Abstract
Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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ATP-Binding Cassette Transporters / antagonists & inhibitors
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ATP-Binding Cassette Transporters / genetics*
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Action Potentials / drug effects
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Action Potentials / physiology
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Animals
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Binding Sites
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Brain / blood supply
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Brain / metabolism
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Calcium Channels / genetics
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Calcium Channels / metabolism
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Chromatin Immunoprecipitation
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Disease Models, Animal
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Endothelial Cells / metabolism
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Glyburide / pharmacology
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Glyburide / therapeutic use
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Hep G2 Cells
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Humans
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Hypoglycemic Agents / pharmacology
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Hypoglycemic Agents / therapeutic use
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Hypoxia-Inducible Factor 1 / antagonists & inhibitors
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Hypoxia-Inducible Factor 1 / genetics
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Hypoxia-Inducible Factor 1 / metabolism*
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Hypoxia-Ischemia, Brain / genetics
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Hypoxia-Ischemia, Brain / metabolism*
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Hypoxia-Ischemia, Brain / prevention & control
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Immunohistochemistry
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KATP Channels / genetics
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KATP Channels / metabolism
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Luciferases / genetics
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Male
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Microvessels / cytology
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Microvessels / metabolism
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Patch-Clamp Techniques
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Plasmids
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Potassium Channels, Inwardly Rectifying / antagonists & inhibitors
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Potassium Channels, Inwardly Rectifying / genetics*
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Promoter Regions, Genetic
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Protein Binding
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Rats
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Rats, Wistar
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Receptors, Drug / antagonists & inhibitors
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Receptors, Drug / genetics*
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Reverse Transcriptase Polymerase Chain Reaction
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Sp1 Transcription Factor / antagonists & inhibitors
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Sp1 Transcription Factor / genetics
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Sp1 Transcription Factor / metabolism*
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Sulfonylurea Receptors
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Transcription, Genetic*
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Transfection
Substances
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ABCC8 protein, human
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ATP-Binding Cassette Transporters
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Abcc8 protein, rat
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Calcium Channels
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Hypoglycemic Agents
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Hypoxia-Inducible Factor 1
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KATP Channels
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Potassium Channels, Inwardly Rectifying
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Receptors, Drug
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Sp1 Transcription Factor
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Sulfonylurea Receptors
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Luciferases
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Glyburide