title: Hemin inhibits NO production by IL-1β-stimulated human astrocytes through induction of heme oxygenase-1 and reduction of p38 MAPK activation.
abstract: BACKGROUND
  Heme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO) by interleukin (IL)-1β-activated human astrocytes.
  
  
  METHODS
  To measure NO production, inducible NO synthase (iNOS), HO-1 expression and mitogen-activated protein (MAP) kinase activation we used hemin as an HO-1 inducer and tin protoporphyrin (SnPP) IX as an inhibitor of HO-1 activity in human astrocyte cultures prior to IL-1β exposure. Transfection of astrocyte cultures was performed using a pLEX expression vector carrying the human HO-1 sequence prior to IL-1β treatment. Supernatants of astrocyte cultures pretreated with inhibitors of p38 MAPK or MEK1/2 prior to IL-1β exposure were collected for NO assay.
  
  
  RESULTS
  IL-1β treatment of astrocytes alone induced undetectable amounts of HO-1 protein by western blot. However, HO-1 mRNA expression was modestly up-regulated in response to IL-1β stimulation. Pretreatment with hemin alone substantially induced both HO-1 mRNA and protein expression, and HO-1 mRNA expression was further enhanced when hemin was combined with IL-1β treatment. In contrast, IL-1β-induced iNOS mRNA expression and NO production were markedly inhibited by hemin treatment. When pretreated with SnPP, the inhibitory effect of hemin on IL-1β-induced NO production and iNOS expression was reversed, suggesting the involvement of HO-1. IL-1β-induced p38 MAPK activation, which is known to be required for NO production, was also down-regulated by hemin.
  
  
  CONCLUSIONS
  These findings support the hypothesis that up-regulation of HO-1 in astrocytes is associated with down-regulation of iNOS expression and thereby NO production, an effect that involves the p38 MAPK signaling pathway, which suggests that this glial cell response could play an important protective role against oxidative stress in the brain.
authors.affiliation: The Center for Infectious Diseases & Microbiology Translational Research, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA. sheng008@umn.edu
authors.forename: Wen S
authors.initials: WS
authors.lastname: Sheng
authors.forename: Shuxian
authors.initials: S
authors.lastname: Hu
authors.forename: Adam R
authors.initials: AR
authors.lastname: Nettles
authors.forename: James R
authors.initials: JR
authors.lastname: Lokensgard
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Vercellotti
authors.forename: R Bryan
authors.initials: RB
authors.lastname: Rock
mesh_terms: D000704:Analysis of Variance; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D053583:Interleukin-1beta; D012333:RNA, Messenger; D009569:Nitric Oxide; D006427:Hemin; D051547:Heme Oxygenase-1; D048051:p38 Mitogen-Activated Protein Kinases
references.citation: Curr Alzheimer Res. 2009 Oct;6(5):424-30
references.pmid: 19874266
references.citation: J Immunol. 2008 Nov 15;181(10):6730-7
references.pmid: 18981090
references.citation: J Infect Dis. 1995 Feb;171(2):516-8
references.pmid: 7844409
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14212-7
references.pmid: 12381793
references.citation: Gut. 2000 Dec;47(6):771-8
references.pmid: 11076874
references.citation: Nat Med. 1998 Sep;4(9):1073-7
references.pmid: 9734404
references.citation: Acta Neuropathol. 2000 Oct;100(4):377-84
references.pmid: 10985695
references.citation: Neurosci Lett. 1996 Jun 7;210(3):205-8
references.pmid: 8805131
references.citation: J Immunol. 2006 Apr 1;176(7):4252-7
references.pmid: 16547262
references.citation: Glia. 1996 Mar;16(3):276-84
references.pmid: 8833198
references.citation: Free Radic Biol Med. 2008 Oct 15;45(8):1103-14
references.pmid: 18706498
references.citation: Neuroreport. 1999 Jun 23;10(9):1849-52
references.pmid: 10501519
references.citation: J Neuroimmunol. 2002 May;126(1-2):180-9
references.pmid: 12020969
references.citation: Neurobiol Dis. 2006 Apr;22(1):76-87
references.pmid: 16378731
references.citation: Clin Pharmacol Ther. 2010 Feb;87(2):187-90
references.pmid: 19956091
references.citation: J Clin Invest. 1999 Jan;103(1):129-35
references.pmid: 9884342
references.citation: J Pharmacol Exp Ther. 1992 Feb;260(2):909-16
references.pmid: 1371161
references.citation: Anesthesiology. 2006 Dec;105(6):1201-10; discussion 5A
references.pmid: 17122583
references.citation: Kidney Int. 1999 May;55(5):1734-9
references.pmid: 10231435
references.citation: J Biol Chem. 2004 Jul 2;279(27):28681-8
references.pmid: 15123630
references.citation: J Am Soc Nephrol. 1999 Dec;10(12):2540-50
references.pmid: 10589693
references.citation: Eur J Biochem. 1999 Sep;264(3):854-61
references.pmid: 10491133
references.citation: J Neurosci Res. 2003 Jul 1;73(1):113-21
references.pmid: 12815715
references.citation: J Exp Med. 1994 Sep 1;180(3):969-76
references.pmid: 7520477
references.citation: Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11141-5
references.pmid: 1280819
references.citation: J Neuroimmune Pharmacol. 2009 Jun;4(2):244-8
references.pmid: 19214751
references.citation: Antioxid Redox Signal. 2009 Mar;11(3):541-54
references.pmid: 18715148
references.citation: Brain Res. 1996 Oct 14;736(1-2):68-75
references.pmid: 8930310
references.citation: Glia. 2005 Jan 15;49(2):211-9
references.pmid: 15390091
references.citation: Brain. 2007 Jun;130(Pt 6):1643-52
references.pmid: 17525142
references.citation: Biochem Pharmacol. 2003 Nov 1;66(9):1821-32
references.pmid: 14563492
references.citation: Mol Cell Neurosci. 1992 Dec;3(6):559-70
references.pmid: 19912899
references.citation: Neurosci Lett. 2000 Mar 17;282(1-2):1-4
references.pmid: 10713382
references.citation: Pediatr Res. 1992 Sep;32(3):324-9
references.pmid: 1408470
references.citation: Biochem Pharmacol. 1999 Jul 15;58(2):227-36
references.pmid: 10423162
references.citation: J Biomed Sci. 2010 Feb 23;17:12
references.pmid: 20175935
references.citation: Biochem Biophys Res Commun. 2004 May 21;318(1):88-94
references.pmid: 15110757
references.citation: J Neuroimmunol. 1993 Jul;46(1-2):19-24
references.pmid: 7689587
references.citation: Nat Med. 1999 Dec;5(12):1403-9
references.pmid: 10581083
references.citation: Trends Immunol. 2003 Aug;24(8):449-55
references.pmid: 12909459
references.citation: Cell Biochem Biophys. 2006;46(1):35-42
references.pmid: 16943622