Identification of a glucocorticoid response element in the rat beta2- adrenergic receptor gene

  • Cornett L
  • Hiller F
  • Jacobi S
 et al. 
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Abstract

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.

Author-supplied keywords

  • *Response Elements
  • Animal
  • Cells, Cultured
  • Glucocorticoids/biosynthesis/*genetics
  • Luciferase/genetics
  • Mutation
  • Plasmids
  • Promoter Regions (Genetics)
  • Rats
  • Receptors, Adrenergic, beta/*genetics/metabolism
  • Receptors, Glucocorticoid/metabolism
  • Support, U.S. Gov't, P.H.S.
  • Transcription Factors/metabolism
  • Transfection

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  • PMID: 9855630

Authors

  • L E Cornett

  • F C Hiller

  • S E Jacobi

  • W Cao

  • D W McGraw

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