Complex human glucocorticoid receptor dim mutations define glucocorticoid induced apoptotic resistance in bone cells

Abstract

A mutation in the D-loop of the second zinc finger of the DNA-binding domain of the human glucocorticoid receptor (hGR), A458T (GRdim), has been suggested to be essential for dimerization and DNA binding of the GR, and genetically altered GRdim mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GRdim mice were reported to be resistant to glucocorticoid-induced apoptosis. To further evaluate the dim mutations in glucocorticoid-induced apoptosis, we stably expressed either the hGRdim (A458T) or the hGRdim4 (A458T, R460D, D462C, and N454D) mutant receptors in human osteosarcoma (U-2 OS) cells that are devoid of hGR and unresponsive to glucocorticoids. We analyzed these cell lines by comparison with a stable expression hGRα U-2 OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGRdim mutation had diminished steroid responsiveness and cells carrying the hGRdim4 mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor αB repression was unaffected by either mutation. Interestingly, both the hGRdim and hGRdim4 receptors readily formed dimers as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGRdim cells were only partially resistant to apoptosis, whereas hGRdim4 cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGRdim4 cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants. © 2012 by The Endocrine Society.