title: 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 (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 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 hGR(dim) mutation had diminished steroid responsiveness and cells carrying the hGR(dim4) mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor κB 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 hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants.
authors.affiliation: National Institute of Environmental Health Sciences/National Institutes of Health, Laboratory of Signal Transduction, Research Triangle Park, North Carolina 27709, USA. jewell@niehs.nih.gov
authors.forename: Christine M
authors.initials: CM
authors.lastname: Jewell
authors.forename: Alyson B
authors.initials: AB
authors.lastname: Scoltock
authors.forename: Brant L
authors.initials: BL
authors.lastname: Hamel
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Yudt
authors.forename: John A
authors.initials: JA
authors.lastname: Cidlowski
mesh_terms: D019943:Amino Acid Substitution; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D003907:Dexamethasone / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010011:Osteocytes / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: D051017:Apoptosis Regulatory Proteins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; C411470:glucocorticoid receptor alpha; D003907:Dexamethasone; D008156:Luciferases