Sequences Downstream of the Glucocorticoid Regulatory Element Mediate Cycloheximide Inhibition of Steroid Induced Expression from the Rat α₁-Acid Glycoprotein Promoter: Evidence for a Labile Transcription Factor

Abstract

The glucocorticoid induction of alpha 1-acid glycoprotein (AGP) RNA in rat hepatoma cells is diminished by inhibiting protein synthesis. We now show that the AGP 5'-flanking region contains a DNA sequence (position -121 to -107), exhibiting a high degree of homology to the glucocorticoid regulatory element (GRE) consensus sequence ACAXXXTGTTCT, which serves to specifically bind purified rat glucocorticoid receptor in vitro. A 15 base pair oligonucleotide representing the AGP GRE confers glucocorticoid responsiveness on a heterologous promoter; such regulation is not diminished by concurrent inhibition of protein synthesis. However, inclusion of the AGP sequences immediately downstream of the AGP GRE (position -106 to -42) renders the hormonal induction sensitive to inhibition of protein synthesis. Furthermore, inclusion of these downstream sequences results in a more pronounced induction mediated by the AGP GRE. In vitro DNase-1 treatment using nuclear extracts prepared from HTC hepatoma cells generate footprints that indicate the presence of DNA-protein interactions spanning the region from -110 to -68 of the AGP gene. We propose that one or more labile factors acting within this domain, immediately downstream of the GRE, is required for efficient transcription at the AGP promoter.

The Glucocorticoid-responsive Gene Cascade

“…On the other hand, especially in the liver, glucocorticoid induction of a number of genes exhibits more or less secondary aspects, i.e. delayed time course and/or sensitivity to protein synthesis inhibitors, as exemplified by genes for ␣ 2u -globulin (8 -10), ␣ 1 -acid glycoprotein (28,46), albumin (47), tryptophan oxygenase (48), phosphoenolpyruvate carboxykinase (49), and ornithine cycle enzymes (6). In mammals, little is known about the transcription factors responsible for secondary activation of target genes.…”

Coactivator TIF1β Interacts with Transcription Factor C/EBPβ and Glucocorticoid Receptor To Induce α1-Acid Glycoprotein Gene Expression

“…In addition to C/EBP␤-binding motifs, a glucocorticoid-responsive element (GRE) also exists between Ϫ120 and Ϫ107 in the 5Ј-flanking region of the agp gene (8,18). Previous reports showed that maximal induction of the agp gene by glucocorticoids also requires another C/EBP␤-binding element located downstream of GRE (34,50,60). The synergistic interaction between cytokines and glucocorticoids has been attributed to protein-protein interactions between C/EBP␤ and the glucocorticoid receptor (GR) (45).…”

“…Previous studies indicated that maximal induction of the agp gene by glucocorticoid requires the downstream C/EBP␤-binding sequences (34,50,60). To further study the TIF1␤-mediated GR induction of the agp gene, we performed transfection assays using agp promoters containing mutated C/EBP␤-binding sites or mutated GRE.…”

A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction.

“…A glucocorticoid-responsive element (GRE) exists between positions -120 and -107 in the 5'-flanking region of this gene. However, maximal induction by glucocorticoid requires another sequence located immediately downstream of this GRE (27,38). Using this latter sequence as a probe (positions -121 to -93 of the AGP promoter), Chang et al isolated AGP/EBP, a mouse homolog of NF-IL6, by direct screening (12 Bacterially expressed NF-IL6 fusion protein was incubated with the end-labeled pAGP-d or pAGP-p oligonucleotide in the absence or in the presence of a 100-fold excess of the unlabeled fragment.…”