title: Human haem oxygenase-1 induction by nitro-linoleic acid is mediated by cAMP, AP-1 and E-box response element interactions.
abstract: Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signalling responses. LNO2 (nitrolinoleic acid) induces expression of HO-1 (haem oxygenase-1), an enzyme that catabolizes haem into products exhibiting potent anti-inflammatory properties. In the present manuscript, the molecular mechanisms underlying HO-1 induction by LNO2 were examined in HAEC (human aortic endothelial cells), HEK-293 (human embryonic kidney 293) cells, and in transcription factor-deficient MEF (mouse embryonic fibroblasts). LNO2 induced HO-1 expression in Nrf2 [NF-E2 (nuclear factor-erythroid 2)-related factor 2]-deficient MEF and in HEK-293 cells transfected with Nrf2-specific shRNA (small-hairpin RNA), supporting the fact that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a -4.5 kb human HO-1 promoter construct, whereas a -4.0 kb construct with deletion of 500 bp from the 5' region was unresponsive. Site-directed mutagenesis of a CRE (cAMP-response element) or of a downstream NF-E2/AP-1 (activating protein-1) element, individually, within this 500 bp region modestly reduced activation of the HO-1 promoter by LNO2. Mutations of both the CRE and the NF-E2/AP-1 site also attenuated LNO2-mediated HO-1 promoter expression, whereas the addition of a third mutation in the proximal E-box sequence completely abolished LNO2-induced HO-1 expression. Chromatin immunoprecipitation assays confirmed CREB (CRE-binding protein)-1 binding to the CRE (located at -4.0 kb) and E-box regions (located at -44 bp) of the human HO-1 promoter. A 3C (Chromosome Conformation Capture) assay of intact cells showed LNO2-induced interactions between the CRE- and E-box- containing regions. These observations indicate that regulation of human HO-1 expression by LNO2 requires synergy between CRE, AP-1 and E-box sequences and involves the participation of CREB-1.
authors.affiliation: Department of Medicine, Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Marcienne M
authors.initials: MM
authors.lastname: Wright
authors.forename: Junghyun
authors.initials: J
authors.lastname: Kim
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hock
authors.forename: Norbert
authors.initials: N
authors.lastname: Leitinger
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Freeman
authors.forename: Anupam
authors.initials: A
authors.lastname: Agarwal
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D024721:E-Box Elements / Q000502:physiology*; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D051547:Heme Oxygenase-1 / Q000096:biosynthesis*; D006801:Humans; D008041:Linoleic Acids / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D009574:Nitro Compounds / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000502:physiology*
chemical_list: C501855:10-nitro-9,12-octadecadienoic acid; D008041:Linoleic Acids; D009574:Nitro Compounds; D018808:Transcription Factor AP-1; D000242:Cyclic AMP; D051547:Heme Oxygenase-1