Troglitazone, a Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Selectively Induces the Early Growth Response-1 Gene Independently of PPARγ
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m208394200
ISSN1083-351X
AutoresSeung Joon Baek, Leigh C. Wilson, Linda C. Hsi, Thomas E. Eling,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoTroglitazone (TGZ) is a peroxisome proliferator-activated receptor γ (PPARγ) ligand that has pro-apoptotic activity in human colon cancer. Although TGZ binds to PPARγ transcription factors as an agonist, emerging evidence suggests that TGZ acts independently of PPARγ in many functions, including apoptosis. Early growth response-1 (Egr-1) transcription factor has been linked to apoptosis and shown to be activated by extracellular signal-regulated kinase (ERK). We investigated whether TGZ-induced apoptosis may be related to Egr-1 induction, because TGZ has been known to induce ERK activity. Our results show that Egr-1 is induced dramatically by TGZ but not by other PPARγ ligands. TGZ affects Egr-1 induction at least by two mechanisms; TGZ increases Egr-1 promoter activity by 2-fold and prolongs Egr-1 mRNA stability by 3-fold. Inhibition of ERK phosphorylation in HCT-116 cells abolishes the Egr-1 induction by TGZ, suggesting its ERK-dependent manner. Further, the TGZ-induced Egr-1 expression results in increased promoter activity using a reporter system containing four copies of Egr-1 binding sites, and TGZ induces Egr-1 binding activity to Egr-1 consensus sites as assessed by gel shift assay. In addition, TGZ induces ERK-dependent phosphorylation of PPARγ, resulting in the down-regulation of PPARγ activity. The fact that TGZ-induced apoptosis is accompanied by the biosynthesis of Egr-1 suggests that Egr-1 plays a pivotal role in TGZ-induced apoptosis in HCT-116 cells. Our results suggest that Egr-1 induction is a unique property of TGZ compared with other PPARγ ligands and is independent of PPARγ activation. Thus, the up-regulation of Egr-1 may provide an explanation for the anti-tumorigenic properties of TGZ. Troglitazone (TGZ) is a peroxisome proliferator-activated receptor γ (PPARγ) ligand that has pro-apoptotic activity in human colon cancer. Although TGZ binds to PPARγ transcription factors as an agonist, emerging evidence suggests that TGZ acts independently of PPARγ in many functions, including apoptosis. Early growth response-1 (Egr-1) transcription factor has been linked to apoptosis and shown to be activated by extracellular signal-regulated kinase (ERK). We investigated whether TGZ-induced apoptosis may be related to Egr-1 induction, because TGZ has been known to induce ERK activity. Our results show that Egr-1 is induced dramatically by TGZ but not by other PPARγ ligands. TGZ affects Egr-1 induction at least by two mechanisms; TGZ increases Egr-1 promoter activity by 2-fold and prolongs Egr-1 mRNA stability by 3-fold. Inhibition of ERK phosphorylation in HCT-116 cells abolishes the Egr-1 induction by TGZ, suggesting its ERK-dependent manner. Further, the TGZ-induced Egr-1 expression results in increased promoter activity using a reporter system containing four copies of Egr-1 binding sites, and TGZ induces Egr-1 binding activity to Egr-1 consensus sites as assessed by gel shift assay. In addition, TGZ induces ERK-dependent phosphorylation of PPARγ, resulting in the down-regulation of PPARγ activity. The fact that TGZ-induced apoptosis is accompanied by the biosynthesis of Egr-1 suggests that Egr-1 plays a pivotal role in TGZ-induced apoptosis in HCT-116 cells. Our results suggest that Egr-1 induction is a unique property of TGZ compared with other PPARγ ligands and is independent of PPARγ activation. Thus, the up-regulation of Egr-1 may provide an explanation for the anti-tumorigenic properties of TGZ. The peroxisome proliferator-activated receptors (PPARs) 1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; Egr-1, early growth response-1; TGZ, troglitazone; ERK, extracellular signal-regulated protein kinase; PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; EMSA, electrophoretic mobility shift assay; BRL, rosiglitazone; PAF, azelaoyl PAF; MAPK, mitogen-activated protein kinase; ARE, A/U-rich elements are transcription factors belonging to the nuclear hormone receptor gene superfamily (1Schoonjans K. Martin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-166Google Scholar). Three isoforms (α, β/δ, and γ) have been identified and are encoded by separate genes. Among them, PPARγ has been further characterized into three subtypes, PPARγ1, PPARγ2, and PPARγ3 (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. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Google Scholar,3Fajas L. Fruchart J.C. Auwerx J. FEBS Lett. 1998; 438: 55-60Google Scholar). Each type plays an important role in cellular differentiation (4Tontonoz P. Singer S. Forman B.M. Sarraf P. Fletcher J.A. Fletcher C.D. Brun R.P. Mueller E. Altiok S. Oppenheim H. Evans R.M. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 237-241Google Scholar), apoptosis (5Yang W.L. Frucht H. Carcinogenesis. 2001; 22: 1379-1383Google Scholar), anti-inflammatory response (6Chang T.H. Szabo E. Cancer Res. 2000; 60: 1129-1138Google Scholar), and lipid metabolism and metabolic disease, such as glucose homeostasis (7Nolan J.J. Ludvik B. Beerdsen P. Joyce M. Olefsky J. N. Engl. J. Med. 1994; 331: 1188-1193Google Scholar). There are several known ligands for PPARγ, including the natural prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), the synthetic anti-diabetic thiazolidinediones, and certain polyunsaturated fatty acids. PPARγ ligands are able to bind to the PPARγ transcription factor, which then forms a heterodimeric complex with retinoid X receptor that functions as a central regulator of differentiation, and modulator of cell growth. Many reports present evidence for anti-tumorigenic activity of PPARγ ligands (5Yang W.L. Frucht H. Carcinogenesis. 2001; 22: 1379-1383Google Scholar, 6Chang T.H. Szabo E. Cancer Res. 2000; 60: 1129-1138Google Scholar, 8Yin F. Wakino S. Liu Z. Kim S. Hsueh W.A. Collins A.R. Van Herle A.J. Law R.E. Biochem. Biophys. Res. Commun. 2001; 286: 916-922Google Scholar, 9Masamune A. Satoh K. Sakai Y. Yoshida M. Satoh A. Shimosegawa T. Pancreas. 2002; 24: 130-138Google Scholar, 10Wakino 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: 47650-47657Google Scholar). Among PPARγ ligands, the anti-tumorigenic activity of troglitazone (TGZ) has been well established. For example, TGZ significantly inhibits 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 (11Kubota T. Koshizuka K. Williamson E.A. Asou H. Said J.W. Holden S. Miyoshi I. Koeffler H.P. Cancer Res. 1998; 58: 3344-3352Google Scholar, 12Sarraf 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: 1046-1052Google Scholar, 13Elstner 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: 8806-8811Google Scholar). However, the molecular mechanism of TGZ effects on anti-tumorigenesis, other than PPARγ activation, is not known. TGZ has specific functions, in addition to being a PPARγ agonist. For example, TGZ up-regulates nitric oxide synthesis (14Hattori Y. Hattori S. Kasai K. Hypertension. 1999; 33: 943-948Google Scholar), induces the p53 pathway (15Okura T. Nakamura M. Takata Y. Watanabe S. Kitami Y. Hiwada K. Eur. J. Pharmacol. 2000; 407: 227-235Google Scholar), inhibits cholesterol biosynthesis (16Wang M. Wise S.C. Leff T. Su T.Z. Diabetes. 1999; 48: 254-260Google Scholar), induces p21 cyclin-dependent kinase inhibitor (17Sugimura A. Kiriyama Y. Nochi H. Tsuchiya H. Tamoto K. Sakurada Y. Ui M. Tokumitsu Y. Biochem. Biophys. Res. Commun. 1999; 261: 833-837Google Scholar), has antioxidant function (18Davies G.F. Khandelwal R.L. Wu L. Juurlink B.H. Roesler W.J. Biochem. Pharmacol. 2001; 62: 1071-1079Google Scholar), and activates extracellular signal-regulated protein kinase (ERK) (19Takeda K. Ichiki T. Tokunou T. Iino N. Takeshita A. J. Biol. Chem. 2001; 276: 48950-48955Google Scholar) in a PPARγ-independent manner. Thus, the molecular mechanism of TGZ-induced anti-tumorigenesis may result from multiple mechanisms. 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. However, many reports have recently suggested Egr-1 as a tumor suppressor gene (20Liu C. Rangnekar V.M. Adamson E. Mercola D. Cancer Gene Ther. 1998; 5: 3-28Google Scholar). Egr-1 activates PTEN (phosphatase and tensin homolog) tumor suppressor gene during UV irradiation (21Virolle T. Adamson E.D. Baron V. Birle D. Mercola D. Mustelin T. de Belle I. Nat. Cell. Biol. 2001; 3: 1124-1128Google Scholar), and re-expression of Egr-1 suppresses the growth of transformed cells both in soft agar and in athymic nude mice (22Liu C. Yao J. Mercola D. Adamson E. J. Biol. Chem. 2000; 275: 20315-20323Google Scholar). Egr-1 is induced very early in the apoptotic process, where it mediates the activation of downstream regulators such as p53 (23Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Google Scholar, 24Muthukkumar S. Han S.S. Rangnekar V.M. Bondada S. J. Biol. Chem. 1997; 272: 27987-27993Google Scholar, 25Nair P. Muthukkumar S. Sells S.F. Han S.S. Sukhatme V.P. Rangnekar V.M. J. Biol. Chem. 1997; 272: 20131-20138Google Scholar). However, Egr-1-induced apoptosis has been reported in p53−/− cells (26Zhang W. Chen S. Exp. Cell Res. 2001; 266: 21-30Google Scholar), indicating that Egr-1 induction occurs in both p53-dependent and p53-independent manners. Moreover, Egr-1 is down-regulated in several types of neoplasia, as well as in an array of tumor cell lines (27Huang R.P. Fan Y. de Belle I. Niemeyer C. Gottardis M.M. Mercola D. Adamson E.D. Int. J. Cancer. 1997; 72: 102-109Google Scholar, 28Huang R.P. Liu C. Fan Y. Mercola D. Adamson E.D. Cancer Res. 1995; 55: 5054-5062Google Scholar). These results indicate that Egr-1 plays a consistent role in growth suppression. Therefore, it is reasonable to think that Egr-1 could be regulated, at least in part by TGZ, because both TGZ and Egr-1 have anti-tumorigenic effects. In the present study, we examine the relationship between TGZ and Egr-1 expression and the effect of TGZ-induced apoptosis. We show that TGZ induces Egr-1 expression by transcriptional and post-transcriptional regulation. Egr-1 induction by TGZ results in the increase of binding affinity and transactivation of the promoter containing Egr-1 consensus sequences, thereby possibly inducing other anti-tumorigenic proteins. Furthermore, Egr-1 induction by TGZ appears to be independent of PPARγ, because other PPARγ ligands do not induce Egr-1. These data provide a novel mechanism for understanding how troglitazone exerts its anti-tumorigenic activity. Human colorectal carcinoma cells, HCT-116, were purchased from ATCC (Manassas, VA) and maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and gentamicin (10 μg/ml). All of HCT-116 cell experiments were done within passage 16. Rosiglitazone (BRL), azelaoyl PAF (PAF), PGJ2, and ciglitazone were purchased from Cayman Chemical (Ann Arbor, MI). TGZ was obtained from Parke-Davis Pharmaceutical Research. PD98059, SB203580, and herbimycin were purchased from Sigma. Egr-1 (sc-110), Egr-2 (sc-190), Egr-3 (sc-191), p53 (sc-263), PTEN (sc-7974), ERK1 (sc-94), ERK2 (sc-154), PPARγ1 (sc-7273), and actin (sc-1615) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-ERK antibody was obtained from Cell Signaling (Beverly, MA). The full-length Egr cDNAs were generated by PCR from human universal QUICK-Clone cDNA (Clontech, Palo Alto, CA) using the following primers: for the Egr-1, 5′-GACACCAGCTCTCCAGCCTGCTCGTCCAGG-3′ (top strand) and 5′-TTCCCTTTAGCAAATTTCAATTGTCCTGGG-3′ (bottom strand); for the Egr-2, 5′-GTGCGAGGAGCAAATGATGACCGCCAAGGC-3′ (top strand) and 5′-CAGCCTGAGTCTCATCTCAAGGTGTCCGGG-3′ (bottom strand); for the Egr-3, 5′-CGGCGGCAGCTCGGGAGTGCTATGACCGGC-3′ (top strand) and 5′-TCTGGGGGCCCGATCCTCAGGCGCAGGTGG-3′ (bottom strand). The amplified products were cloned into pCR2.1 TOPO vector (Stratagene, CA) and followed by cloning into pCDNA3.1/NEO expression vector (Invitrogen). The pEBS14luc construct was generously provided by Dr. Gerald Thiel (University of Bari, Bari, Italy). The Egr-1 promoter (−1260 to +35) linked to the luciferase gene was cloned by PCR from human genomic DNA (Promega, Madison, WI) using the following primers: 5′-CGGCTCGAGCGGGAGGAGGAGCGAGGAGGCGGCGG-3′ (top strand; XhoI site is underlined) and 5′-CCCAAGCTTGGGCGGCGGCGGCTCCCCAAGTTCTGCGGC-3′ (bottom strand; HindIII site is underlined). After PCR amplification, the fragment was digested with XhoI andHindIII and ligated into pGLBasic3 luciferase vector. All plasmids were sequenced for verification. The DNA content for sub-G1 population was determined by flow cytometry. HCT-116 cells were plated at 3 × 105 cells/well in 6-well plates, incubated for 16 h, and then treated with TGZ at different time points in the presence of serum. The cells (attached and floating cells) were then harvested, washed with phosphate-buffered saline, fixed by the slow addition of cold 70% ethanol to a total of 1 ml, and stored at 4 °C overnight. The fixed cells were pelleted, washed with ethanol (50%, then 30%), followed by phosphate-buffered saline, and stained in 1 ml of 20 μg/ml propidium iodide containing 1 mg/ml RNase in phosphate-buffered saline for 20 min. 7,500 cells were examined by flow cytometry using BD Biosciences FACSort equipped with CellQuest software by gating on an area versus width dot plot to exclude cell debris and cell aggregates. Apoptosis was measured by the level of sub-diploid DNA contained in cells following treatment with compounds using CellQuest software. Soft agar assays were performed to compare the clonogenic potential of HCT-116 cells in semisolid medium. HCT-116 cells were resuspended at 10,000 cells in 2 ml of 0.4% agarose-containing TGZ in McCoy's 5A medium and plated on top of 1 ml of 0.8% agarose in 6-well plates. Plates were incubated for 2–3 weeks at 37 °C. Cell colonies were visualized by staining with 0.5 ml of p-iodonitrotetrazolium violet staining (Sigma). 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 0.5 μg of promoter linked to luciferase, 0.5 μg of expression vector, and 0.05 μg of pRL-null (Promega) were transfected by LipofectAMINE (Invitrogen) 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 using a dual luciferase assay kit (Promega). For the PPARγ ligand treatments, cells were treated with PPARγ ligand in the absence of serum for 24 h and assayed for luciferase activity. When reaching 60–80% confluence in 10-cm plates, the cells were grown in the absence of serum for 24 h and then treated with either vehicle or TGZ (5 μm) for 2 h. The transcription inhibitor, actinomycin D (5 μg/ml), was treated at the indicated time points. Total RNAs were isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For Northern blot analysis, 20 μg of total RNA was denatured at 55 °C for 15 min and separated in a 1.0% agarose gel containing 2.2 m formaldehyde and 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 using random primer extension (DECAprimeII kit; Ambion). The probes used were full-length human Egr-1 fragment. After 4 h of incubation at 65 °C, the blots were washed once with 2× SSC/0.1% SDS at room temperature and twice with 0.1× SSC/0.1% SDS at 65 °C. Messenger RNA abundance was estimated by intensities of the hybridization bands of autoradiographs using Scion Image (Scion Image Co.). Equivalent loading of RNA samples was confirmed by hybridizing the same blot with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase probe, which recognizes RNA of ∼1.3 kb. The level of protein expression was evaluated using Western blot analysis with Egr-1, Egr-2, Egr-3, PPARγ1, PTEN, and phospho-ERK antibodies. Cells were grown to 60–80% confluency in 10-cm plates followed by 24 h of additional growth in the absence of serum. Cells were treated with indicated compounds, and total cell lysates were isolated using 0.1 mTris/pH 8.0 containing proteinase inhibitors (Sigma). After sonication of samples, proteins (30 μg) were separated by SDS-PAGE and transferred for 1 h onto nitrocellulose membrane (Schleicher & Schuell). The blots were blocked overnight with 5% skim milk in TBS-T (Tris-buffered saline/Tween 0.05%), and probed with each antibody for 2 h at room temperature. After washing with TBS-T, 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). Western analysis for p53 was described previously (29Baek S.J. Wilson L.C. Eling T.E. Carcinogenesis. 2002; 23: 425-432Google Scholar). Nuclear extracts were prepared as described previously (30Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Google Scholar). For the gel shift assay, double stranded oligonucleotides were end-labeled with [γ-32P]ATP by T4 polynucleotide kinase (New England Biolabs). Assays were performed by incubating 10 μg of nuclear extracts in the binding buffer (Geneka Biotechnology) containing 200,000 cpm of labeled probe for 20 min at room temperature. To assure the specific binding of transcription factors to the probe, the probe was chased by 50-fold molar excess of cold wild type or mutant oligonucleotide. For the supershift experiments, Egr-1 antibody (Geneka Biotechnology) was incubated with nuclear extracts on ice for 30 min before adding to the binding reaction. Samples were then electrophoresed on 5% nondenaturing polyacrylamide gels with 0.5× TBE (Tris/borate/EDTA), and gels were dried and subjected to autoradiography. HCT-116 cells were grown in the absence of serum for 24 h and treated with 5 μm TGZ or vehicle for 3 h. Nuclei were isolated as described previously (30Baek S.J. Horowitz J.M. Eling T.E. J. Biol. Chem. 2001; 276: 33384-33392Google Scholar).In vitro nuclear run-on transcription was carried out using 1 × 107 nuclei and 250 μCi of [α-32P]UTP in transcription-optimized buffer (P118A;Promega). The reaction was performed at 30 °C for 30 min. Labeled transcripts were purified using TRIzol reagent (Invitrogen) according to manufacturer's protocol. A total of 1 × 107 cpm elongated nascent RNA per assay was hybridized for 24 h at 65 °C to filter-immobilized 5 μg of plasmid DNA. The filters were washed with 2× SSC/0.1% SDS for 20 min, followed by 0.1× SSC/0.1% SDS for 40 min. The autoradiographs were subjected to densitometric analysis using Scion Image. We examined the correlation between TGZ-induced apoptosis and TGZ-induced anti-tumorigenesis in HCT-116 cells. Anti-tumorigenic activity was measured by soft agar assay, whereas apoptosis was measured by flow cytometry. As shown in Fig.1 A, TGZ treatment in HCT-116 cells dramatically inhibited the growth of HCT-116 cells on soft agar. The growth inhibition by TGZ was concentration-dependent, and 5 μm TGZ completely inhibited the growth of HCT-116 cells in soft agar assays. To determine whether TGZ induces cell cycle arrest and/or apoptosis in HCT-116 cells, flow cytometry analysis was performed (Fig. 1 B). Apoptosis and G1 cell cycle arrest were observed as early as 12 h after treatment with 5 μm TGZ. The stimulation of apoptosis and G1cell cycle arrest by TGZ are consistent with previous publications (8Yin F. Wakino S. Liu Z. Kim S. Hsueh W.A. Collins A.R. Van Herle A.J. Law R.E. Biochem. Biophys. Res. Commun. 2001; 286: 916-922Google Scholar,31Kitamura S. Miyazaki Y. Shinomura Y. Kondo S. Kanayama S. Matsuzawa Y. Jpn. J. Cancer Res. 1999; 90: 75-80Google Scholar) reporting that TGZ induces apoptosis and cell cycle arrest in human colon and breast cancer cells. One logical mechanism by which TGZ exerts anti-tumorigenesis is the up-regulation of anti-tumorigenic proteins. To address this question, we measured the expression of known anti-tumorigenic proteins, Egr-1, PTEN, and p53. HCT-116 cells were treated with 5 μm TGZ at indicated time points, and Western analysis was performed. As shown in Fig. 2 A, Egr-1 is induced dramatically within 3 h, and longer treatment, up to 24 h, results in a decrease of Egr-1 expression. TGZ-induced Egr-1 expression was also seen in Northern analysis using HCT-116 cells treated with 5 μm TGZ for 2 h (data not shown). We also measured PTEN and p53 tumor suppressor gene expression. PTEN is only marginally induced at an early time point, whereas p53 expression is not altered by TGZ in HCT-116 cells. Taken together, these results suggest that Egr-1 induction by TGZ may be pivotal to the anti-tumorigenic activity of TGZ. Because TGZ is a PPARγ ligand, we next compared TGZ to other PPARγ ligands with regard to Egr-1 induction. HCT-116 cells were treated with several PPARγ ligands, BRL, PGJ2, PAF, ciglitazone, and 13-hydroxyoctadecadienoic acid, for 3 h. These PPARγ ligands are reported to bind and activate PPARγ transcription factor (32Davies 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: 16015-16023Google Scholar, 33Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Google Scholar). Consistent with previous data, Egr-1 induction was seen in TGZ-treated cells, but poor or no induction was observed in other PPARγ ligand-treated cells (Fig. 2 B), suggesting that Egr-1 induction by TGZ may be independent of PPARγ. In addition, the expression of other Egr family proteins, Egr-2 and Egr-3, was not altered by TGZ and other PPARγ ligands, indicating that the effect of TGZ is specific for Egr-1. To address whether Egr-1 protein, produced by TGZ-treated HCT-116 cells, has potential binding activity to the Egr-1 consensus sequences, EMSA was performed. Because Egr-1 protein increases in the presence of TGZ, the protein should bind to the Egr-1 consensus binding site. As shown in Fig.3 A, oligonucleotides containing two copies of Egr-1 binding sites were generated and used as a probe. Nuclear extracts prepared from 5 μm TGZ treatment at different time points (Fig. 3 B, lanes 2–5) and the recombinant proteins, Egr-1, Egr-2, and Egr-3, generated by in vitro translation (Fig. 3 B,lanes 6–8), were used for the EMSA. Using nuclear extracts from TGZ-treated HCT-116 cells and a probe corresponding to the Egr-1 binding site, results show multiple DNA·protein complexes with a mobility shift (Fig. 3 B, arrows α and β). Compared with in vitro synthesized Egr proteins, the shifted band α correspond to Egr-1 proteins, whereas β indicates Egr-3 proteins. Interestingly, a shifted band representing Egr-1 was seen only at the 3-h time point, whereas Egr-3 was constitutively expressed during the time course. A shifted band representing Egr-2 was not detected in TGZ-treated HCT-116 cells. These results are consistent with Egr-1 induction at the 3-h time point after TGZ treatment as assessed by Western analysis. These bands represent a specific protein binding to the Egr-1 sequence elements, because complex formation was diminished by the addition of 50 molar excess of non-radiolabeled identical competitor but not by addition of the identical oligonucleotide in which the Egr-1 sites were point-mutated (Fig.3 C, lanes 2 and 3). To confirm that Egr-1 binds to these sites, we performed a gel shift assay in the presence of Egr-1 antibody to demonstrate supershifting. As shown in Fig. 3 C, the Egr-1 antibody supershifted the band (lanes 4 and 6). This indicates that the shifted bands (SS) contain Egr-1 protein. As a control, we examined Egr-1 binding affinity using nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated K562 cells and observed similar results as with the nuclear extracts from TGZ-treated HCT-116 cells (Fig. 3 C, lanes 5 and6). It has been shown that Egr-1 is induced by 12-O-tetradecanoylphorbol-13-acetate-treated K562 cells (34Cheng T. Wang Y. Dai W. J. Biol. Chem. 1994; 269: 30848-30853Google Scholar). Taken together, TGZ specifically induces Egr-1, and the induced Egr-1 can bind to the Egr-1 consensus sequence as assessed by EMSA. The transactivation activity of Egr-1 in TGZ-treated HCT-116 cells was determined using an Egr-1-responsive reporter. The plasmid pEBS14luc contained four copies of Egr-1 response elements linked to the basal promoter followed by a luciferase reporter gene (Fig.4 A) (35Cibelli G. Policastro V. Rossler O.G. Thiel G. J. Neurosci. Res. 2002; 67: 450-460Google Scholar). To determine whether the expression of Egr-1, Egr-2, and Egr-3 proteins could activate the luciferase reporter, the plasmid pEBS14luc was transfected into HCT-116 cells, in combination with empty, Egr-1, Egr-2, or Egr-3 expression vectors. As shown in Fig. 4 B, the luciferase activity was increased by overexpression of Egr-1, Egr-2, and Egr-3 compared with vector-transfected cells, suggesting that overexpression of Egr family proteins is able to bind and transactivate the reporter vector. Subsequently, to determine whether TGZ induced Egr-1 expression would also transactivate the reporter vector containing Egr-1 binding sites, we transfected the reporter vector and treated with varying concentrations of TGZ. Indeed, TGZ induced luciferase activity in a concentration-dependent manner, with 4- and 12-fold induction observed with 10 and 20 μm TGZ treatment, respectively. Because TGZ is a PPARγ ligand, we tested whether other PPARγ ligands would also induce luciferase activity. We examined BRL, PGJ2, and PAF, which have been known to bind to PPARγ. TGZ is the strongest Egr-1 inducer of luciferase activity (Fig.4 D), although the other compounds are better PPARγ ligands in terms of PPARγ binding activity. However, in this system, 10 μm TGZ is required to see any significant luciferase induction, compared with 5 μm TGZ being used for Western and Northern analyses. This system is an artificial construct and may require higher concentration of TGZ to see effects. Taken together with previous results, these data demonstrate that TGZ not only induces Egr-1 expression and binding activity but also transactivates Egr-1 responsive genes. We performed nuclear run-on experiments to examine whether the Egr-1 induction by TGZ is regulated at a transcriptional level. HCT-116 cells were treated with serum-free medium for 24 h followed by treatment with TGZ or vehicle for 3 h. Radioactive-labeled nascent transcripts were analyzed by hybridization to immobilized DNAs. Egr-1 gene transcription at 3 h after TGZ treatment was increased 2-fold, as determined by triplicate independent experiments. The gene for Sp1 transcription factor was used as an internal control, because Sp1 expression is not altered by TGZ treatment in HCT-116 cells (data not shown). Thus, Egr-1 transcripts were induced by TGZ at the transcriptional level. Fig.5 A is a representative autoradiogram of three experiments. To confirm whether TGZ induces Egr-1 at the transcription level, the Egr-1 promoter was cloned into the luciferase reporter gene. A plasmid, pEgr1260/LUC, was transfected into HCT-116 cells, and several PPARγ ligands were treated. As shown in Fig. 5 B, TGZ enhances Egr-1 promoter activity 2-fold, whereas the other PPARγ ligands do not enhance promoter activity significantly. Taken together with the nuclear run-on experiments, these results suggest that TGZ induces Egr-1 expression at the transcription level by at least 2-fold. However, these data do not fully explain the dramatic induction shown in Western analysis (Fig.2), indicating that TGZ may be involved with other mechanisms of Egr-1 induction. Because TGZ affects minimum induction at the Egr-1 promoter, and Egr-1 proteins were dramatically induced in HCT-116 cells, it is possible that TGZ may increase Egr-1 mRNA stability. HCT-116 cells were treated with either TGZ or vehicle for 2 h and then 5 μg/ml of actinomycin D was added at the indicated time points. Fig.6, A and Bdemonstrates that the half-life of Egr-1 mRNA in control HCT-116 cells was ∼15 min compared with 48 min in TGZ-treated cells. These results suggest that an increase in stability of Egr-1 mRNA is also a major factor in the induction of Egr-1 proteins by TGZ. Therefore, TGZ-induced Egr-1 promoter activity and increased Egr-1 mRNA stability may explain the dramatic induction of Egr-1 at the protein level. TGZ activates ERK1/2 activity in smooth muscle cells (19Takeda K. Ichiki T. Tokunou T. Iino N. Takeshita A. J. Biol. Chem. 2001; 276: 48950-48955Google Scholar), and the activated ERK pathway induces Egr-1 activity (36Sakaue M. Adachi H. Dawson M. Jetten A.M. Cell Death Differ. 2001; 8: 411-424Google Scholar, 37Kaufmann K. Bach K. Thiel G. Biol. Chem. 2001; 382: 1077-1081Google Scholar). In addition, ERK phosphorylates PPARγ, which results in the inactivation of PPARγ activity (38Reginato M.J. Krakow S.L. Bailey S.T. Lazar M.A. J. Biol. Chem. 1998; 273: 1855-1858Google Scholar, 39Hu E.
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