Human glucocorticoid receptor cDNA contains sequences sufficient for receptor down-regulation

Abstract

Glucocorticoid receptors are ligand-dependent transcription factors that are subject to down-regulation by their cognate ligand; however, the mechanisms mediating this physiological response are not completely understood. Since analysis of the human glucocorticoid receptor (hGR) cDNA sequence revealed the presence of sequences with homology to both positive and negative glucocorticoid regulatory elements, we have examined the potential of hGR to bind to the hGR cDNA by Southwestern blot analysis. The data revealed that glucocorticoid receptors exhibited specific binding to their own cDNA. To determine whether this binding was of functional significance in the down-regulation of glucocorticoid receptors, we analyzed the effect of glucocorticoids on hGR protein levels from COS 1 cells transfected with an hGR cDNA expression vector. These transfected cells produced intact hGR that were capable of ligand-dependent regulation of a co-transfected glucocorticoid-responsive reporter gene. Glucocorticoid treatment of hGR-transfected cells resulted in down-regulation of hGR (assayed by both glucocorticoid binding capacity and hGR protein levels) within 24 h of steroid administration. To determine if the glucocorticoid-induced down-regulation of transfected hGR was compatible with effects at the levels of receptor gene expression and RNA stability, we examined hGR mRNA steady state levels. Reductions from 2- to 6-fold were observed in hGR mRNA levels following glucocorticoid treatment of transfected COS 1 cells. This down-regulation of transfected hGR mRNA could not be attributed to either the Rous sarcoma virus promoter, which drives hGR expression, or to other sequences present in the vector plasmid since transcription of a related plasmid containing a chloramphenicol acetyltransferase gene in place of the hGR cDNA was not regulated by glucocorticoids. Down-regulation of hGR mRNA by glucocorticoids in transfected cells occurred in a time- and dose-dependent manner that is consistent with a glucocorticoid receptor-mediated process. Glucocorticoid-induced down-regulation of hGR mRNa steady state levels was not observed in COS 1 cells transfected with cDNAs encoding mutant hGR (defective in either steroid or DNA binding), which indicates that functional steroid and DNA binding domains of the expressed hGR were required for down-regulation. Interestingly, treatment of transfected COS 1 cells with the glucocorticoid antagonist RU486 also resulted in down-regulation of transfected hGR mRNA. Deletion analysis revealed that the region of the hGR cDNA that was responsible in part for the observed down-regulation in response to glucocorticoid was contained within a 1-kilobase restriction fragment (from base pair +527 to +1526).(ABSTRACT TRUNCATED AT 400 WORDS)