B. Singer S. Fletcher C. Spiegelman B.M. Nat. Med. 1998; 4: 1046-1052Crossref PubMed Scopus (929) Google Scholar, 17Elstner E. Muller C. Koshizuka K. Williamson E.A. Park D. Asou H. Shintaku P. Said J.W. Heber D. Koeffler H.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8806-8811Crossref PubMed Scopus (760) Google Scholar, 18Elstner E. Williamson E.A. Zang C. Fritz J. Heber D. Fenner M. Possinger K. Koeffler H.P. Breast Cancer Res. Treat. 2002; 74: 155-165Crossref PubMed Scopus (80) Google Scholar). Furthermore, TGZ and PGJ2 affect several pathways in a PPARγ-independent manner. TGZ up-regulates nitric oxide synthesis (19Hattori Y. Hattori S. Kasai K. Hypertension. 1999; 33: 943-948Crossref PubMed Scopus (77) Google Scholar), induces the p53 pathway (20Okura T. Nakamura M. Takata Y. Watanabe S. Kitami Y. Hiwada K. Eur. J. Pharmacol. 2000; 407: 227-235Crossref PubMed Scopus (104) Google Scholar), inhibits cholesterol biosynthesis (21Wang M. Wise S.C. Leff T. Su T.Z. Diabetes. 1999; 48: 254-260Crossref PubMed Scopus (108) Google Scholar), and has antioxidant function (22Davies S.S. Pontsler A.V. Marathe G.K. Harrison K.A. Murphy R.C. Hinshaw J.C. Prestwich G.D. Hilaire A.S. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 2001; 276: 16015-16023Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), whereas PGJ2 induces apoptosis (23Nikitakis N.G. Siavash H. Hebert C. Reynolds M.A. Hamburger A.W. Sauk J.J. Br. J. Cancer. 2002; 87: 1396-1403Crossref PubMed Scopus (54) Google Scholar) and affects signaling pathways that utilize ERK1/2 or NF-κB (24Eligini S. Banfi C. Brambilla M. Camera M. Barbieri S.S. Poma F. Tremoli E. Colli S. Thromb. Haemostasis. 2002; 88: 524-532Crossref PubMed Scopus (38) Google Scholar) independent of PPARγ. In addition, we have recently demonstrated that TGZ induces the early growth response gene EGR-1 independently of the PPARγ transcription factor (25Baek S.J. Wilson L.C. Hsi L.C. Eling T.E. J. Biol. Chem. 2003; 278: 5845-5853Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). However, the molecular mechanism by which TGZ and PGJ2 exhibit antitumorigenesis, other than by PPARγ activation, is not known. The EGR-1 transcription factor (also known as NGFI-A, TIS8, krox-24, and zif268) is a member of the immediate-early gene family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to signals such as mitogens, growth factors, and stress stimuli. EGR-1 has been proposed as a tumor suppressor gene (26Liu C. Rangnekar V.M. Adamson E. Mercola D. Cancer Gene Ther. 1998; 5: 3-28PubMed Google Scholar, 27Calogero A. Arcella A. De Gregorio G. Porcellini A. Mercola D. Liu C. Lombari V. Zani M. Giannini G. Gagliardi F.M. Caruso R. Gulino A. Frati L. Ragona G. Clin. Cancer Res. 2001; 7: 2788-2796PubMed Google Scholar). EGR-1 activates the PTEN (phosphatase and tensin homolog) tumor suppressor gene during UV irradiation (28Virolle T. Adamson E.D. Baron V. Birle D. Mercola D. Mustelin T. de Belle I. Nat. Cell Biol. 2001; 3: 1124-1128Crossref PubMed Scopus (352) Google Scholar), and re-expression of EGR-1 suppresses the growth of transformed cells both in soft agar and in athymic nude mice (29Liu C. Yao J. Mercola D. Adamson E. J. Biol. Chem. 2000; 275: 20315-20323Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). EGR-1 induction is both p53-dependent and p53-independent (30Nair P. Muthukkumar S. Sells S.F. Han S.S. Sukhatme V.P. Rangnekar V.M. J. Biol. Chem. 1997; 272: 20131-20138Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 31Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 32Muthukkumar S. Han S.S. Rangnekar V.M. Bondada S. J. Biol. Chem. 1997; 272: 27987-27993Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Moreover, EGR-1 is down-regulated in several types of neoplasia as well as in an array of tumor cell lines (33Huang R.P. Liu C. Fan Y. Mercola D. Adamson E.D. Cancer Res. 1995; 55: 5054-5062PubMed Google Scholar, 34Huang R.P. Fan Y. de Belle I. Niemeyer C. Gottardis M.M. Mercola D. Adamson E.D. Int. J. Cancer. 1997; 72: 102-109Crossref PubMed Scopus (218) Google Scholar). These results suggest that EGR-1 has a role in growth suppression. The nonsteroidal anti-inflammatory drug-activated gene NAG-1 was identified from an indomethacin-induced gene library (35Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). NAG-1 (also known as MIC-1, GDF-15, placental transforming growth factor-β (TGF-β), and PLAB) represents a divergent member of the TGF-β superfamily. NAG-1 has antitumorigenic and pro-apoptotic activities as assessed by in vivo and in vitro assays (35Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar, 36Tan M. Wang Y. Guan K. Sun Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 109-114Crossref PubMed Scopus (230) Google Scholar, 37Li P.X. Wong J. Ayed A. Ngo D. Brade A.M. Arrowsmith C. Austin R.C. Klamut H.J. J. Biol. Chem. 2000; 275: 20127-20135Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 38Baek S.J. Wilson L.C. Eling T.E. Carcinogenesis. 2002; 23: 425-434Crossref PubMed Scopus (172) Google Scholar). The expression of NAG-1 in human colon tissue was seen only in the tips of the villi, where apoptosis occurs (39Kim K.S. Baek S.J. Flake G.P. Loftin C.D. Calvo B.F. Eling T.E. Gastroenterology. 2002; 122: 1388-1398Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Although the expression of NAG-1 is regulated by several nonsteroidal anti-inflammatory drugs independent of cyclooxygenase (40Baek S.J. Wilson L.C. Lee C.H. Eling T.E. J. Pharmacol. Exp. Ther. 2002; 301: 1126-1131Crossref PubMed Scopus (116) Google Scholar), it is also regulated by several antitumorigenic compounds, including resveratrol (38Baek S.J. Wilson L.C. Eling T.E. Carcinogenesis. 2002; 23: 425-434Crossref PubMed Scopus (172) Google Scholar), genistein (41Wilson L. Baek S.J. Call A. Eling T. Int. J. Cancer. 2003; 105: 747-753Crossref PubMed Scopus (105) Google Scholar), and the retinoid 6-(3-(1-adamantyl)-4-hydroxyphenyl)-2-naphthalene carboxylic acid (42Newman D. Sakaue M. Koo J.S. Kim K.S. Baek S.J. Eling T. Jetten A.M. Mol. Pharmacol. 2003; 63: 557-564Crossref PubMed Scopus (53) Google Scholar). We have previously reported the cloning and characterization of the 3.5-kb NAG-1 promoter (43Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Although Sp1 and chicken ovalbumin upstream promoter transcription factor-1 are essential factors in the regulation of the basal level of NAG-1 expression, compound-induced NAG-1 expression at the transcriptional level has not been fully characterized. In this study, we examine the relationship between PPARγ ligands and NAG-1 expression. PPARγ ligands, including TGZ and PGJ2, induce NAG-1 expression in human colorectal cancer cells. We found that TGZ induces EGR-1 expression, followed by induction of NAG-1 at the transcription level, whereas PGJ2 does not induce EGR-1. Rather, NAG-1 seems to be induced by PGJ2 through the PPARγ transcription factor because a PPARγ antagonist inhibited NAG-1 expression. EGR-1 induction by TGZ appears to be independent of PPARγ because other PPARγ ligands did not induce EGR-1, and PPARγ-binding sites are not located in the TGZ response element in the NAG-1 promoter. These data suggest that the expression of NAG-1 provides a novel mechanism for understanding how TGZ exerts its antitumorigenic activity. Cell Lines and Reagents—Human colorectal carcinoma cells (HCT-116) were purchased from American Type Culture Collection (Manassas, VA) and maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and gentamycin (10 μg/ml). Rosiglitazone (Invitrogen), PGJ2, WY-14643, 13-hydroxyoctadecadienoic acid, and the PPARγ antagonist GW9662 were purchased from Cayman Chemical Co., Inc. (Ann Arbor, MI). All-trans-retinoic acid (RA), 9-cis-RA, and retinol were purchased from Sigma. Recombinant human TGF-β1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). TGZ was obtained from Parke-Davis. Anti-EGR-1 (sc-110) and anti-actin (sc-1615) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-NAG-1 antibody was described previously (43Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Construction of Plasmids—The full-length EGR-1 cDNA in the pcDNA3 expression vector was described previously (25Baek S.J. Wilson L.C. Hsi L.C. Eling T.E. J. Biol. Chem. 2003; 278: 5845-5853Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The luciferase constructs containing the NAG-1 promoter and Sp1 in the pcDNA3 expression vector were generated previously (43Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The pNAG133/+70 constructs were previously generated (38Baek S.J. Wilson L.C. Eling T.E. Carcinogenesis. 2002; 23: 425-434Crossref PubMed Scopus (172) Google Scholar). The pNAG41/+70 construct was generated using primers 5′-AAGTCCGGGGACTATAAAGGCCGGTCCGGC-3′ (sense) and 5′-TGAGAGCCATTCACCGTCCTGAGTTC-3′ (antisense). After PCR, the fragment was cloned into the TA vector (Invitrogen), sequenced, and further cloned into the pGLBasic3 vector digested with XhoI and HindIII restriction enzymes. The NGFI-A-binding protein NAB1 cDNA in the expression vector was cloned by PCR from the IMAGE:843249 clone (Invitrogen) using primers 5′-TCCAGAGTAATGGCTGCGGCC-3′ (sense) and 5′-ATCACAGCTATCTTGAATCTTC-3′ (antisense). The amplified products were cloned into the pCR2.1/TOPO vector (Invitrogen), followed by cloning into the pcDNA3.1/NEO expression vector. Transfection and Luciferase Assay—HCT-116 cells were plated in 6-well plates at 2 × 105 cells/well in McCoy's 5A medium supplemented with 10% fetal bovine serum. After growth for 16 h, plasmid mixtures containing 1 μg of NAG-1 promoter linked to luciferase and 0.1 μg of pRL-null (Promega, Madison, WI) were transfected with LipofectAMINE (Invitrogen) according to the manufacturer's protocol. For the cotransfection experiment, plasmid mixtures containing 0.5 μg of promoter linked to luciferase, 0.5 μg of expression vector, and 0.1 μg of pRL-null were transfected with LipofectAMINE according to the manufacturer's protocol. After 48 h of transfection, the cells were harvested in 1× luciferase lysis buffer, and luciferase activity was determined and normalized to the pRL-null luciferase activity with a dual luciferase assay kit (Promega). For PPARγ ligand treatments, the cells were treated with the ligand in the absence of serum for 24 h and then assayed for luciferase activity. Western Blot Analysis—The level of protein expression was evaluated by Western blot analysis with anti-EGR-1 and anti-NAG-1 antibodies. Cells were grown to 60-80% confluency in 10-cm plates, followed by 16 h of additional growing in the absence of serum. After treatment with the indicated compounds, total cell lysates were isolated using precipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS). After sonication of samples, lysate proteins (30 μg) were separated by SDS-PAGE and transferred for 1 h onto nitrocellulose membrane (Schleicher & Schüll). The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline and Tween 0.05% and probed with each antibody for 2 h at room temperature. After washing with Tris-buffered saline and Tween 0.05%, the blots were treated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed several times. Proteins were detected by the enhanced chemiluminescence system (Amersham Biosciences). Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared as described previously (43Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). For the gel shift assay, double-stranded oligonucleotides (Invitrogen) were end-labeled with [γ-32P]ATP by T4 polynucleotide kinase (New England Biolabs Inc., Beverly, MA). Assays were performed by incubating 10 μg of nuclear extracts in binding buffer (Geneka Biotechnology) containing 2 × 105 cpm of labeled probe for 20 min at room temperature. To assure the specific binding of transcription factors, the probe was chased with 1-, 10-, and 50-fold molar excesses of unlabeled wild-type oligonucleotide. For the supershift experiments, anti-EGR-1 antibody (Geneka Biotechnology) was incubated with nuclear extracts on ice for 30 min before addition to the binding reaction. Samples were then electrophoresed on 5% nondenaturing polyacrylamide gels with 0.5× Tris borate/EDTA, and gels were dried and subjected to autoradiography. RNA Isolation and Northern Blot Analysis—After reaching 60-80% confluency in 10-cm plates, the cells were treated at the indicated concentrations with PPARγ ligands in the absence of serum. For the cycloheximide experiment, the cells were treated with 5 μg/ml compound for 30 min prior to TGZ treatment. Total RNAs were isolated with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Ten μg of total RNA was denatured at 55 °C for 15 min, separated on a 1.2% agarose gel containing 2.2 m formaldehyde, and then transferred to Hybond-N membrane (Amersham Biosciences). After fixing the membrane by UV, blots were prehybridized in hybridization solution (Rapid-Hyb buffer, Amersham Biosciences) for 1 h at 65 °C, followed by hybridization with cDNA labeled with [α-32P]dCTP by random primer extension (DECAprimeII kit, Ambion Inc., Austin, TX). The probes used were full-length NAG-1 fragments. After 4 h of incubation at 65 °C, the blots were washed once with 2× SSC and 0.1% SDS at room temperature and twice with 0.1× SSC and 0.1% SDS at 65 °C. mRNA abundance was estimated from the intensities of the hybridization bands of autoradiographs with a Scion Image (Scion Corp.). Equivalent loading of RNA samples was confirmed by hybridizing the same blot with a 32P-labeled β-actin probe, which recognizes an mRNA of ∼2 kb. PPARγ Ligands PGJ2 and TGZ Induce NAG-1 Expression by Different Pathways—PPARγ ligands have an antitumorigenic activity that is either dependent on binding to the ligand to PPRE or independent of PPARγ transcriptional binding (25Baek S.J. Wilson L.C. Hsi L.C. Eling T.E. J. Biol. Chem. 2003; 278: 5845-5853Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 44Kim Y. Suh N. Sporn M. Reed J.C. J. Biol. Chem. 2002; 277: 22320-22329Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 45Zhang J. Fu M. Zhao L. Chen Y.E. Biochem. Biophys. Res. Commun. 2002; 298: 128-132Crossref PubMed Scopus (29) Google Scholar, 46Nencioni A. Lauber K. Grunebach F. Brugger W. Denzlinger C. Wesselborg S. Brossart P. Exp. Hematol. 2002; 30: 1020-1028Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). One mechanism by which PPARγ ligands exert antitumorigenesis may involve the transcriptional up-regulation of antitumorigenic proteins. We measured PTEN and p53 tumor suppressor gene expression. The PTEN protein is only marginally induced, whereas the level of p53 is not altered by TGZ in HCT-116 cells (25Baek S.J. Wilson L.C. Hsi L.C. Eling T.E. J. Biol. Chem. 2003; 278: 5845-5853Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Interestingly, NAG-1, which has antitumorigenic activity, was significantly induced by the PPARγ ligands. As shown in Fig. 1A, PGJ2 and TGZ, which are both PPARγ ligands, induced NAG-1 mRNA in a concentration-dependent manner (3-fold at 1 and 5 μm, respectively). HCT-116 cells were also treated with 1 μm PGJ2 or 5 μm TGZ for different times. Both PGJ2 and TGZ induced NAG-1 protein expression as early as 6 h (Fig. 1B), and a marked increase in NAG-1 was observed at 24 and 48 h, indicating that PGJ2 and TGZ induce NAG-1 expression in a dose- and time-dependent manner. In addition, the PPARα ligand WY-14643 did not induce NAG-1 expression at concentrations up to 100 μm (data not shown), indicating that induction of NAG-1 is specific for this PPARγ ligand. We then examined whether NAG-1 induction by PPARγ ligands is dependent on the PPARγ transcription factor in HCT-116 cells expressing intact PPARγ (25Baek S.J. Wilson L.C. Hsi L.C. Eling T.E. J. Biol. Chem. 2003; 278: 5845-5853Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). HCT-116 cells were treated with a combination of PPARγ ligands and/or GW9662, a selective PPARγ inhibitor. Western analyses suggest that the PPARγ antagonist suppressed the PGJ2-induced NAG-1 expression, but did not suppress TGZ-induced NAG-1 expression. These findings suggest that TGZ-induced NAG-1 expression may be PPARγ-independent, whereas PGJ2 increased NAG-1 expression through activation of PPARγ (Fig. 2).Fig. 2Effect of a PPARγ antagonist on PGJ2-and TGZ-induced NAG-1 expression. The quiescent cells were pretreated with or without the PPARγ antagonist GW9662 (1 μm) for 30 min prior to the addition of either TGZ (5 μm) or PGJ2 (1 μm). After 24 h, total proteins were isolated for Western blot analysis. Equal loading was confirmed by determining actin immunoreactivity. The relative NAG-1 levels normalized by actin are shown at the bottom.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NAG-1 Promoter Activity and PPARγ Ligands—To evaluate the importance of cis-acting elements in conferring PPARγ-inducible NAG-1 expression, the 3.5-kb NAG-1 promoter and other deletion constructs were transfected into HCT-116 cells and then treated with either PGJ2 or TGZ. As an internal control, the plasmid pRL-null was used to determine the transfection efficiency. As shown in Fig. 3A, a large increase in luciferase activity was observed after TGZ treatment for all NAG-1 promoter constructs. However, in contrast, an increase in luciferase activity was not observed with PGJ2 treatment. In fact, the response of the different constructs to PGJ2 appeared to be the same as that to the vehicle. The pGLBasic3 promoterless vector was transfected into HCT-116 cells as a negative control, and no significant luciferase activity was observed with either PGJ2 or TGZ treatment. These data suggest the presence of a positive TGZ response element in the 3.5-kb NAG-1 promoter, but the absence of a PGJ2 response element. These results also indicate that TGZ and PGJ2 induce NAG-1 expression by different mechanisms, which is consistent with the finding from the Western analysis experiment in which we used a PPARγ antagonist (Fig. 2). To investigate whether this promoter region is responsive to other ligands, pNAG133/LUC-transfected cells were treated with the ligands all-trans-RA, 9-cis-RA, retinol, rosiglitazone, 13-hydroxyoctadecadienoic acid, PGJ2, WY-14643, and TGF-β1. As shown in Fig. 3B, TGZ increased luciferase activity, but the other ligands did not. This result supports the notion that TGZ increases the transcriptional activity of NAG-1 by a mechanism that does not utilize the nuclear receptor PPARγ. TGZ Response Element Is Located in Region -73 to -51 of the NAG-1 Promoter—A p53 site that is controlled by several dietary antitumorigenic compounds is present in the NAG-1 promoter at position +43 (38Baek S.J. Wilson L.C. Eling T.E. Carcinogenesis. 2002; 23: 425-434Crossref PubMed Scopus (172) Google Scholar). TGZ is known to induce apoptosis by either a p53-dependent or p53-independent pathway (47Bae M.A. Rhee H. Song B.J. Toxicol. Lett. 2003; 139: 67-75Crossref PubMed Scopus (50) Google Scholar, 48Chung S.H. Onoda N. Ishikawa T. Ogisawa K. Takenaka C. Yano Y. Hato F. Hirakawa K. Jpn. J. Cancer Res. 2002; 93: 1358-1365Crossref PubMed Scopus (54) Google Scholar). To examine the importance of p53 in TGZ-induced NAG-1 expression and to further define the TGZ response element, we generated two constructs containing the p53 site at position +43, pNAG133/+70 and pNAG41/+70. These constructs were transfected into HCT-116 cells, which were treated with vehicle or TGZ. As shown in Fig. 4A, the p53 site is not responsible for TGZ-induced NAG-1 expression because pNAG41/+70-transfected cells did not exhibit an increase in luciferase activity in the presence of TGZ. Thus, the TGZ response element is located between positions -133 and -41 in the NAG-1 promoter. There are two Sp1 sites (Sp1-B and Sp1-C) in this region of the NAG-1 promoter, which play a pivotal role in basal level expression (43Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In addition, these Sp1-B and Sp1-C sites overlap with putative EGR-1-binding sites (Fig. 4B). To demonstrate a functional role for the Sp-1 or EGR-1 site in TGZ-induced NAG-1 expression, we generated point/deletion mutation clones in the Sp1 and EGR-1 sites (Fig. 4B). The analysis of all mutant constructs revealed a dramatic reduction of luciferase activity compared with the wild-type construct, indicating that the TGZ response element may be located in region -73 to -51 region of the NAG-1 promoter. Furthermore, both Sp1 and EGR-1 may be involved in TGZ-induced NAG-1 expression. Sp1 and TGZ Induce NAG-1 Expression—Because region -73 to -51 of the promoter contains two Sp1 sites and two EGR-1 sites, the corresponding transcription factors might bind and transactivate the TGZ-induced NAG-1 expression. To evaluate the importance of these sites, Sp1 and EGR-1 expression vectors were generated and c