Hemin inhibits NO production by IL-1β-stimulated human astrocytes through induction of heme oxygenase-1 and reduction of p38 MAPK activation.
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.
Sheng
Wen S
WS
The Center for Infectious Diseases & Microbiology Translational Research, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA. sheng008@umn.edu
Hu
Shuxian
S
Nettles
Adam R
AR
Lokensgard
James R
JR
Vercellotti
Gregory M
GM
Rock
R Bryan
RB
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*
D053583:Interleukin-1beta; D012333:RNA, Messenger; D009569:Nitric Oxide; D006427:Hemin; D051547:Heme Oxygenase-1; D048051:p38 Mitogen-Activated Protein Kinases
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