Identification of a glucocorticoid response element in the rat beta2-adrenergic receptor gene.
Regulation of beta2-adrenergic receptor (beta2AR) levels by glucocorticoids is a physiologically important mechanism for altering beta2AR responsiveness. Glucocorticoids increase beta2AR density by increasing the rate of beta2AR gene transcription, but the cis-elements involved have not been well characterized. We now show that one of six potential glucocorticoid response elements (GREs) in the 5'-flanking region of the rat beta2AR gene is necessary for glucocorticoid-dependent stimulation of receptor gene expression. Using a nested set of deletion fragments of the rat beta2AR gene 5'-flanking region fused to a luciferase reporter gene, glucocorticoid-dependent induction of reporter gene expression in HepG2 cells was localized to a region between positions -643 and -152, relative to the transcription initiation site. In electrophoretic mobility shift assays, a double-stranded oligonucleotide incorporating a near-consensus GRE from this region (positions -379 to -365) formed complexes with the human recombinant glucocorticoid receptor, as well as with nuclear protein from dexamethasone-treated HepG2 cells. Mutation of a single base within this GRE sequence greatly diminished interaction of the mutated oligonucleotide with the human recombinant glucocorticoid receptor. The functional activity of the GRE was characterized using a luciferase reporter construct driven by a minimal thymidine kinase promoter. In HepG2 cells transfected with constructs containing the GRE, dexamethasone increased reporter gene expression approximately 3-fold, whereas a dexamethasone effect was not observed with constructs lacking the GRE. Taken together, these findings show that a GRE located at positions -379 to -365 in the 5'-flanking region of the rat beta2AR gene mediates glucocorticoid stimulation of beta2AR gene transcription.
Cornett
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Division of Critical and Pulmonary Care Medicine, Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. cornettlawrencee@exchange.uams.edu
Hiller
F C
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Jacobi
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Cao
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McGraw
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D000818:Animals; D002478:Cells, Cultured; D005938:Glucocorticoids / Q000096:biosynthesis / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000235:genetics* / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D005938:Glucocorticoids; D011943:Receptors, Adrenergic, beta; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D008156:Luciferases