Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1α-linked induction of P450 2C11 epoxygenase in astrocytes

Mingyue Liu, Nabil Alkayed

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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 epoxyeico-satrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1α, 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-1α were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1α 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-1α protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1α 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-1α-linked upregulation of P450 2C11.

Original languageEnglish (US)
Pages (from-to)939-948
Number of pages10
JournalJournal of Cerebral Blood Flow and Metabolism
Volume25
Issue number8
DOIs
StatePublished - Aug 2005

Fingerprint

Hypoxia-Inducible Factor 1
Astrocytes
Ischemic Preconditioning
Electrophoretic Mobility Shift Assay
Cytochrome P-450 Enzyme System
Messenger RNA
Proteins
DNA
Brain
Arachidonic Acid
Northern Blotting
Cell Death
Up-Regulation
Ischemia
Western Blotting
Stroke
Binding Sites
Oxygen
Glucose
Acids

Keywords

  • Arachidonic acid
  • Astrocytes
  • EET
  • Hypoxia preconditioning (HPC)
  • Hypoxia-inducible factor (HIF)
  • Oxygen glucose deprivation (OGD)
  • P450 2C11 epoxygenase
  • Tolerance

ASJC Scopus subject areas

  • Endocrinology
  • Neuroscience(all)
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{31dae2e0cccd4a7f977a9f939afb2d16,
title = "Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1α-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 epoxyeico-satrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1α, 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-1α were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1α 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-1α protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1α 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-1α-linked upregulation of P450 2C11.",
keywords = "Arachidonic acid, Astrocytes, EET, Hypoxia preconditioning (HPC), Hypoxia-inducible factor (HIF), Oxygen glucose deprivation (OGD), P450 2C11 epoxygenase, Tolerance",
author = "Mingyue Liu and Nabil Alkayed",
year = "2005",
month = "8",
doi = "10.1038/sj.jcbfm.9600085",
language = "English (US)",
volume = "25",
pages = "939--948",
journal = "Journal of Cerebral Blood Flow and Metabolism",
issn = "0271-678X",
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T1 - Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1α-linked induction of P450 2C11 epoxygenase in astrocytes

AU - Liu, Mingyue

AU - Alkayed, Nabil

PY - 2005/8

Y1 - 2005/8

N2 - 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 epoxyeico-satrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1α, 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-1α were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1α 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-1α protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1α 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-1α-linked upregulation of P450 2C11.

AB - 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 epoxyeico-satrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1α, 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-1α were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1α 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-1α protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1α 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-1α-linked upregulation of P450 2C11.

KW - Arachidonic acid

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KW - EET

KW - Hypoxia preconditioning (HPC)

KW - Hypoxia-inducible factor (HIF)

KW - Oxygen glucose deprivation (OGD)

KW - P450 2C11 epoxygenase

KW - Tolerance

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U2 - 10.1038/sj.jcbfm.9600085

DO - 10.1038/sj.jcbfm.9600085

M3 - Article

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AN - SCOPUS:23044439770

VL - 25

SP - 939

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JO - Journal of Cerebral Blood Flow and Metabolism

JF - Journal of Cerebral Blood Flow and Metabolism

SN - 0271-678X

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