Glucocorticoids have a broad array of life-sustaining functions and play important roles in the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Changes of tissue responsiveness to glucocorticoids may produce pathologic states and influence both the course of a disease and its response to therapy. We investigated the pathophysiologic mechanisms of the familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations of the glucocorticoid receptor (GR) gene. We analyzed the functional defects of pathologic mutant receptors GRV571A, GRD641V, GRV729I, GRGRI559N and GRI747M by examining their nuclear shuttling, DNA-binding activity, interaction with p160 coactivators and transdominant negative activity on the wild type receptor, and compared observed mutants? defects with the clinical phenotypes of the patients. We also found a novel pathologic heterozygote mutation that replaces leucine at amino acid 773 of the GR with proline. This mutant demonstrated reduced transactivation activity due to its inability to form a ligand-dependent transactivation surface in its ligand-binding domain and behaved as a dominant negative inhibitor of the normal receptor, thereby producing clinical pathology at the heterozygotic condition. To discover intracellular molecules influencing tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screenings. GR specifically bound one of the orphan nuclear receptors, chicken ovalbumin-upstream promoter-transcription factor II (COUP-TFII). GR and COUP-TFII mutually affected each other's transcriptional activity on the promoters of several genes involved in intermediary metabolism. We also found that the G protein coupled receptor downstream mediator guanine nucleotide-binding protein (G protein) beta and the transforming growth factor beta/bone morphogenetic protein-induced SMAD6 repressor specifically interacted with the GR. Both Gbeta and SMAD6 proteins suppressed GR-induced transactivation of glucocorticoid-responsive promoters. Contrary to dogma, the former protein, with its partner molecule G gamma, co-migrated with the GR into the nucleus in response to glucocorticoids, and was attracted to glucocorticoid response elements (GREs) located in the promoter regions of glucocorticoid-responsive genes. These results indicate that the G protein beta/gamma complexes may suppress the transcriptional activity of the GR by interfering with the GR-associated transcriptosomes on GR-regulated promoters. In contrast, SMAD6 suppressed GR transcriptional activity by attracting histone deacetylases, functioning as a corepressor similarly to other classic molecules, like the nuclear receptor corepressors (NCoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT). In addition to the above proteins, we found that the autoimmune regulator protein (AIRE), whose mutations cause the autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy (APECED) syndrome, interacted with the GR and enhanced its transcriptional activity. AIRE has four LXXLL signature motifs through which classic nuclear receptor coactivators interact with ligand-activated receptor molecules. Destruction of these motifs in AIRE disturbed its interaction with the GR and inactivated its enhancing effect on GR transactivation, indicating that AIRE may act as a tissue-specific (thymus) coactivator of GR by using such signature motifs. We are also working on Brx, a Rho type guanine nucleotide exchange factor, which activates the Rho family of small G proteins by converting them from an inactive GDP-bound to an active GTP-bound form. This protein has a nuclear receptor-interacting domain in its C-terminal portion. We have demonstrated that Brx enhanced GR transcriptional activity by activating and closely attracting small G proteins to the GR. Endogenous Brx is necessary for the action of glucocccorticoids in splenic lymphocytes and mediates lysophosphatidic acid (LPA)-induced potentiation of GR transactivation. We are investigating the roles of GRbeta and transcriptional coactivator TRAP220 in regulating tissue sensitivity to glucocorticoids. The former is a splicing variant of the classic receptor GRalpha, while the latter is a component of the huge coactivator protein complex DRIP/TRAP. We developed a stable cell line that over-expresses GFP-GRbeta and obtained TRAP220 KO cells from Dr. R. Roeder?s laboratory. We are examining the impact of their expression/depletion on glucocorticoid-induced transcriptional activity by using regular DNA microarray chips. To elucidate endogenous genes that attract GR to their promoter region, we have developed CpG microarray chips, which include the promoter regions of over 10,000 endogenous genes. We performed chromatin immunoprecipitation assays using antibodies against the GR and will treat the chips with the harvested DNA to discover endogenous promoters that attract GR. Patients with the acquired immunodeficiency syndrome (AIDS), caused by infection with the Human Immunodeficiency Virus type-1 (HIV-1), have several clinical manifestations compatible with increased tissue sensitivity to glucocorticoids. We focused on one of the HIV-1-encoding accessory proteins, Vpr, and demonstrated that this viral polypeptide acts as a coregulator of the GR. We further examined the mechanism of Vpr-induced coactivation of GR transactivation focusing on its interaction with host nuclear receptor coactivators p300/CBP, and addressed the biological significance of Vpr coactivator activity by testing it on the production/secretion of glucocorticoid-responsive interleukin-12 and other gene products. Our findings may explain several manifestations of AIDS, including immunosuppression related to a T-helper 1/T-helper 2 shift, myopathy, muscle weakness/atrophy and obesity-related insulin resistance with metabolic and cardiovascular manifestations.
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