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• W1966197860 abstract "Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, protects against oxidative stress, and shows potent anti-inflammatory effects. Oxidized phospholipids, which are generated during inflammation and apoptosis, modulate the inflammatory response by inducing the expression of several genes including HO-1. Here we investigated the signaling pathways and transcriptional events involved in the induction of HO-1 gene expression by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in human umbilical vein endothelial cells. OxPAPC up-regulated HO-1 mRNA and protein in a time- and concentration-dependent manner, whereas pro-inflammatory agents like TNF-α and lipopolysaccharide did not significantly induce HO-1 expression in human umbilical vein endothelial cells. Signaling pathways involved in the OxPAPC-mediated HO-1 induction included protein kinases A and C, as well as the mitogen-activated protein kinases p38 and ERK. The cAMP-responsive element-binding protein (CREB) was phosphorylated via these pathways in response to OxPAPC treatment and expression of a dominant-negative mutant of CREB inhibited OxPAPC-induced activity of a human heme oxygenase-1 promoter-driven luciferase reporter construct. We identified a cAMP-responsive element and a Maf recognition element to be involved in the transcriptional activation of the HO-1 promoter by OxPAPC. In gel shift assays we observed binding of CREB to the cAMP-responsive element after OxPAPC treatment. Induction of HO-1 expression by lipid oxidation products via CREB may represent a feedback mechanism to limit inflammation and associated tissue damage. Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, protects against oxidative stress, and shows potent anti-inflammatory effects. Oxidized phospholipids, which are generated during inflammation and apoptosis, modulate the inflammatory response by inducing the expression of several genes including HO-1. Here we investigated the signaling pathways and transcriptional events involved in the induction of HO-1 gene expression by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in human umbilical vein endothelial cells. OxPAPC up-regulated HO-1 mRNA and protein in a time- and concentration-dependent manner, whereas pro-inflammatory agents like TNF-α and lipopolysaccharide did not significantly induce HO-1 expression in human umbilical vein endothelial cells. Signaling pathways involved in the OxPAPC-mediated HO-1 induction included protein kinases A and C, as well as the mitogen-activated protein kinases p38 and ERK. The cAMP-responsive element-binding protein (CREB) was phosphorylated via these pathways in response to OxPAPC treatment and expression of a dominant-negative mutant of CREB inhibited OxPAPC-induced activity of a human heme oxygenase-1 promoter-driven luciferase reporter construct. We identified a cAMP-responsive element and a Maf recognition element to be involved in the transcriptional activation of the HO-1 promoter by OxPAPC. In gel shift assays we observed binding of CREB to the cAMP-responsive element after OxPAPC treatment. Induction of HO-1 expression by lipid oxidation products via CREB may represent a feedback mechanism to limit inflammation and associated tissue damage. Heme oxygenase-1 (HO-1) 1The abbreviations used are: HO-1heme oxygenase-1LPSlipopolysaccharidePAPC1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholineOxPAPCoxidized PAPCPKAprotein kinase APKCprotein kinase CERKextracellular signal-regulated kinasePPARperoxisome proliferator-activated receptorNFATnuclear factor of activated T cellsEgr-1early growth response factor-1MAREMaf recognition elementHUVEChuman umbilical vein endothelial cellsCREBcAMP-responsive element-binding proteinMAPKmitogen-activated protein kinaseCREcAMP-responsive elementBisIbisindolylmaleimide ICDCcinnamoyl-3,4-dihydroxy-a-cyanocinnamateSCSsupplemented calf serumRTreverse transcriptasednCREBdominant-negative CREB.1The abbreviations used are: HO-1heme oxygenase-1LPSlipopolysaccharidePAPC1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholineOxPAPCoxidized PAPCPKAprotein kinase APKCprotein kinase CERKextracellular signal-regulated kinasePPARperoxisome proliferator-activated receptorNFATnuclear factor of activated T cellsEgr-1early growth response factor-1MAREMaf recognition elementHUVEChuman umbilical vein endothelial cellsCREBcAMP-responsive element-binding proteinMAPKmitogen-activated protein kinaseCREcAMP-responsive elementBisIbisindolylmaleimide ICDCcinnamoyl-3,4-dihydroxy-a-cyanocinnamateSCSsupplemented calf serumRTreverse transcriptasednCREBdominant-negative CREB. is the rate-limiting enzyme of heme catabolism, catalyzing the breakdown of heme into biliverdin, iron, and carbon monoxide (1Maines M.D. Ann. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2185) Google Scholar). The phenotype of humans and mice deficient of HO-1 suggests an important physiological role of HO-1 in the regulation of the inflammatory process (2Kawashima A. Oda Y. Yachie A. Koizumi S. Nakanishi I. Hum. Pathol. 2002; 33: 125-130Crossref PubMed Scopus (210) Google Scholar, 3Yachie A. Niida Y. Wada T. Igarashi N. Kaneda H. Toma T. Ohta K. Kasahara Y. Koizumi S. J. Clin. Invest. 1999; 103: 129-135Crossref PubMed Scopus (1073) Google Scholar, 4Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10925-10930Crossref PubMed Scopus (1089) Google Scholar, 5Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924Crossref PubMed Scopus (844) Google Scholar). Indeed, HO-1 is highly up-regulated in acute and chronic inflammation (6Willis D. Moore A.R. Willoughby D.A. J. Pathol. 2000; 190: 627-634Crossref PubMed Scopus (63) Google Scholar, 7Wang L.J. Lee T.S. Lee F.Y. Pai R.C. Chau L.Y. Am. J. Pathol. 1998; 152: 711-720PubMed Google Scholar). HO-1 not only provides protection against oxidative stress (4Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10925-10930Crossref PubMed Scopus (1089) Google Scholar, 8Nath K.A. Balla G. Vercellotti G.M. Balla J. Jacob H.S. Levitt M.D. Rosenberg M.E. J. Clin. Invest. 1992; 90: 267-270Crossref PubMed Scopus (592) Google Scholar, 9Bauer M. Bauer I. Antioxid. Redox. Signal. 2002; 4: 749-758Crossref PubMed Scopus (212) Google Scholar) but also possesses potent anti-inflammatory properties (10Willis D. Moore A.R. Frederick R. Willoughby D.A. Nat. Med. 1996; 2: 87-90Crossref PubMed Scopus (704) Google Scholar, 11Otterbein L.E. Bach F.H. Alam J. Soares M. Tao L.H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1796) Google Scholar, 12Lee T.S. Chau L.Y. Nat. Med. 2002; 8: 240-246Crossref PubMed Scopus (889) Google Scholar), which seem to be mediated by its products biliverdin (13Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Invest. 1997; 100: 1209-1216Crossref PubMed Scopus (254) Google Scholar, 14Vachharajani T.J. Work J. Issekutz A.C. Granger D.N. Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1613-H1617Crossref PubMed Google Scholar) and carbon monoxide (11Otterbein L.E. Bach F.H. Alam J. Soares M. Tao L.H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1796) Google Scholar, 15Otterbein L.E. Zuckerbraun B.S. Haga M. Liu F. Song R. Usheva A. Stachulak C. Bodyak N. Smith R.N. Csizmadia E. Tyagi S. Akamatsu Y. Flavell R.J. Billiar T.R. Tzeng E. Bach F.H. Choi A.M. Soares M.P. Nat. Med. 2003; 9: 183-190Crossref PubMed Scopus (436) Google Scholar, 16Sarady J.K. Otterbein S.L. Liu F. Otterbein L.E. Choi A.M. Am. J. Respir. Cell Mol. Biol. 2002; 27: 739-745Crossref PubMed Scopus (122) Google Scholar, 17Song R. Ning W. Liu F. Ameredes B.T. Calhoun W.J. Otterbein L.E. Choi A.M. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284: L50-L56Crossref PubMed Scopus (39) Google Scholar, 18Chapman J.T. Otterbein L.E. Elias J.A. Choi A.M. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 281: L209-L216Crossref PubMed Google Scholar, 19Sato K. Balla J. Otterbein L. Smith R.N. Brouard S. Lin Y. Csizmadia E. Sevigny J. Robson S.C. Vercellotti G. Choi A.M. Bach F.H. Soares M.P. J. Immunol. 2001; 166: 4185-4194Crossref PubMed Scopus (422) Google Scholar). Carbon monoxide inhibits the expression of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and, in addition, increases the expression of anti-inflammatory cytokines (11Otterbein L.E. Bach F.H. Alam J. Soares M. Tao L.H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1796) Google Scholar). In this respect HO-1 seems to play a key role in the resolution of inflammation (10Willis D. Moore A.R. Frederick R. Willoughby D.A. Nat. Med. 1996; 2: 87-90Crossref PubMed Scopus (704) Google Scholar, 20Willoughby D.A. Moore A.R. Colville-Nash P.R. Gilroy D. Int. J. Immunopharmacol. 2000; 22: 1131-1135Crossref PubMed Scopus (111) Google Scholar, 21Seed M.P. Willoughby D.A. Inflamm. Res. 1997; 46: 279-281Crossref PubMed Scopus (15) Google Scholar, 22Willis D. Inflamm. Res. 1995; 44: 218-220Crossref PubMed Scopus (22) Google Scholar), which is pivotal to limit tissue damage resulting from oxidation of DNA, proteins, and membrane lipids by free radicals generated by inflammatory cells (23Heinecke J.W. Toxicology. 2002; 177: 11-22Crossref PubMed Scopus (99) Google Scholar, 24Shackelford R.E. Kaufmann W.K. Paules R.S. Environ. Health Perspect. 1999; 107: 5-24Crossref PubMed Scopus (222) Google Scholar, 25Lang J.D. McArdle P.J. O'Reilly P.J. Matalon S. Chest. 2002; 122: 314S-320SAbstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 26Savenkova M.L. Mueller D.M. Heinecke J.W. J. Biol. Chem. 1994; 269: 20394-20400Abstract Full Text PDF PubMed Google Scholar, 27Zhang R. Brennan M.L. Shen Z. MacPherson J.C. Schmitt D. Molenda C.E. Hazen S.L. J. Biol. Chem. 2002; 277: 46116-46122Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). heme oxygenase-1 lipopolysaccharide 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidized PAPC protein kinase A protein kinase C extracellular signal-regulated kinase peroxisome proliferator-activated receptor nuclear factor of activated T cells early growth response factor-1 Maf recognition element human umbilical vein endothelial cells cAMP-responsive element-binding protein mitogen-activated protein kinase cAMP-responsive element bisindolylmaleimide I cinnamoyl-3,4-dihydroxy-a-cyanocinnamate supplemented calf serum reverse transcriptase dominant-negative CREB. heme oxygenase-1 lipopolysaccharide 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidized PAPC protein kinase A protein kinase C extracellular signal-regulated kinase peroxisome proliferator-activated receptor nuclear factor of activated T cells early growth response factor-1 Maf recognition element human umbilical vein endothelial cells cAMP-responsive element-binding protein mitogen-activated protein kinase cAMP-responsive element bisindolylmaleimide I cinnamoyl-3,4-dihydroxy-a-cyanocinnamate supplemented calf serum reverse transcriptase dominant-negative CREB. Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which is an integral part of cellular membranes and lipoproteins, leads to the generation of intermediate oxidation products (OxPAPC), some of which are potent bioactive substances (28Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). We have shown that OxPAPC profoundly modulates the fate of an inflammatory response by limiting LPS-induced inflammation (29Bochkov V.N. Kadl A. Huber J. Gruber F. Binder B.R. Leitinger N. Nature. 2002; 419: 77-81Crossref PubMed Scopus (320) Google Scholar). In endothelial cells OxPAPC induces the adhesion of monocytes (28Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar) and activates, probably via yet non-identified G-protein-coupled receptors (30Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. Berliner J.A. Vora D.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12010-12015Crossref PubMed Scopus (226) Google Scholar), protein kinases A (PKA) (30Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. Berliner J.A. Vora D.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12010-12015Crossref PubMed Scopus (226) Google Scholar) and C (PKC) (31Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Blood. 2002; 99: 199-206Crossref PubMed Scopus (177) Google Scholar), as well as the ERK pathway (31Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Blood. 2002; 99: 199-206Crossref PubMed Scopus (177) Google Scholar). OxPAPC induces genes like tissue factor (31Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Blood. 2002; 99: 199-206Crossref PubMed Scopus (177) Google Scholar), monocyte chemoattractant protein-1, and interleukin-8 (32Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) via transcription factors such as peroxisome proliferator-activated receptor (PPAR)-α (33Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. Nagy L. Circ. Res. 2000; 87: 516-521Crossref PubMed Scopus (268) Google Scholar), nuclear factor of activated T cells (NFAT), and early growth response factor-1 (Egr-1) (31Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Blood. 2002; 99: 199-206Crossref PubMed Scopus (177) Google Scholar). It has been shown recently that OxPAPC is also an inducer of HO-1 gene transcription in vitro (13Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Invest. 1997; 100: 1209-1216Crossref PubMed Scopus (254) Google Scholar) and in vivo (34Kadl A. Huber J. Gruber F. Bochkov V.N. Binder B.R. Leitinger N. Vasc. Pharmacol. 2002; 38: 219-227Crossref PubMed Scopus (90) Google Scholar). The induction of HO-1 expression is controlled primarily at the transcriptional level and has been studied extensively in the mouse promoter (35Choi A.M. Alam J. Am. J. Respir. Cell Mol. Biol. 1996; 15: 9-19Crossref PubMed Scopus (999) Google Scholar). DNA motifs alternatively termed as Maf recognition elements (MAREs), NF-E2 sites, stress responsive elements, or antioxidant-responsive elements have been shown to be crucial for the activation of mouse HO-1 gene expression in response to numerous stimuli (36Gong P. Hu B. Stewart D. Ellerbe M. Figueroa Y.G. Blank V. Beckman B.S. Alam J. J. Biol. Chem. 2001; 276: 27018-27025Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 37Li N. Venkatesan M.I. Miguel A. Kaplan R. Gujuluva C. Alam J. Nel A. J. Immunol. 2000; 165: 3393-3401Crossref PubMed Scopus (256) Google Scholar, 38Alam J. Killeen E. Gong P. Naquin R. Hu B. Stewart D. Ingelfinger J.R. Nath K.A. Am. J. Physiol. 2002; 284: F743-F752Google Scholar, 39Gong P. Stewart D. Hu B. Vinson C. Alam J. Arch. Biochem. Biophys. 2002; 405: 265-274Crossref PubMed Scopus (55) Google Scholar, 40Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 41Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (497) Google Scholar) and are also present in the human promoter (42Kitamuro T. Takahashi K. Ogawa K. Udono R.F. Takeda K. Furuyama K. Nakayama M. Sun J. Fujita H. Hida W. Hattori T. Shirato K. Igarashi K. Shibahara S. J. Biol. Chem. 2003; PubMed Google Scholar). Different members of the basic-leucine zipper (bZIP) family of transcription factors, with Nrf2 as a central regulator, are able to bind to this element (36Gong P. Hu B. Stewart D. Ellerbe M. Figueroa Y.G. Blank V. Beckman B.S. Alam J. J. Biol. Chem. 2001; 276: 27018-27025Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 38Alam J. Killeen E. Gong P. Naquin R. Hu B. Stewart D. Ingelfinger J.R. Nath K.A. Am. J. Physiol. 2002; 284: F743-F752Google Scholar, 39Gong P. Stewart D. Hu B. Vinson C. Alam J. Arch. Biochem. Biophys. 2002; 405: 265-274Crossref PubMed Scopus (55) Google Scholar, 40Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 41Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (497) Google Scholar). Nevertheless the mechanism of the OxPAPC-mediated induction of HO-1 gene expression remains elusive. In the present study, we investigated in detail the mechanism of OxPAPC-induced HO-1 gene expression in human umbilical vein endothelial cells (HUVEC). We show that the HO-1 induction by OxPAPC depends on the cAMP-responsive element-binding protein (CREB) and is mediated via signaling pathways including PKA, PKC, p38MAPK, and ERK1/2, which promote the phosphorylation of CREB. By using luciferase promoter reporter constructs and mutational analysis, we demonstrate that a regulatory region, located 4 kb upstream the transcription start site, is responsible for the induction of the HO-1 transcription by OxPAPC and that a cAMP-responsive element (CRE) and a Maf recognition element, both of which are present in this region, are involved responsive elements. Finally we show in gel shift assays that OxPAPC increases binding of several nuclear complexes, some of which include CREB, to the human HO-1 promoter. Materials—TNF-α was purchased from Genzyme (Cambridge, MA), and l-α-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and M199 were from Sigma. PD98059, SB203580, H89, and bisindolylmaleimide I (BisI) were obtained from Calbiochem. CDC (cinnamoyl-3,4-dihydroxy-a-cyanocinnamate) and AACOCF3 (arachidonyltrifluoromethyl ketone) were from Biomol. Polyclonal antibodies against HO-1 were from Stressgen. Antibodies against NFATc, CREB/ATF1, Nrf1, Nrf2, MafF/G/K, and c-Fos were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against phospho-CREB was obtained from New England Biolabs (Beverly, MA). Peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences. Cell Culture—HUVEC were cultured at 37 °C and 5% CO2 in M199 containing 20% supplemented calf serum (SCS), 1 unit/ml heparin, 50 μg/ml bovine endothelial cell growth supplement (Technoclone, Vienna, Austria), 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Experiments were performed using cells at passage 4. In all experiments, before addition of the stimulus, HUVEC were incubated in M199 containing 1% SCS for 4 h, except in the experiment shown in Fig. 1D, where medium containing 5% SCS was used to provide HUVEC with enough serum-derived soluble CD14 to respond to LPS. Transfection and Enzyme Assays—Cells were seeded in 6-well plates and transfected after 48 h at ∼80% confluence. Transient transfections were performed using the LipofectAMINE Plus reagent (Invitrogen) according to the protocol provided by the manufacturer. Cells were incubated with a transfection mixture containing 1.5 μg of total DNA (including 0.25 μg of PRL-SV40 vector as transfection-efficiency control), 6 μl of Plus reagent, and 4 μl of LipofectAMINE in a total volume of 1 ml of M199 for 130 min. After 48 h, cells were starved for 4 h and stimulated for, unless otherwise stated, 14 h in M199 containing 1% SCS. Luciferase activity of the cell lysates was determined using a Dual-Luciferase Reporter Assay system (Promega). Firefly activity was then normalized to SV40 Renilla activity. Plasmids—A bacterial artificial chromosome clone containing the human HO-1 gene and the complete 5′regulatory region was a kind gift from Dr. Markus Exner (Clinical Institute of Medical and Chemical Laboratory Diagnostics, General Hospital of Vienna, Vienna, Austria). A 4.9-kb SacI-XhoI fragment containing the human HO-1 promoter including the previously described cadmium-responsive elements (43Takeda K. Ishizawa S. Sato M. Yoshida T. Shibahara S. J. Biol. Chem. 1994; 269: 22858-22867Abstract Full Text PDF PubMed Google Scholar) was cloned into the pGL3 basic vector (Promega) to obtain hHO4.9luc. Plasmid hHO4.9luc was simultaneously digested at the SacI site and at a series of restriction endonuclease sites located in the promoter, blunt-ended, and re-circularized to generate a set of 5′-nested deletions. The end points of the deletion constructs and the enzymes used were as follows: -3870 (XbaI), -2245 (HindIII), -1435 (EcoRI), and -302 (PstI). The vector over-expressing the dominant-negative CREB (pCMV-CREB133) was purchased from Clontech (Palo Alto, CA). Lipid Oxidation—PAPC was oxidized by exposure of dry lipid to air for 72 h. The extent of oxidation was monitored by positive ion electrospray mass spectrometry as described previously (28Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Quantitative Real-time RT-PCR—RNA was isolated using Trizol reagent (Invitrogen). Nine hundred ng of total RNA were reverse-transcribed with murine leukemia virus reverse transcriptase using the Gene Amp RNA PCR kit (Applied Biosystems, Foster City, CA) and oligo(dT) (16Sarady J.K. Otterbein S.L. Liu F. Otterbein L.E. Choi A.M. Am. J. Respir. Cell Mol. Biol. 2002; 27: 739-745Crossref PubMed Scopus (122) Google Scholar) primers. The mRNA sequences of the investigated genes were obtained from GenBank™. The primers for β-2 microglobulin were described previously (44Wellmann S. Taube T. Paal K. Graf V.E. Geilen W. Seifert G. Eckert C. Henze G. Seeger K. Clin. Chem. 2001; 47: 654-660Crossref PubMed Scopus (84) Google Scholar). The primers for E-selectin and HO-1 were designed using the PRIMER3 software from the Whitehead Institute for Biomedical Research (Cambridge, MA). The following forward (F) and reverse (R) primers were used: HO-1: F, 5′-AAGATTGCCCAGAAAGCCCTGGAC-3′; R, 5′-AACTGTCGCCACCAGAAAGCTGAG-3′; E-selectin: F, 5′-GGTTTGGTGAGGTGTGCTC-3′; R, 5′-TGATCTTTCCCGGAACTGC-3′. Quantitative real-time RT-PCR was performed using LightCycler technology (Roche Diagnostics) and the Fast Start SYBR Green I kit for amplification and detection. In all assays, cDNA was amplified using a standardized program (10-min denaturing step; 55 cycles of 5 s at 95 °C, 15 s at 65 °C, and 15 s at 72 °C; melting point analysis in 0.1 °C steps; final cooling step). Each LightCycler capillary was loaded with 1.5 μl of DNA Master Mix, 1.8 μl of MgCl2 (25 mm), 10.1 μl of H2O, 0.4 μl of 10 μm stock of each primer. The final amount of cDNA per reaction corresponded to 2.5 ng of total RNA used for reverse transcription. Quantification of target gene expression was performed using a mathematical model by Pfaffl (45Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24586) Google Scholar). The expression of the target molecule was normalized to the expression of β-2 microglobulin. Electrophoretic Mobility Shift Assay—Nuclear extracts from HUVEC were prepared as described previously (46Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar), except that phosphatase inhibitor mixture 1 + 2 (Sigma) were added. The protein concentration was determined using the Coomassie protein assay reagent (Pierce) with bovine serum albumin as a standard. To prepare probes for the electrophoretic mobility shift assay, 100 pmol of each complementary single-stranded oligonucleotide (VBC-Genomics, Vienna, Austria) were annealed in 20 μl of H2O to obtain a double-stranded oligonucleotide with the sequences 5′-GCTGCATTTCTGCTGCGTCATGTTTGGGAG-3′ (HO-CRE) or 5′-CTAGATTTTGCTGAGTCACCAGTGC-3′ (MARE). 1 μg of nuclear extracts were incubated initially for 10 min at room temperature in 20 μl containing 18 mm HEPES-KOH (pH 7.9), 80 mm KCl, 2 mm MgCl2, 10 mm dithiothreitol, 10% glycerol, 0.2 mg/ml bovine serum albumin, and 1.5 μg of poly(dI-dC). The mixture was then incubated for an additional 20 min after adding 105 cpm of the [γ-32P]ATP-labeled probe, with or without an unlabeled competitor or antibody for supershift. In antibody supershift assays, 2 μl (4 μg) of antibody were added to the reaction mixture. The mixture was electrophoresed on a 6.5% polyacrylamide gel in a 0.5% TBE buffer. The gel was dried, and the radioactivity was visualized and quantified using a PhosphorImager and ImageQuant software (Amersham Biosciences). Western Blot Analysis—After stimulation, HUVEC were lysed in Laemmli buffer, and proteins were separated by electrophoresis in 12% SDS-polyacrylamide gels. Proteins were blotted onto polyvinylidene difluoride membranes and, after blocking with 5% dry milk/0.1% Tween 20, incubated with primary antibodies in the same solution. Bound antibodies were detected by anti-IgG conjugated with peroxidase and subsequent chemiluminescent detection. Site-directed Mutagenesis—The CRE and MARE of the HO-1 promoter were mutated by a PCR-based technique using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the protocol provided by the manufacturer. For each mutation two complementary primers containing the mutation were synthesized (VBC-Genomics). The sequences of the primers used to mutate the HO-CRE site were F, 5′-CCCTCGTGCAGCTGCATTTCTGCGTAGATCTGTTTGGGAGGGGGG-3′ and R, 5′-CCCCCCTCCCAAACAGATCTACGCAGAAATGCAGCTGCACGAGGG-3′. To mutate the MARE in the HO-1 promoter, primers with the sequences F, 5′-GGCGGATTTTGCTAGATTTTGCGTAGGTACCAGTGCCTCCTCAGC-3′ and R, 5′-GCTGAGGAGGCACTGGTACCTACGCAAAATCTAGCAAAATCCGCC-3′ were used. Expression of HO-1 in HUVEC Is Induced by OxPAPC but Not by TNF-α and LPS—HO-1 mRNA expression and HO-1 protein levels after OxPAPC stimulation were determined by quantitative real time RT-PCR and by Western blot, respectively. OxPAPC increased levels of HO-1 mRNA and protein in HUVEC in a concentration- and time-dependent manner starting at 25 μg/ml (Fig. 1, A and B). In contrast to OxPAPC, native PAPC did not affect HO-1 expression. To investigate the effect of OxPAPC on the human HO-1 promoter activity, we cloned the proximal 4.9 kb of the human HO-1 promoter into the PGL-3 vector to obtain a HO-1 promoter-driven luciferase reporter construct (hHO4.9luc). hHO4.9luc activity was induced by OxPAPC in a concentration-dependent manner (Fig. 1C), whereas native PAPC had no effect. Among potential HO-1-stimulating agents that are involved in inflammation are substances like TNFα, LPS, or H2O2 (4Poss K.D. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10925-10930Crossref PubMed Scopus (1089) Google Scholar, 47Camhi S.L. Alam J. Wiegand G.W. Chin B.Y. Choi A.M. Am. J. Respir. Cell Mol. Biol. 1998; 18: 226-234Crossref PubMed Scopus (125) Google Scholar, 48Terry C.M. Clikeman J.A. Hoidal J.R. Callahan K.S. Am. J. Physiol. 1998; 274: H883-H891Crossref PubMed Google Scholar). To compare the effect of these agents on the induction of HO-1 gene expression in endothelial cells, we stimulated HUVEC with OxPAPC, TNF-α, H2O2, or LPS. Then mRNA levels of HO-1 and E-selectin, which is known to be strongly induced by TNF-α and LPS, were determined by quantitative real time RT-PCR. Treatment of HUVEC with OxPAPC increased levels of HO-1 mRNA 17-fold (Fig. 1D). At concentrations where TNF-α and LPS strongly induced E-selectin expression, we did not detect a significant rise in HO-1 mRNA levels, whereas 400 μm H2O2 increased the expression of HO-1 mRNA ∼1.5-fold. Neither OxPAPC nor H2O2 induced a significant elevation of E-selectin mRNA levels (Fig. 1D). These results indicate the activation of distinct signaling pathways by OxPAPC and the pro-inflammatory stimuli TNF-α and LPS in endothelial cells. In accordance with these findings, we have shown previously that OxPAPC does not activate the NF-κB pathway in endothelial cells (31Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Blood. 2002; 99: 199-206Crossref PubMed Scopus (177) Google Scholar), although OxPAPC shares a set of target genes with TNF-α and LPS, such as tissue factor, interleukin-8, and monocyte chemoattractant protein-1 (31Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Blood. 2002; 99: 199-206Crossref PubMed Scopus (177) Google Scholar, 32Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 49Brown Z. Gerritsen M.E. Carley W.W. Strieter R.M. Kunkel S.L. Westwick J. Am. J. Pathol. 1994; 145: 913-921PubMed Google Scholar). Induction of HO-1 Expression by OxPAPC Is Mediated by PKA, PKC, p38MAPK, and ERK—To address the role of individual signaling pathways in HO-1 gene regulatio" @default.
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• W1966197860 title "Oxidized Phospholipids Induce Expression of Human Heme Oxygenase-1 Involving Activation of cAMP-responsive Element-binding Protein" @default.
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