title: Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes.
abstract: The brain's adaptive response to ischemic preconditioning (IPC) is mediated in part via hypoxia inducible factor (HIF)-responsive genes. We previously showed that IPC induces cytochrome P450 2C11 expression in the brain, associated with protection from stroke. Cytochrome P450 2C11 is an arachidonic acid (AA) epoxygenase expressed in astrocytes, which metabolizes AA to epoxyeicosatrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1alpha, and that the P450 epoxygenase pathway contributes to enhanced astrocyte tolerance to ischemia-like injury induced by oxygen-glucose deprivation (OGD). Primary cultured astrocytes were incubated under normoxic or hypoxic conditions for 1, 3, 6, 24, or 48 h, and protein levels of P450 2C11 and HIF-1alpha were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1alpha to 2C11 promoter was evaluated using electrophoretic mobility shift assay (EMSA) with 2C11 promoter DNA containing putative HIF-binding sites. Levels of 2C11 mRNA and protein were significantly increased starting at 3 and 6 h of hypoxia, respectively. The increase in 2C11 expression was preceded by an increase in HIF-1alpha protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1alpha in nuclear extracts from astrocytes. HPC and EETs reduced astrocyte cell death, and P450 epoxygenase inhibition prevented protection by HPC. We conclude that HPC induces tolerance in astrocytes, at least in part, via HIF-1alpha-linked upregulation of P450 2C11.
authors.affiliation: Department of Anesthesiology and Critical Care, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Mingyue
authors.initials: M
authors.lastname: Liu
authors.forename: Nabil J
authors.initials: NJ
authors.lastname: Alkayed
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis*; D001253:Astrocytes / Q000201:enzymology* / Q000473:pathology*; D015152:Blotting, Northern; D015153:Blotting, Western; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D000072467:Cytochrome P450 Family 2; D004537:Eicosanoic Acids / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000502:physiology; D000860:Hypoxia / Q000503:physiopathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007150:Immunohistochemistry; D019194:Ischemic Preconditioning; D009687:Nuclear Proteins / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D039181:Steroid 16-alpha-Hydroxylase / Q000096:biosynthesis*; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D004537:Eicosanoic Acids; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; D005947:Glucose