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• W2087535194 abstract "Activating transcription factor 3 (ATF3) is a transcriptional repressor that is rapidly induced in cells exposed to a wide range of stress stimuli. To clarify the role of ATF3 in determining cell fate, we overexpressed it in human umbilical vein endothelial cells (HUVECs) by adenovirus-mediated gene transfer. ATF3 protected these cells from tumor necrosis factor (TNF)-α-induced apoptosis, as measured by flow cytometric analysis, trypan blue exclusion assay, and cleavage of procaspase 3 and poly(ADP-ribose) polymerase. Northern blot and nuclear run on assay showed that the transcription of tumor suppressor gene p53 was down-regulated in the ATF3-overexpressing cells. In the transient expression assay, ATF3 suppressed the p53 gene promoter activity through its specific binding to an atypical AP-1 element, PF-1 site, in the p53 gene promoter. Furthermore, the cell-protecting effect of ATF3 was remarkably reduced inp53-deficient cells. These results demonstrate that overexpression of ATF3 suppresses TNF-α-induced cell death of HUVECs, at least in part, through down-regulating the transcription ofp53 gene. ATF3 may function as a cell survival factor of endothelial cells during vascular inflammation and atherogenesis. Activating transcription factor 3 (ATF3) is a transcriptional repressor that is rapidly induced in cells exposed to a wide range of stress stimuli. To clarify the role of ATF3 in determining cell fate, we overexpressed it in human umbilical vein endothelial cells (HUVECs) by adenovirus-mediated gene transfer. ATF3 protected these cells from tumor necrosis factor (TNF)-α-induced apoptosis, as measured by flow cytometric analysis, trypan blue exclusion assay, and cleavage of procaspase 3 and poly(ADP-ribose) polymerase. Northern blot and nuclear run on assay showed that the transcription of tumor suppressor gene p53 was down-regulated in the ATF3-overexpressing cells. In the transient expression assay, ATF3 suppressed the p53 gene promoter activity through its specific binding to an atypical AP-1 element, PF-1 site, in the p53 gene promoter. Furthermore, the cell-protecting effect of ATF3 was remarkably reduced inp53-deficient cells. These results demonstrate that overexpression of ATF3 suppresses TNF-α-induced cell death of HUVECs, at least in part, through down-regulating the transcription ofp53 gene. ATF3 may function as a cell survival factor of endothelial cells during vascular inflammation and atherogenesis. tumor necrosis factor-α human umbilical vein endothelial cell phosphate-buffered saline phenylmethylsulfonyl fluoride fluorescence-activated cell sorter glutathioneS-transferase activator protein-1 p53 factor-1 Jun dimerization partners-2 cAMP response element-binding protein cAMP response element basic region and leucine zipper poly(ADP-ribose) polymerase c-Jun NH2-terminal kinase Non-adhesive and non-thrombotic feature of vascular endothelial surface is essential to maintaining physiological homeostasis of vascular function. When this function is perturbed, a cascade of thrombogenic and atherogenic reaction may occur. Injured endothelial cells become procoagulant and promote the formation of thrombi. They also attract phagocytes and produce cytokines and growth factors that act on adjacent vascular cells to promote their growth. Thus, endothelial cell death may play a pivotal role in the pathogenesis of vascular diseases such as vascular inflammation, thrombosis, and atherosclerosis (1Karsan A. Harlan J.M. J. Atheroscler. Thromb. 1996; 3: 75-80Crossref PubMed Scopus (55) Google Scholar). Tumor necrosis factor (TNF)1-α is a cytokine produced by many cell types, including macrophages, monocytes, lymphoid cells, and fibroblasts, in response to inflammation, infection, and various environmental stimuli (2Tracey K.J. Cerami A. Annu. Rev. Cell Biol. 1993; 9: 317-343Crossref PubMed Scopus (761) Google Scholar, 3Vandenabeele P. Declercg W. Beyaert R. Fiers W. Trends Cell Biol. 1995; 5: 392-399Abstract Full Text PDF PubMed Scopus (739) Google Scholar). This cytokine is associated with a variety of cellular defense responses and activation of beneficial and cell-protecting genes. Simultaneously, TNF-α also causes a myriad of lethal effects such as septic shock, tissue injury, inflammation, and cachexia, and these effects may be associated with apoptosis in susceptible cells. Although intact endothelial cells are rather resistant to TNF-α, they clearly undergo apoptotic cell death when exposed to TNF-α (4Robaye B. Mosselmans R. Fiers W. Dumont J.E. Galand P. Am. J. Pathol. 1991; 138: 447-453PubMed Google Scholar, 5Polunovsky V.A. Wendt C.H. Ingbar D.H. Peterson M.S. Bitterman P.B. Exp. Cell Res. 1994; 214: 584-594Crossref PubMed Scopus (272) Google Scholar, 6Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). This becomes more significant in the presence of a low amount of RNA or protein synthesis inhibitors such as actinomycin D (5Polunovsky V.A. Wendt C.H. Ingbar D.H. Peterson M.S. Bitterman P.B. Exp. Cell Res. 1994; 214: 584-594Crossref PubMed Scopus (272) Google Scholar, 6Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Thus, TNF-α is considered to play roles in the pathogenesis and progression of vascular diseases. In TNF-α-induced apoptosis, factors such as TRADD, FADD/MORT, RIP, FLICE/MACH, and TRAFs associate with cell surface receptor TNFR1 in initiating the TNF-α-induced signaling pathway (7Liu Z-g. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 8Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4559) Google Scholar). In contrast, downstream events connecting the initial receptor binding and the final apoptotic process are relatively unknown, although the sphingomyelin pathway and ceramide are reported to be involved. Tumor suppressor protein p53 is activated in response to DNA damage or a wide range of stress stimuli. This leads to growth arrest and provides sufficient time for cells to repair damage. Alternatively, p53 can trigger apoptosis and thus eliminate cells that have been damaged beyond repair. In TNF-α-induced apoptosis, p53 is also implicated in the death process. For instance, p53 is involved in cytotoxic activity of TNF-α in c-Myc-expressing cells (9Klefstrom J. Arighi E. Littlewood T. Jaattela M. Saksela E. Evan G.I. Alitalo K. EMBO J. 1997; 16: 7382-7392Crossref PubMed Scopus (107) Google Scholar), and its loss of function is associated with resistance of MCF7 human breast carcinoma cells to TNF-α (10Cai Z. Capoulade C. Moyret-Lalle C. Amor-Gueret M. Feunteun J. Larsen A.K. Bressac-de Paillerets B. Chouaib S. Oncogene. 1997; 15: 2817-2826Crossref PubMed Scopus (82) Google Scholar). Furthermore, p53 is activated in TNF-induced apoptosis of human promonocytic cells (11Yeung M.C. Lau A.S. J. Biol. Chem. 1998; 273: 25198-25202Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) or ME-180 cells (12Donato N.J. Perez M. J. Biol. Chem. 1998; 273: 5067-5072Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Many studies show that translational and post-translational events are major regulatory steps in p53 activation (13Ewen M. Miller S. Biochim. Biophys. Acta. 1996; 1242: 181-184PubMed Google Scholar). Recently, however, it has been shown that c-jun null cells express elevated levels ofp53 mRNA, and reintroduction of a constitutive c-jun allele represses p53 transcription (14Schreiber M. Kolbus A. Piu F. Szabowski A. Mohle-Steinlein U. Tian J. Karin M. Angel P. Wagner E.F. Genes Dev. 1999; 13: 607-619Crossref PubMed Scopus (467) Google Scholar,15Shaulian E. Schreiber M. Piu F. Beeche M. Wagner E.F. Karin M. Cell. 2000; 103: 897-907Abstract Full Text Full Text PDF PubMed Google Scholar). Furthermore, AP-1 repressor protein JDP-2 inhibits UV-mediated apoptosis through down-regulation of p53 (16Piu F. Aronheim A. Katz S. Karin M. Mol. Cell. Biol. 2001; 21: 3012-3024Crossref PubMed Scopus (56) Google Scholar). Therefore, the transcriptional regulation of p53 also contributes to its accumulation in response to stress. However, at this moment, our knowledge regarding one or more transcription factors that control the expression of p53 gene in response to stress stimuli is rather limited. Activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily, is a bZip transcription factor (17Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar, 18Hsu J.-C. Laz T. Mohn K.L. Taub R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3511-3515Crossref PubMed Scopus (120) Google Scholar). It forms a homodimer that represses transcription from promoters with ATF sites (17Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar), TNF-α-induced E-selectin gene expression (19Nawa T. Nawa T.M. Cai Y. Zhang C. Uchimura I. Narumi S. Numano F. Kitajima S. Biochem. Biophys. Res. Commun. 2000; 87: 565-576Google Scholar), and arsenite-responsive activation of GADD153 gene (20Fawcett T.W. Martindale J.L. Guyton K.Z. Hai T. Holbrook N.J. Biochem. J. 1999; 339: 135-141Crossref PubMed Scopus (368) Google Scholar). Heterodimers of ATF3 with c-Jun and JunB, on the other hand, activate transcription in transient transfection assays (21Hsu J.-C. Bravo R. Taub R. Mol. Cell. Biol. 1992; 12: 4654-4665Crossref PubMed Scopus (158) Google Scholar). ATF3 therefore can repress or activate target genes by forming homo- or heteromeric complexes. ATF3 is rapidly induced by ischemia-coupled reperfusion in heart and kidney (22Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (258) Google Scholar, 23Yin T. Sandhu G. Wplfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. Chem. 1997; 272: 19943-19950Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) and several stimuli such as anisomycin (17Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar), anti-cancer drugs (24Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.-N.T. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (151) Google Scholar), genotoxic agents, or ionizing radiation (25Amundson S.A. Bittner M. Chen Y. Trent J. Meltzer P. Fornace A.J. Oncogene. 1999; 18: 3666-3672Crossref PubMed Scopus (298) Google Scholar), which can all induce cell cycle arrest and apoptotic cell death. We previously reported that ATF3 is rapidly induced in human vascular endothelial cells in response to TNF-α, oxidized low density lipoprotein, and homocysteine (26Nawa T. Nawa T.M. Adachi T.M. Uchimura I. Shimokawa R. Fujisawa K. Tanaka A. Numano F. Kitajima S. Atherosclerosis. 2001; 161: 281-291Abstract Full Text Full Text PDF Scopus (64) Google Scholar, 27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). However, it remains unclear whether ATF3 functions as proapoptotic or antiapoptotic factor or which one or more target genes are regulated by ATF3 in this process. In this study, we overexpressed ATF3 in human umbilical vein endothelial cells (HUVECs) by adenovirus-mediated gene transfer and found that ATF3 protected them from TNF-α-induced apoptosis. Furthermore, ATF3 was shown to repress the transcription of thep53 gene. This suggests that ATF3 functions as a cell-survival factor through down-regulation of p53 at the transcriptional level. ATF3 could be a novel therapeutic target in preventing pathogenesis and progression of vascular diseases. Recombinant human TNF-α was purchased from Genzyme (Cambridge, MA), and actinomycin D was obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit anti-ATF3 (C-19), monoclonal anti-p53, and anti-glutathione S-transferase (GST) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-CPP32 antibody was from Transduction Laboratories (Lexington, KY), and monoclonal anti-poly(ADP-ribose) polymerase (PARP) antibody was purchased from R&D systems (Minneapolis, MN). Expression plasmid for human ATF3, pCI-ATF3, was as described previously (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). GST-ATF3 fusion protein was expressed inEscherichia coli using pGEX4T-1 vector and purified as in protocol from Amersham Biosciences. HUVECs were obtained and grown in EGM-2 medium (Clonetics Corp., San Diego, CA) as described (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). Cells of passages 2–3 were used in this study to minimize age-dependent variation in the level of apoptosis. For transient expression, plasmid DNA was vortex-mixed with SuperFect (Qiagen, Chatsworth, CA) and transfected into cells as previously described (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). Human osteosarcoma Saos-2 cells with p53null allele and human breast cancer MCF-7 cells with wild typep53 allele were cultured in Dulbecco's minimum essential medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Adenovirus vector encoding the human ATF3 gene (AdATF3) was constructed by using an Adenovirus Expression Vector kit obtained from Takara (Otsu, Japan). Briefly, a blunt-ended cDNA fragment encoding ATF3 was subcloned into theSwaI site of the E1-deleted region of cassette cosmid vector pAxCAwt, and co-transfected into 293 cells with DNA-terminal protein complex (28Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (787) Google Scholar). Recombinant virus positive for ATF3 was screened by immunoblot analysis using anti-ATF3 antibody and cloned by limiting dilution. Adenovirus encoding β-galactosidase (AdLacZ) was a generous gift from Dr. Saito of Tokyo University, Japan. These adenoviruses were plaque-purified, and their titers were determined by titration in 293 cells. For adenovirus-mediated gene transfer, HUVECs were exposed to adenoviral vectors at the indicated of multiplicity of infection (m.o.i.) and cultured for the indicated time. HUVECs (3 × 105 cells) were infected with AdATF3 or AdLacZ viruses for 48 h and treated with 10 ng/ml TNF-α and 0.1 μg/ml actinomycin D. Both floating and attached cells were combined, washed once with PBS, and fixed with 70% ice-cold ethanol. After staining with 50 μg/ml propidium iodide, the DNA content of the cells was analyzed by using a FACScan (Becton Dickinson). Apoptotic cells were quantitated by the percentage of cells with a subG1 DNA content. HUVECs were infected with AdATF3 or AdLacZ viruses for 48 h and treated with the indicated concentration of TNF-α in the presence of 0.1 μg/ml actinomycin D for 18 h. Both attached and floating cells were combined and stained with 0.2% trypan blue. Viable cells were counted and expressed as a proportion of cells treated with actinomycin D alone. HUVECs (3 × 105 cells) treated as indicated were washed in PBS, resuspended in 50 μl of lysis buffer (50 mm Hepes-KOH, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1.5 mm MgCl2, 1 mmEGTA, 0. 1 mm PMSF, 10 μg/ml each leupeptin and aprotinin, 200 μm sodium vanadate, 100 mmNaF, and 10% glycerol), and incubated on ice for 10 min. The cells were centrifuged at 10,000 rpm for 10 min, and the supernatant was taken as whole cell extract. The amounts of protein were measured by Lowry method using bovine serum albumin as standard (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Whole cell extracts (20 μg of protein) were separated on an SDS-PAGE, transferred onto a nitrocellulose membrane, and subjected to Western blot using the protocol of ECL kit (Amersham Biosciences) as described previously (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). Total RNA was isolated by the acid guanidinium method using Isogen (Nippon Gene, Japan), fractionated on a formaldehyde-agarose gel, transferred to a Hybond-N membrane, and hybridized to random-primed cDNA probe for the human p53 gene as described (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). The membrane was exposed and analyzed by Bas 2500 Bio-image analyzer (Fujifilm Co., Tokyo, Japan). DNA fragment for the human p53 cDNA, 2.0 kb, was radiolabeled with [α-32P]dCTP (6000 Ci/mmol,Amersham Biosciences) using a random primer-labeling kit from Takara and used as probe. HUVECs (1 × 107cells) were infected with AdATF3 or AdLacZ viruses for 48 h, and their nuclei were prepared and frozen in liquid nitrogen as described previously (30Noda A. Toma-Aiba Y. Fujiwara Y. Oncogene. 2000; 19: 21-31Crossref PubMed Scopus (24) Google Scholar). Elongation of nascent RNA chains was initiated by mixing the nuclei with 100 μl of reaction buffer (10 mmTris-HCl, pH 8.0; 5 mm MgCl2; 300 mm KCl; 0.5 mm each of ATP, CTP, and GTP; and 100 μCi of [α-32P]UTP (3000 Ci/mmol, AmershamBiosciences) and incubating at 30 °C for 30 min. RNA synthesis was terminated by incubating with 5 μg/ml RNase-free DNase I (Roche Molecular Biochemicals) at 30 °C for 10 min. The mixture was then digested with proteinase K (200 μg/ml) in 10 mm Tris-HCl, pH 7.4, 5 mm EDTA, and 1% SDS at 50 °C for 1 h, and radiolabeled nuclear transcripts were separated from unincorporated nucleotides on Sephadex G-50 column equilibrated with 10 mmTris-HCl, pH 7.5, 1 mm EDTA, and 1% SDS. The labeled RNA was boiled for 3 min, chilled on ice, and hybridized to human glyceraldehyde-3-phosphate dehydrogenase cDNA, humanp53 cDNA, and plasmid pBR322 (10 μg each) immobilized on a Hybond-N membrane at 42 °C for 48 h. The membrane filters were analyzed by Bas 2500 Bio-image analyzer (Fujifilm). Reporter plasmid containing a 2.4-kb fragment of the human p53 gene promoter, pLuc-2400, was as described (30Noda A. Toma-Aiba Y. Fujiwara Y. Oncogene. 2000; 19: 21-31Crossref PubMed Scopus (24) Google Scholar). The 5′-deletion mutants of reporter genes were prepared by PCR-amplifying and subcloning of DNA fragments from −460, −290, and −110 to +6 into the XhoI and BglII sites of PicaGene pGVB2 (Toyobo, Osaka, Japan), to produce pLuc-460, -290, and -110, respectively. pLuc-460mPF-1 and pLuc-460mCRE-BP1 containing the mutant PF-1 site from −184 to −177 and the mutant CRE-BP1 site from −402 to −395, respectively, were prepared from pLuc-460 using a QuikChange mutagenesis kit from Stratagene (La Jolla, CA). The mutant PF-1 and CRE-BP1 sites contained the sequence of 5′-TGAATTC-3′ and 5′-TTTGGGAA-3′ (mutated nucleotides underlined) compared with wild type PF-1 sequence, 5′-TGACTCT-3′, and CRE-BP1 sequence, 5′-TTACGGAA-3′, respectively. HUVECs (3 × 105 cells in a 35-mm dish) were transfected with 1 μg of human p53 reporter plasmid along with pCIATF3 as described (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). At 48 h post-transfection, cells were harvested and washed once in phosphate-buffered saline, and cell extracts were assayed for basal p53 promoter activity. For the activated p53 promoter activity, cells were further stimulated by 10 ng/ml TNF-α in the presence of 0.1 μg/ml actinomycin D for another 10 h. Both firefly and sea pansy luciferase activities were measured using a dual luciferase reporter assay system according to the manufacture's protocol (Promega, Madison, WI). pRL-TK (Toyo Ink, Tokyo, Japan) containing the sea pansy luciferase gene was used as an internal control of transfection and expression. Nuclear extracts were prepared from the AdATF3 or AdLacZ-infected HUVECs (2 × 107 cells) as described (27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar). Recombinant GST-ATF3 fusion protein was expressed in E. coli and purified as described under “Experimental Procedures.” Nuclear extract protein (2 μg) or GST-ATF3 protein (0.5 μg) were incubated in 20 μl of binding buffer (10 mm HEPES-KOH, pH 7.9, 60 mm KCl, 0.5 mm EDTA, 5 mm MgCl2, 0.1 mm PMSF, 5 mm β-mercaptoethanol) containing 0.5 μg of poly(dI-dC) and 0.5 ng of radiolabeled DNA probe at room temperature for 30 min. For supershift assays, anti-ATF3 antibody (0.1 μg) was added and incubated for another 30 min. DNA probe was obtained by annealing 0.1 μg each of sense and antisense oligonucleotides for PF-1 site, 5′-GCGAGAATCCTGACTCTGCACC-3′ (31Ginsberg D. Oren M. Yaniv M. Piette J. Oncogene. 1990; 5: 1285-1290PubMed Google Scholar, 32Reisman D. Loging W.T. Semin. Cancer Biol. 1998; 8: 317-324Crossref PubMed Scopus (54) Google Scholar), and radiolabeled with 25 μCi of [γ-32P]ATP (6000 Ci/mmol) and polynucleotide kinase. Mutant oligonucleotide used for the competition experiment contained substitutions in the normal sequence of 5′-TGACTCT-3′ to 5′-TGAATTC-3′. Binding mixture was applied onto a 5% non-denatured polyacrylamide slab gel in Tris borate EDTA buffer. After electrophoresis, the gel was dried on a 3MM Whatman paper and visualized by Fuji Bas 2500 image analyzer. Multiple comparisons were evaluated by analysis of variance followed by Scheffe's post hoc test. Data are presented as mean ± S.D. Statistical significance was assigned at the level of p < 0.05. To study the functional role of ATF3 in endothelial cell apoptosis, we expressed ATF3 in HUVECs by infecting cells with adenoviral expression vector AdATF3. Fig. 1 showed that the increasing titer of AdATF3 expressed ATF3 protein in a dose-dependent manner, while AdLacZ, which encoded β-galactosidase, did not express ATF3 and served as control. The expression of ATF3 did not change the cell viability for at least 4 days after infection, suggesting that overexpression of ATF3 by itself was not capable of causing apoptosis. We then treated HUVECs by TNF-α in the presence of actinomycin D, under which HUVECs undergo significant apoptotic cell death (5Polunovsky V.A. Wendt C.H. Ingbar D.H. Peterson M.S. Bitterman P.B. Exp. Cell Res. 1994; 214: 584-594Crossref PubMed Scopus (272) Google Scholar, 6Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). This treatment caused a rapid induction of ATF3 expression as significantly as AdATF3 (Fig. 1 and Ref. 26Nawa T. Nawa T.M. Adachi T.M. Uchimura I. Shimokawa R. Fujisawa K. Tanaka A. Numano F. Kitajima S. Atherosclerosis. 2001; 161: 281-291Abstract Full Text Full Text PDF Scopus (64) Google Scholar). Next, the effect of ATF3 expression on TNF-α-induced apoptosis was examined. As shown in theupper panel of Fig.2 A, FACS analysis revealed that the number of cells at the subG1 region, i.e.hypodiploid, increased after treatment with TNF-α for 6 and 18 h in the AdLacZ-infected cells. Under this condition, overexpression of ATF3 remarkably suppressed the number of subG1 cells (Fig.2 A, lower panel). This inhibition of apoptosis was observed by ∼3 times higher level of ATF3 than in the TNF-treated HUVECs (compare the ATF3 expression at m.o.i. of 50 with that at 6 h of TNF-treated cells in Fig. 1). These data indicate that ATF3 may protect HUVECs from TNF-α-induced cell death.Figure 2TNF-α-induced cell death is inhibited in AdATF3-infected HUVECs. A, HUVECs were infected with AdLacZ (upper panel) or AdATF3 (lower panel) at an m.o.i. of 50. At 48 h post-infection, cells were stimulated with 10 ng/ml TNF-α in the presence of 0.1 μg/ml actinomycin D. After further incubation for 6 and 18 h, cells were subjected to FACS analysis as under “Experimental Procedures.”B, cell viability was assayed by trypan blue dye exclusion method. HUVECs infected with AdATF3 (○), AdLacZ (■), or uninfected control (▵), were stimulated by increasing amounts of TNF-α in the presence of 0.1 μg/ml actinomycin D, and cell viability was expressed as a percentage to that treated with actinomycin D only. Results are the mean ± S.D. of three independent experiments. C, HUVECs treated as in A were examined by phase contrast microscopy. Cells uninfected (panels a and d) or infected with AdLacZ (panels c and f) became round and detached from the dish after treatment with 10 ng/ml TNF-α in the presence of 0.1 μg/ml actinomycin D for 18 h. In contrast, most of cells that were infected with AdATF3 appeared normal and remained attached to dish (panels b ande).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we further examined the effect of ATF3 on cell viability in the TNF-α-treated HUVECs. For this purpose, HUVECs infected with AdATF3 or AdLacZ were treated with TNF-α, and their viability was determined. As shown in Fig. 2 B, treatment with increasing amounts of TNF-α decreased cell viability of the control and AdLacZ-infected HUVECs, as measured by trypan blue exclusion assay. On the other hand, overexpression of ATF3 significantly prevented the decrease of cell viability. These cells were also examined on microscopy. Most of the uninfected control and AdLacZ-infected HUVECs exhibited a round shape change and became detached from the dish (Fig. 2 C, panels d andf), indicative of cell death. In contrast, the AdATF3 cells retained the normal shape and remained attached to the dish (Fig.2 C, panel e). Thus, it is indicated that ATF3 prevents the decrease of cell viability of HUVECs induced by TNF-α. The caspase family of cysteine proteases is implicated in the apoptotic process of numerous cells, and its target substrate such as PARP is proteolytically cleaved. To obtain biochemical evidence for the protective effect of ATF3 in the TNF-α-induced apoptosis, we examined the activation and cleavage of procaspase 3 and PARP in HUVECs that overexpressed ATF3. Fig.3 (upper panel) showed that procaspase 3 was activated to yield a 16-kDa fragment in the uninfected control and AdLacZ-infected cells after exposure to TNF-α, whereas its activation was suppressed in the AdATF3-infected cells (upper panel). PARP was cleaved after TNF-α treatment in the control and AdLacZ cells and this cleavage was also suppressed in the ATF3-expressing cells (Fig. 3, lower panel). These data indicate that ATF3 suppresses the activation and cleavage of procaspase 3 and PARP in the TNF-α-stimulated HUVECs. The data above suggested that the expression of proapoptotic or antiapoptotic genes may be affected by ATF3, which leads to suppression of endothelial cell death in response to TNF-α. As a first step to elucidate the mechanism of action of ATF3, we examined for such genes whose regulation is correlated with inhibition of apoptosis. Fig.4 A showed that p53mRNA was significantly down-regulated in the AdATF3-infected HUVECs, although it was not affected in the AdLacZ cells. In Fig.4 B, we performed nuclear run-on assay to determine whether the reduced level of p53 mRNA was due to the decreased transcription. Results showed that the p53 transcription was significantly suppressed in the AdATF3-infected cells. This effect appeared to be specific, because AdLacZ cells showed no such suppression and the activity of glyceraldehyde-3-phosphate dehydrogenase gene transcription was comparable between these cells. These data clearly shows that overexpression of ATF3 down-regulatesp53 mRNA at the level of transcription. We next studied the expression ofp53 mRNA of the AdATF3-infected HUVECs in response to TNF-α. Fig. 5 A showed thatp53 mRNA was induced by ∼2.3-fold in the AdLacZ cells at 2 h post-stimulation by TNF-α. In the AdATF3-cells, basal level of p53 mRNA was remarkably reduced, and its activation was almost abolished at 2 h post-stimulation, although it appeared to slightly increase at 24 h post-stimulation. The activation of p53 protein of these cells was also examined. As shown in Fig. 5 B, p53 protein was rapidly accumulated in the AdLacZ cells in response to TNF-α. In the AdATF3 cells, both basal and stimulated level of p53 protein was significantly reduced compared with that of AdLacZ cells. However, the extent of activation by TNF-α appeared comparable between AdLacZ and AdATF3 cells, 2.98- and 2.85-fold, respectively, suggesting that stability of p53 protein was not affected by ATF3. These data together indicate that the expression of ATF3 down-regulates both basal and stimulated level ofp53 mRNA and this leads to decreased activation of p53 protein. Transcriptional regulation of p53 gene contributes to its activation in response to stress stimuli (14Schreiber M. Kolbus A. Piu F. Szabowski A. Mohle-Steinlein U. Tian J. Karin M. Angel P. Wagner E.F. Genes Dev. 1999; 13: 607-619Crossref PubMed Scopus (467) Google Scholar, 15Shaulian E. Schreiber M. Piu F. Beeche M. Wagner E.F. Karin M. Cell. 2000; 103: 897-907Abstract Full Text Full Text PDF PubMed Google Scholar, 16Piu F. Aronheim A. Katz S. Karin M. Mol. Cell. Biol. 2001; 21: 3012-3024Crossref PubMed Scopus (56) Google Scholar). Because the data above suggested that p53 gene transcription might be regulated by ATF3, we transfected the human p53reporter gene, pLuc-2400, into HUVECs and examined whether ATF3 affected the p53 promoter activity. Fig.6 A showed that the promoter activity of pLuc-2400 was suppressed by c" @default.
• W2087535194 created "2016-06-24" @default.
• W2087535194 creator A5020248828 @default.
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• W2087535194 title "Transcriptional Repressor Activating Transcription Factor 3 Protects Human Umbilical Vein Endothelial Cells from Tumor Necrosis Factor-α-induced Apoptosis through Down-regulation ofp53 Transcription" @default.
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