Increasing expression of heme oxygenase-1 by proteasome inhibition protects astrocytes from heme-mediated oxidative injury.
Hemin is released from hemoglobin after CNS hemorrhage, and may contribute to cell loss in surrounding tissue. Heme oxygenase-1 (HO-1) is induced by these injuries, and may have an effect on cell viability. In a prior study, we reported that increasing HO-1 expression by adenoviral gene transfer prior to hemin exposure attenuated oxidative stress and cell death in astrocytes. However, rapid gene transfer to the CNS may not be feasible. HO-1 expression is controlled by a stress-responsive transcription factor, Nrf2, which is a labile protein that is subject to proteasomal degradation. In this study, we hypothesized that preventing degradation of Nrf2 with a lipid-soluble proteasome inhibitor would increase HO-1 expression and protect astrocytes from hemin. Treatment of cortical astrocyte cultures with 1 microM MG-132 resulted in a rapid increase in Nrf2, to a level that was five-fold that of vehicle-treated cultures by 2 h. This was followed by a three to six-fold increase in HO-1 expression that persisted through the 16 h observation period. Exposure of cultures to 30 microM or 60 microM hemin for 8 h resulted in death, as measured by LDH release, of 39+/-3.0 or 67.5+/-5.9% of astrocytes. Pre-treatment with MG-132 prevented approximately half of this injury. Cytoprotection persisted at 24 h, and was also observed when injury was assessed via the MTT assay. Astrocyte protein oxidation produced by hemin was also significantly attenuated by MG-132 pre-treatment. These results suggest that increasing HO-1 expression with a proteasome inhibitor protects astrocytes from heme-mediated oxidative injury. This pharmacological approach may provide a mechanism for rapidly upregulating HO-1 in astrocytes after CNS hemorrhage.
Chen
Jing
J
Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, 239 Thompson Building, Philadelphia, PA 19107, USA.
Regan
Raymond F
RF
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D051547:Heme Oxygenase-1; D006427:Hemin / Q000633:toxicity*; D007976:Leupeptins / Q000494:pharmacology*; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D051267:NF-E2-Related Factor 2; D018384:Oxidative Stress; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D007976:Leupeptins; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D015534:Trans-Activators; D006427:Hemin; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde