Complex Human Glucocorticoid Receptor dim Mutations Define Glucocorticoid Induced Apoptotic Resistance in Bone Cells

A mutation in the D-loop of the second zinc finger of the DNA-binding domain of the human glucocorticoid receptor (hGR), A458T (GR dim ), has been suggested to be essential for dimerization and DNA binding of the GR, and genetically altered GR dim mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GR dim 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 hGR dim (A458T) or the hGR dim4 (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 hGR U-2 OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGR dim mutation had diminished steroid responsiveness and cells carrying the hGR dim4 mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor B repression was unaffected by either mutation. Interestingly, both the hGR dim and hGR dim4 receptors readily formed dimers as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGR dim cells were only partially resistant to apoptosis, whereas hGR dim4 cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGR dim4 cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGR. These studies challenge conclusions drawn from previous studies of GR dim mutants. (Molecular Endocrinology 26: 244 -256, 2012) G lucocorticoid hormone signaling is essential for an organism's homeostasis and is critical for modulating the response to inflammation and to both physiological and psychological stress. The vast majority of this hormone's effects are mediated through the glucocorticoid receptor (GR). This receptor, when activated by hormone, acts as a transcription factor regulating genes involved in multiple signaling pathways and is critical for both cell survival and cell death. Mice with targeted disruption of the murine GR do not survive postpartum due to a number of defects, among these atelectasis of the lung, impaired gluconeogenesis, adrenal gland hypertrophy, repression of the hypothalamic pituitary axis, and disregulated apoptosis of thymocytes. The range of phenotypic abnormalities observed in this model is reflective of the nearly ubiquitous expression of the GR across tissues. The importance of this receptor system is also broadened by medical use of synthetic glucocorticoids in treating conditions, including ectopic dermatitis, allergies