Regulation of glucocorticoid receptor expression in cultured fibroblasts from a patient with familial glucocorticoid resistance

Abstract

The thermolabile glucocorticoid receptor (GR) in fibroblasts from a patient with familial glucocorticoid resistance (FGR) was characterized by solution hybridization, Northern blot analysis and Western immunoblotting using an hGR and cRNA probe and a GR specific monoclonal antibody. Specific DNA binding was measured by binding of cytosolic GR to mouse mammary tumour virus (MMTV) DNA. Northern blot analysis of total cellular RNA isolated from the fibroblasts showed hybridization of the hGR probe to 7.0 and 6.1 kb RNA species. Basal expression of hGR mRNA was 1.8 times higher in fibroblasts derived from the patient compared to control fibroblasts as assayed by solution hybridization. Even though nonsignificant, dexamethasone treatment maximally caused at 60% down-regulation of GR mRNA in normal fibroblasts after 12 h but only a 40% down-regulation in fibroblasts from the patient. In both cases, the initial mRNA values were restored after 72 h. No difference in GR mRNA stability was observed between fibroblasts from the patient and from controls. The induction of the glucocorticoid-regulated gene metallothionein IIA (MTIIA) by dexamethasone and cadmium sulphate was studied at different temperatures using a cRNA probe for human MTIIA. At elevated temperatures, cadmium sulphate but not dexamethasone increased MTIIA mRNA levels approximately three-fold in fibroblasts from the patient, whereas in normal fibroblasts regardless of temperature both cadmium sulphate and dexamethasone increased MTIIA mRNA levels approximately three- and two-fold, respectively. Cytosolic GR from FGR-fibroblasts showed an increased specific binding to MMTV DNA at 4 degrees C. These data support our previous findings of a thermolabile GR, probably due to a defect intrinsic to the GR protein, in this patient with primary cortisol resistance and indicate a compensatory mechanism at the transcriptional level of GR expression. The data also indicate a receptor defect affecting specific DNA binding in vitro.