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• W2053143976 abstract "Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) present in tobacco smoke and tar, have been implicated in the development of atherosclerosis as well as cancer. Increased expression of cyclooxygenase-2 (COX-2) has been detected both in atherosclerotic lesions and in epithelial cancers. To determine whether polycyclic aromatic hydrocarbons might directly affect COX expression in vascular cells, we investigated the effects of B[a]P on COX-2 expression in human and rat arterial smooth muscle cells (SMC). Treatment with B[a]P increased levels of COX-2 protein and mRNA and enhanced prostaglandin synthesis. Nuclear runoff assays and transient transfections revealed increased COX-2gene transcription after treatment with B[a]P. Experiments were done to define the signaling mechanism by which B[a]P induced COX-2. B[a]P caused a rapid increase in phosphorylation of extracellular signal-regulated kinase (ERK); pharmacologic inhibition of mitogen-activated protein kinase kinase blocked B[a]P-mediated induction of COX-2. Depletion of the intracellular antioxidant, glutathione, with buthionine sulfoximine significantly increased B[a]P-mediated induction of COX-2 while exposure to N-acetylcysteine, a precursor of glutathione, suppressed the induction of COX-2 by B[a]P. Several lines of evidence suggest that the induction of COX-2 by B[a]P is mediated, at least in part, by NF-κB. Treatment with B[a]P increased binding of NF-κB to DNA. Moreover, B[a]P-mediated stimulation of COX-2 promoter activity was blocked when a construct containing a mutagenized NF-κB site was used. Pharmacological inhibitors of NF-κB blocked the induction of COX-2 protein and the stimulation of COX-2 promoter activity by B[a]P. Taken together, these data are likely to be important for understanding the atherogenic effects of tobacco smoke. Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) present in tobacco smoke and tar, have been implicated in the development of atherosclerosis as well as cancer. Increased expression of cyclooxygenase-2 (COX-2) has been detected both in atherosclerotic lesions and in epithelial cancers. To determine whether polycyclic aromatic hydrocarbons might directly affect COX expression in vascular cells, we investigated the effects of B[a]P on COX-2 expression in human and rat arterial smooth muscle cells (SMC). Treatment with B[a]P increased levels of COX-2 protein and mRNA and enhanced prostaglandin synthesis. Nuclear runoff assays and transient transfections revealed increased COX-2gene transcription after treatment with B[a]P. Experiments were done to define the signaling mechanism by which B[a]P induced COX-2. B[a]P caused a rapid increase in phosphorylation of extracellular signal-regulated kinase (ERK); pharmacologic inhibition of mitogen-activated protein kinase kinase blocked B[a]P-mediated induction of COX-2. Depletion of the intracellular antioxidant, glutathione, with buthionine sulfoximine significantly increased B[a]P-mediated induction of COX-2 while exposure to N-acetylcysteine, a precursor of glutathione, suppressed the induction of COX-2 by B[a]P. Several lines of evidence suggest that the induction of COX-2 by B[a]P is mediated, at least in part, by NF-κB. Treatment with B[a]P increased binding of NF-κB to DNA. Moreover, B[a]P-mediated stimulation of COX-2 promoter activity was blocked when a construct containing a mutagenized NF-κB site was used. Pharmacological inhibitors of NF-κB blocked the induction of COX-2 protein and the stimulation of COX-2 promoter activity by B[a]P. Taken together, these data are likely to be important for understanding the atherogenic effects of tobacco smoke. benzo[a]pyrene B[a]P-diolexpoxide cyclooxygenase extracellular signal-regulated kinase enhanced chemiluminescence mitogen-activated protein kinase nuclear factor κB prostaglandin E2 6-keto-prostaglandin F1a phorbol 12-myristate 13-acetate Dulbecco's modified Eagle's medium fetal bovine serum smooth muscle cell buthionine sulfoximine N-acetylcysteine pyrrolidinedithiocarbamate Cardiovascular disease remains one of the major causes of death in industrialized nations. Many studies have suggested that tobacco smoking is a major contributor to cardiovascular disease (1.Kannel W.B. Am. Heart J. 1981; 101: 319-328Crossref PubMed Scopus (253) Google Scholar, 2.Jacobs D.R. Adachi H. Mulder I. Kromhout D. Menotti A. Nissine K. Blackburn H. Arch. Intern. Med. 1999; 159: 733-740Crossref PubMed Scopus (210) Google Scholar, 3.Hanrahan J.P. Sherman C.B. Bresnetz E.A. Emmons K.M. Mannino D.M. Am. J. Respir. Crit. Care Med. 1996; 153: 861-865Crossref PubMed Scopus (205) Google Scholar). Smoking promotes platelet hyperreactivity, accelerates atherosclerotic plaque formation, and increases ischemic tissue damage. Benzo[a]pyrene (B[a]P),1 a polycyclic aromatic hydrocarbon present in tobacco smoke, is metabolized in tissue to mutagenic derivatives which form DNA adducts within target cells (4.Bond J.A. Yang H.L. Majesky M.W. Benditt E.P. Juchau M.R. Toxicol. Appl. Pharmacol. 1980; 52: 323-325Crossref PubMed Scopus (46) Google Scholar, 5.Qu S. Stacey N.H. Carcinogenesis. 1996; 17: 53-59Crossref PubMed Scopus (34) Google Scholar, 6.Denissenko M. Pao A. Tang M. Pfeifer G. Science. 1996; 274: 430-432Crossref PubMed Scopus (1504) Google Scholar). Considerable experimental evidence suggests that B[a]P accelerates smooth muscle proliferation and promotes atherosclerosis in animals ranging from chickens to rats (7.Albert R.E. Vanderlaan M. Burns F.J. Nishizumi M. Cancer Res. 1977; 37: 2232-2235PubMed Google Scholar, 8.Bond J.A. Kocan R.M. Benditt E.P. Juchau M.R. Life Sci. 1979; 25: 425-430Crossref PubMed Scopus (45) Google Scholar, 9.Hough J.L. Baird M.B. Sfeir G.T. Pacini C.S. Darrow D. Wheelock C. Arterioscler. Thromb. 1993; 13: 1721-1727Crossref PubMed Google Scholar, 10.Ou X. Ramos K.S. Toxicology. 1992; 74: 243-258Crossref PubMed Scopus (35) Google Scholar, 13.Izzotti A. De flora S. Petrilli G.L. Gallagher A. Rojas M. Alexandrow K. Bartsch H. Lewtas J. Cancer Epidemiol. Biomark. Prev. 1995; 4: 111-115PubMed Google Scholar). In addition, we and others have detected B[a]P-related DNA adducts in atherosclerotic arteries (11.Zhang Y.-J. Weksler B.B. Wang L.Y. Schwartz J. Santella R.M. Atherosclerosis. 1998; 140: 325-331Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 12.Izzotti A. D'Agostini F. Bagnasco M. Scatolini L. Rovida A. Balansky R.M. Cesarone C.F. De Flora S. Cancer Res. 1994; 54 (suppl.): 1994-1998Google Scholar, 13.Izzotti A. De flora S. Petrilli G.L. Gallagher A. Rojas M. Alexandrow K. Bartsch H. Lewtas J. Cancer Epidemiol. Biomark. Prev. 1995; 4: 111-115PubMed Google Scholar). In some systems, B[a]P up-regulates the expression of proto-oncogenes, such as c-Ha-ras and c-myc, that favor cell proliferation (14.Ramos K.S. Zhang Y. Sadhu D.N. Chapkin R.S. Arch. Biochem. Biophys. 1996; 15: 213-222Crossref Scopus (32) Google Scholar). Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the oxygenation of arachidonic acid to prostaglandin endoperoxides which are converted enzymatically into prostaglandins (PGs) and thromboxane A2 that play both physiologic and pathologic roles in vascular function. COX is capable of cooxidizing B[a]P; the peroxyl radicals formed during arachidonate metabolism catalyze the epoxidation of B[a]P to its mutagenic products (15.Guthrie J. Robertson L.G.C. Zeiger E. Boyd J.A. Eling T.E. Cancer Res. 1982; 42: 1620-1623PubMed Google Scholar, 16.Marnett L.J. Reed G.A. Dennison D.J. Biochem. Biophys. Res. Commun. 1978; 82: 210-216Crossref PubMed Scopus (106) Google Scholar, 17.Eling T.E. Curtis J.F. Pharmacol. Ther. 1992; 53: 261-273Crossref PubMed Scopus (74) Google Scholar). Two distinct isoforms of COX have been identified (18.Smith W.L. Garavito R.M. De Witt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1847) Google Scholar). COX-1 is constitutively expressed at low levels in most tissues. In contrast, COX-2, the product of a related inducible gene, is absent from most normal tissues but is expressed in response to proliferative and inflammatory stimuli such as growth factors and cytokines (19.Jones D.A. Carlton D.P. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar, 20.Herschman H.R. Biochim. Biophys. Acta. 1996; 1299: 125-140Crossref PubMed Scopus (1163) Google Scholar, 21.Pritchard K.A. O'Banion M.K. Miano J.M. Vlasic N. Bhatia U.G. Young D.A. Stemerman M.B. J. Biol. Chem. 1994; 269: 8504-8509Abstract Full Text PDF PubMed Google Scholar, 22.Sheng H. Shao J. O'Mahony C.A. Lamps L. Albo D. Isakson P.C. Berger D. DuBois R. Beauchamp R.D. Oncogene. 1999; 18: 855-867Crossref PubMed Scopus (66) Google Scholar, 23.Rimarachin J.A. Jacobson J.A. Szabo P. Maclouf J. Creminon C. Weksler B.B. Arterioscler. Thromb. 1994; 14: 1021-1031Crossref PubMed Google Scholar, 24.Beasley D. Am. J. Physiol. 1999; 276: H1369-H1378PubMed Google Scholar, 25.Kelley D.J. Mestre J.R. Subbaramaiah K. Sacks P.G. Schantz S.P. Tanabe T. Inoue H. Ramonetti J.T. Dannenberg A.J. Carcinogenesis. 1997; 18: 795-799Crossref PubMed Scopus (148) Google Scholar). Overexpression of COX-2 in epithelial cells inhibits apoptosis and increases the invasiveness of malignant cells, favoring tumorigenesis and metastasis (22.Sheng H. Shao J. O'Mahony C.A. Lamps L. Albo D. Isakson P.C. Berger D. DuBois R. Beauchamp R.D. Oncogene. 1999; 18: 855-867Crossref PubMed Scopus (66) Google Scholar, 26.Tsujii M. DuBois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2137) Google Scholar, 27.Liu X.Y. Yao S. Kirschenbaum A. Levine A.C. Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar, 28.Sheng H.M. Shao J.Y. Morrow J.D. Beauchamp R.D. DuBois R.N. Cancer Res. 1998; 58: 362-366PubMed Google Scholar, 29.Boolbol S.K. Dannenberg A.J. Chadburn A. Martucci C. Guo X.J. Ramonetti J.T. Abren-Goris M. Newmark H.J. Lipkin M.L. DeCosse J.J. Bertagnolli M.M. Cancer Res. 1996; 56: 3785-3789Google Scholar, 30.Kargman S.L. O'Neill G.P. Vickers P.J. Evans J.F. Mancini J.A. Jothy S. Cancer Res. 1995; 55: 2556-2559PubMed Google Scholar, 31.Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrebach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar). COX-2 in activated human monocytes generates the isoprostane 8-epi-PGF2, which is mitogenic and vasoactive, leading to cellular proliferation and vasoconstriction (32.Pratico D. FitzGerald G.A. J. Biol. Chem. 1996; 271: 8919-8924Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). COX-2 is expressed in atherosclerotic lesions (33.Baker C. Hall R. Evans T. Pomerance A. Maclouf J. Creminon C. Yacoub M.H. Polak J.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 646-655Crossref PubMed Scopus (290) Google Scholar), increases after vascular injury (21.Pritchard K.A. O'Banion M.K. Miano J.M. Vlasic N. Bhatia U.G. Young D.A. Stemerman M.B. J. Biol. Chem. 1994; 269: 8504-8509Abstract Full Text PDF PubMed Google Scholar, 23.Rimarachin J.A. Jacobson J.A. Szabo P. Maclouf J. Creminon C. Weksler B.B. Arterioscler. Thromb. 1994; 14: 1021-1031Crossref PubMed Google Scholar), and has been detected in myocardium of patients with congestive heart failure (34.Wong S.C.Y. Eng B. Fukuchi M. Melnyk P. Rodger I. Giaid A. Circulation. 1998; 98: 100-103Crossref PubMed Scopus (272) Google Scholar). Aspirin, a COX inhibitor, can reduce the risks of cardiovascular disease (35.Speir E., Yu, Z.-X. Ferrans V.J. Huang E.-S. Epstein S.E. Circ. Res. 1998; 83: 210-216Crossref PubMed Scopus (145) Google Scholar, 36.Hennekens C.H. Annu. Rev. Public Health. 1997; 18: 37-49Crossref PubMed Scopus (34) Google Scholar). These data all suggest that COX-2 may participate in the genesis of atherosclerosis. This study is designed to explore the effect of B[a]P on COX-2 in human arterial vascular smooth muscle cells (SMC) from atherosclerotic lesions and in rat aortic SMC. We show that exposure to B[a]P increases COX-2 gene expression and protein production and that these effects, which are augmented by oxidant stress, are mediated by ERK1/2-MAPK signaling and the transcription factor NF-κB. DMEM medium was obtained from BioWhittaker (Walkersville, MA); fetal bovine serum was from Gemini-Bio-Products (Calabasas, CA). NS 398 (N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide), COX-2 polyclonal antibody, COX-2 standard, and enzyme immunoassay reagents were from Cayman Co. (Ann Arbor, MI). Human COX-2 cDNA was a gift from Dr. Stephen M. Prescott (University of Utah). Specific rat COX-2 primers and β-actin primers were made by Genosys (The Woodlands, TX). FuGENE™ 6 transfection reagent and anti-[rabbit IgG]-peroxidase were from Roche Molecular Biochemicals. Nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). Bisacrylamide solution, ammonium persulfate, and non-fat dry milk were from Bio-Rad (Hercules, CA). Lipofectin reagent, Opti-MEM I reduced serum medium, dNTPs, Moloney murine leukemia virus reverse transcriptase, and Taq DNA polymerase were from Life Technologies, Inc. Random hexamers were from Perkin Elmer. Enhanced chemiluminescence (ECL) reagents were from Amersham Life Science. Enhanced luciferase assay kits were from Analytical Luminescence Laboratories (San Diego, CA). PD98059 and BAY 11–7085 were from BIOMOL (Plymouth Meeting, PA). SB203580 was from Calbiochem (La Jolla, Ca). Polyclonal antibodies to ERK1/2-MAPK (K23), NF-κB p65, and NF-κB p50 were from Santa Cruz Biotechnology. Polyclonal antibody to phospho-ERK1/2-MAPK (Thr-202/Tyr-204) was from New England Biolabs, Inc. (Beverly, MA). B[a]P, Tri Reagent, and all other chemicals and biochemicals were from Sigma. Fisher-344 rats were from the National Institutes of Health Aging Institute Colony. Rat SMC (rSMC), explanted from the aortas of 18-month-old Fisher-344 rats, were maintained in DMEM supplemented with 5% FBS, 1× minimal essential medium vitamins, 10 mm HEPES, 3 mm glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml). These rSMC were previously shown to have atherogenic potential and enhanced proliferative capacity (37.McCaffrey T. Nicholson A. Szabo P. Weksler M. Weksler B.B. J. Exp. Med. 1988; 167: 163-174Crossref PubMed Scopus (88) Google Scholar). Passage 10–14 rSMC were grown to 80% confluence and then growth arrested by incubation in 0.5% FBS/DMEM for 48 h prior to use. Human arterial SMC isolated in our laboratory from atherosclerotic carotid artery lesions under Institutional Review Board-approved protocols (E12 cells, hSMC) were cultured in the same medium detailed above and were incubated in 0.5% FBS/DMEM for 48 h prior to use. In studies of both cell types, medium was removed following growth arrest and replaced by fresh 0.5% FBS/DMEM to which B[a]P (0.5–10 μm) was added for 18–24 h to rSMC or for 6 h to hSMC prior to harvest of cells. SMC were washed twice and homogenized in lysis buffer (150 mm NaCl, 100 mm Tris, pH 8.0, 1% Tween 20, 1 mm EDTA, and 1 mmphenylmethylsulfonyl fluoride). Protein concentrations of lysates were determined by the method of Lowry. Proteins (50 μg/lane) were separated on a denaturing 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked in 3% non-fat dry milk in phosphate-buffered saline containing 0.1% Triton X-100. For detection of COX-2, polyclonal anti-COX-2 antibody (1:1,000) was incubated with the membranes overnight at 4 °C. Secondary antibody linked to horseradish peroxidase was used at 1:10,000, and signals were visualized by the ECL technique using Kodak x-ray film. For detection of ERK1/2-MAPK total protein, the primary antibody was rabbit polyclonal anti-ERK1/2 antibody (1:1,000), whereas for detection of phosphorylated ERK1/2-MAPK, an antibody that recognizes only the phosphorylated forms of ERK1/2-MAPK was used. The effects of different treatments on total COX activity of SMC were determined in intact cells cultured in 24-well plates and stimulated with B[a]P for 6 h, following which medium was removed and prewarmed serum-free medium containing 10 μm sodium arachidonate was added to stimulate maximum prostaglandin release. After 15 min, this medium was collected to measure formation of 6-keto-PGF1α, the stable metabolite of PGI2, or prostaglandin E2(PGE2), by specific enzyme immunoassay as described previously (23.Rimarachin J.A. Jacobson J.A. Szabo P. Maclouf J. Creminon C. Weksler B.B. Arterioscler. Thromb. 1994; 14: 1021-1031Crossref PubMed Google Scholar). To investigate the role of oxidant stress on B[a]P-induced COX-2 protein synthesis, growth-arrested rSMC were pretreated with 100 μml-buthionine sulfoximine (BSO) for 24 h and then stimulated with B[a]P for 18 h or were incubated at the same time with both B[a]P and 200 μm N-acetylcysteine (NAC) for 18 h. Cells were then harvested for Western analysis as above. Total cellular RNA was extracted from cells using Tri Reagent according to the manufacturer's instructions. Briefly, Tri Reagent (1 ml/T-25 flask) was added to the tissue culture flasks for 10 min. Cell lysates were harvested and centrifuged at 12,000 × g for 15 min at 4 °C after adding 1/10 volume of chloroform. The upper aqueous layer was mixed with an equal volume of isopropanol to precipitate RNA. RNA pellets were washed with 70% ethanol, dried, and dissolved in DEPC-treated water. RNA concentrations were determined spectrophotometrically. cDNA was reverse-transcribed from 1 μg total cellular RNA using random hexamer primers and murine leukemia virus reverse transcriptase. One μg of cDNA was amplified for 30 cycles using the following rat COX-2 gene-specific primers (38.Feng L. Sun W. Xia Y. Tang W.W. Chanmugam P. Soyoola E. Wilson C.B. Hwang D. Arch. Biochem. Biophys. 1993; 307: 361-368Crossref PubMed Scopus (456) Google Scholar): 5′-ACTTGCCTCACTTTGTTGAGTCATTC-3′ (sense) and 5′-TTTGATTAGTACTGTAGGGTTAATG-3′ (antisense). The cycling parameters were the following: 30 s at 94 °C for denaturation, 30 s at 60 °C for primer annealing, and 1 min at 72 °C for polymerization. Meanwhile, the same amount of cDNA was amplified for 20 cycles using specific β-actin primers: 5′-GAGACCTTCAACACCCC-3′ (sense) and 5′-GTGGTGGTGAAGCTGTAGCC-3′ (antisense). The products were visualized after electrophoresis on a 2% agarose gel containing ethidium bromide. Ten μg of total cellular RNA per lane were electrophoresed in a formaldehyde-containing 1.2% agarose gel and transferred to a nitrocellulose membrane. After baking, membranes were prehybridized in a solution containing 50% formamide, 5× sodium chloride/sodium phosphate/EDTA buffer (SSPE), 5× Denhardt's solution, 0.1% SDS, and 100 μg/ml salmon sperm DNA for 3 h and hybridized in the above solution containing 32P-labeled COX-2 cDNA probes for 16 h at 42 °C. Following hybridization, the membranes were washed twice for 20 min in 2× SSPE and 0.1% SDS at room temperature, twice for 20 min in the same solution at 55 °C, and twice for 20 min in 0.1× SSPE and 0.1% SDS at 55 °C. Washed membranes were then exposed to x-ray film. To verify the equivalency of RNA loading in the different lanes, the membranes were rehybridized with a probe for 18 S rRNA. The signal level of the bands was quantified densitometrically. Serum-deprived hSMC plated in T150 cell culture dishes were grown to 80% confluence, serum-starved for 48 h, and then stimulated with 1 μm B[a]P for 3 h. Nuclei were isolated and stored in liquid nitrogen until use for transcription assays as described previously (39.Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429PubMed Google Scholar). Briefly, nuclei were incubated in reaction buffer containing 100 μCi of uridine 5′-[α-32P]triphosphate and unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts were isolated. COX-2 and 18S rRNA cDNAs were immobilized onto nitrocellulose membranes and prehybridized overnight. Hybridization was carried out at 42 °C for 24 h using 5 × 105 cpm of labeled nascent RNA transcripts. The filters were washed twice with 2× SSC buffer at 55 °C for 1 h and were then treated with 10 mg/ml RNase A in 2× SSC at 37 °C for 30 min, dried, and autoradiographed. Human SMC were seeded at a density of 1 × 105 cells/well in 6-well culture dishes and grown for 24 h in medium containing 5% FBS.COX-2 promoter-luciferase plasmid DNA (40.Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and pSV-β-galactosidase were co-transfected into cells using FuGENE™ 6 transfection reagent according to the manufacturer's protocol. After 6 h of incubation, the cells were maintained in 5% FBS/DMEM for another 24 h and then stimulated with B[a]P for 3 h. Cells were lysed and luciferase activity was measured in the cellular extracts using an enhanced luciferase assay kit. Luciferase activity was normalized to β-galactosidase activity (41.Ausubel, F. M. et al. (eds) (1995) Current Protocols in Molecular Biology, Vol. 2, Ch. 9.7B, John Wiley & Sons, Inc., New YorkGoogle Scholar). Human vascular smooth muscle cells were treated with B[a]P in DMEM containing 0.5% FBS for 2 h. Cells were harvested, and nuclear extracts were prepared as described previously (42.Zhang F. Subbaramaiah K. Altorki N. Dannenberg A.J. J. Biol. Chem. 1998; 273: 2424-2428Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). For binding studies, an NF-κB consensus oligonucleotide was used with the following sequence: 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (sense) and 3′-TCAACTCCCCTGAAAGGGTCCG-5′ (antisense). A mutant NF-κB oligonucleotide was obtained from Santa Cruz Biotechnology Inc. for additional binding studies. The complementary oligonucleotides were annealed in 20 mmTris (pH 7.6), 50 mm NaCl, 10 mmMgCl2, and 1 mm dithiothreitol. The annealed oligonucleotides were phosphorylated at the 5′-end with [γ-32P]ATP and T4 polynucleotide kinase. The binding reaction was performed by incubating 2 μg of nuclear protein in 20 mm HEPES (pH 7.9), 10% glycerol, 300 μg of bovine serum albumin, and 1 μg of poly(dI-dC) in a final volume of 10 μl for 10 min at 25 °C. The labeled NF-κB consensus oligonucleotide was added to the reaction mixture and allowed to incubate for an additional 20 min at 25 °C. To detect an antibody supershift, 2 μl of antibody to NF-κB p65 or p50 were added to the reaction mixture for 30 min at 25 °C. The samples were electrophoresed on a 4% nondenaturing polyacrylamide gel. The gel was then dried and subjected to autoradiography at −80 °C. Data are presented as mean ± S.D., and statistical comparisons were made using the Student'st test for paired observations or analysis of variance for multiple comparisons. Significance was defined at the p< 0.05 level. B[a]P treatment of hSMC produced a dose-dependent induction of COX-2 protein, maximal at 1 μm B[a]P (Fig.1 A). Treatment of rSMC with B[a]P also produced a dose- and time-dependent induction of COX-2 protein (Fig. 1, B and C, respectively.) Maximal effects were observed at 5 μmB[a]P and 18 h, respectively. To determine if B[a]P-induced COX-2 enzyme was functional, prostaglandin production was measured. Synthesis of PGE2, the main prostaglandin produced by hSMC, increased by 2–3-fold; this increase was blocked by 2 μm NS 398, a known specific COX-2 inhibitor (data not shown). Serum-deprived rSMC similarly treated with B[a]P increased prostaglandin production (mainly PGI2) up to 8-fold in a dose-dependent manner, with maximal production in response to 1–5 μmB[a]P (data not shown). Northern blot analysis was done to determine whether B[a]P induced COX-2 mRNA. Steady state levels of COX-2 mRNA increased after 1–3 h of exposure to B[a]P in both types of vascular SMC (Fig. 2). To determine if B[a]P-mediated induction of COX-2 mRNA involved increased transcription, de novo COX-2 mRNA synthesis was determined by nuclear runoff assay in hSMC treated for 3 h with 1 μm B[a]P. Fig.3 A shows that B[a]P stimulated the transcription of COX-2. This increase was similar in extent to that induced by PMA, a known inducer of COX-2 gene expression. In complementary studies, transient transfections were performed with a human COX-2promoter-luciferase reporter construct (−1432/+59) demonstrating that exposure to B[a]P resulted in more than a 100% increase in COX-2 promoter activity (Fig. 3 B).Figure 3B[a]P increasesCOX-2 transcription and promoter activity. A, nuclear run-off. Nuclei were isolated from hSMC treated for 3 h with 0.01% Me2SO, 1 μmB[a]P, or 10 ng/ml PMA. Nuclear run-off assays were performed as described under “Materials and Methods.” B[a]P treatment increased COX-2 transcription in hSMC to a similar degree as did PMA. B, COX-2 promoter activity. hSMC were cotransfected with 1.8 μg/ml COX-2promoter construct (−1432/+59) ligated to luciferase and 0.2 μg/ml pSV-β-galactosidase. Twenty-four hours after transfection, cells were treated with or without 1 μm B[a]P for 3 h, and cell lysates were prepared to measure luciferase activity. The values expressed have been normalized to co-transfected β-galactosidase activity. Exposure to B[a]P significantly increased COX-2 promoter activity. (n = 6, mean ± S.D.) *, p < 0.01, compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In preliminary dose-ranging experiments, we established that 100 μm BSO and 200 μm NAC induced, respectively, a maximal decrease or increase in intracellular levels of glutathione (data not shown). Serum-deprived rSMC either were pretreated for 24 h with 100 μm BSO and then with 5 μm B[a]P or were co-incubated with B[a]P and NAC for 18 h. Western blot analysis revealed that BSO alone caused a small increase in COX-2 protein (Fig.4 A). Markedly increased levels of COX-2 protein were detected in SMC pretreated with BSO and then exposed to B[a]P (Fig. 4 A). Conversely, NAC treatment blocked B[a]P-mediated induction of COX-2 protein (Fig. 4 B.) To define the mechanism of B[a]P-mediated COX-2 induction in SMC, we examined the effect of B[a]P on ERK1/2 MAP kinase activation. As shown in Fig.5 A (5 left lanes), B[a]P induced ERK1/2 phosphorylation in a time-dependent manner, with maximal activity appearing 20–30 min after initiating exposure to B[a]P. Amounts of ERK1/2 MAPK protein were unaffected by B[a]P during the same time interval (Fig. 5 B, 5 left lanes). To investigate whether ERK1/2 MAPK mediated the induction of COX-2 by B[a]P, PD98059, a specific inhibitor of MAPK kinase, was used to block activation of ERK1/2 MAPK. Treatment of SMC with 20 μm PD98059 decreased B[a]P-mediated phosphorylation of ERK1/2 MAPK (Fig.5 A). However, neither B[a]P nor PD98059 altered the total amounts of ERK1/2 MAPK (Fig. 5 B). Treatment of rSMC with 20 μmPD98059 also decreased B[a]P-mediated induction ofCOX-2 mRNA (Fig. 6 A), COX-2 protein (Fig.6 B), and prostaglandin synthesis (Fig. 6 C). The decline in both basal and B[a]P-stimulated PG synthesis in the presence of PD98059 can be attributed to the known inhibitory effect of PD98059 on enzymatic activity of COX-1 and COX-2 (43.Borsch-Haubold A.G. Pasque S. Watson S.P. J. Biol. Chem. 1998; 273: 28766-28772Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), in addition to suppression of any increase in COX-2 protein. In contrast, SB203580, an inhibitor of p38 MAPK, had little effect on B[a]P-mediated induction of COX-2 protein although it also decreased COX-2 enzymatic activity (data not shown). Activation of NF-κB signaling can stimulateCOX-2 transcription. Therefore, to study whether NF-κB binding mediates B[a]P-induced COX-2 gene transcription, electrophoretic mobility shift assays were performed. NF-κB binding activity increased in SMC treated with B[a]P (Fig. 7, Aand B). The specificity of the binding was confirmed by using excess unlabeled NF-κB oligonucleotides (Fig.7 A) and an oligonucleotide containing a mutant NF-κB (Fig. 7 B). Supershift analyses identified both NF-κB p65 and p50 proteins in the binding complex (Fig. 7 B). The functional importance of NF-κB in mediating the induction of COX-2 by B[a]P was then tested by transient transfections. B[a]P treatment led to a 2-fold increase in COX-2 promoter activity. Stimulation by B[a]P was blocked by mutating the NF-κB (−223/−214) site in the 5′-region of the COX-2 promoter (Fig.7 C). Furthermore, inhibitors of NF-κB activation, Bay 11–7085 and pyrrolidinedithiocarbamate (PDTC), blocked B[a]P-mediated induction of COX-2 promoter activity (Fig. 8 A) and COX-2 protein (Fig.8 B).Figure 8NF-κB inhibitors block B[a]P-induction of COX-2. A, COX-2 promoter activity. hSMC were transfected with a human COX-2promoter (−1432/+59) construct ligated to luciferase. After transfection, cells were pretreated with NF-κB inhibitors, 0.1 mm PDTC, or 5 μm BAY 11–7085, for 1 h before exposure to 1 μm B[a]P for 3 h. Luciferase activity represents data that have been normalized to cotransfected β-galactosidase activity. Both NF-κB inhibitors blocked B[a]P-induced COX-2 promoter activity. (Mean ± S.D.,n = 6.) *, p < 0.01, compared with untreated cells. Data are from one of two separate experiments.B, Western blot. hSMC were pretreated with 0.1 mm PDTC or 5 μm BAY 11–7085 for 1 h before being stimulated with B[a]P (1 μm) for 6 h. COX-2 protein was determined by Western analysis. NF-κB inhibitors PDTC and BAY 11–7085 blocked B[a]P-mediated induction of COX-2 protein by 97% and 96%, respectively. Data presented are from one of two experiments with similar findings.View Large Image Figure V" @default.
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• W2053143976 title "Benzo[a]pyrene Induces the Transcription of Cyclooxygenase-2 in Vascular Smooth Muscle Cells" @default.
• W2053143976 cites W1503877672 @default.
• W2053143976 cites W1536684354 @default.
• W2053143976 cites W1544157645 @default.
• W2053143976 cites W1587059515 @default.
• W2053143976 cites W1648593433 @default.
• W2053143976 cites W1811191752 @default.
• W2053143976 cites W1964797510 @default.
• W2053143976 cites W1973699080 @default.
• W2053143976 cites W1976056213 @default.
• W2053143976 cites W1976385326 @default.
• W2053143976 cites W1976690335 @default.
• W2053143976 cites W1977258455 @default.
• W2053143976 cites W1978701756 @default.
• W2053143976 cites W1980896096 @default.
• W2053143976 cites W1982194889 @default.
• W2053143976 cites W1983601831 @default.
• W2053143976 cites W1985888026 @default.
• W2053143976 cites W1989480480 @default.
• W2053143976 cites W1996995529 @default.
• W2053143976 cites W2001750055 @default.
• W2053143976 cites W2002754915 @default.
• W2053143976 cites W2013014413 @default.
• W2053143976 cites W2015336992 @default.
• W2053143976 cites W2026628125 @default.
• W2053143976 cites W2029303498 @default.
• W2053143976 cites W2037775191 @default.
• W2053143976 cites W2042928324 @default.
• W2053143976 cites W2043707891 @default.
• W2053143976 cites W2044063704 @default.
• W2053143976 cites W2050703121 @default.
• W2053143976 cites W2068019939 @default.
• W2053143976 cites W2068319865 @default.
• W2053143976 cites W2070567048 @default.
• W2053143976 cites W2070609164 @default.
• W2053143976 cites W2078363670 @default.
• W2053143976 cites W2082728629 @default.
• W2053143976 cites W2083917519 @default.
• W2053143976 cites W2085807260 @default.
• W2053143976 cites W2090292194 @default.
• W2053143976 cites W2092690876 @default.
• W2053143976 cites W2093585097 @default.
• W2053143976 cites W2095495320 @default.
• W2053143976 cites W2112336272 @default.
• W2053143976 cites W2113231420 @default.
• W2053143976 cites W2120946197 @default.
• W2053143976 cites W2136785799 @default.
• W2053143976 cites W2146311014 @default.
• W2053143976 cites W2159973565 @default.
• W2053143976 cites W2314805921 @default.
• W2053143976 cites W4213066848 @default.
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