We have investigated the pathophysiologic mechanism of familial/sporadic generalized glucocorticoid resistance syndrome, a prototype of glucocorticoid resistance caused by mutations in the glucocorticoid receptor (GR) gene. We found three new heterozygotic cases with mutations in the GR gene (GR V423A, GR V575G and GR H726R) in collaboration with Dr. Evangelia Charmandari, the Univ. of Athens Medical School, Athens, Greece. We have almost completed molecular analysis on the functional defects of the former two mutations and are now preparing manuscripts. We have completed the computer-based 3-dimensional structural analysis for the ligand-binding domain of all pathologic GR mutations ever reported in addition to these newly identified mutations. We are also writing a manuscript based on these findings. We encountered a 7-year old boy with mild resistance to glucocorticoids, androgens and thyroid hormones. Array-based comparative genomic hybridization analysis showed that this patient has 1.1 Mb size heterozygotic 16p11.2 microdeletion, while the siRNA-based screening and subsequent molecular analyses revealed that heterozygotic deletion of the ZNF764 gene by his microdeletion is responsible for his multi-hormone resistance, as this protein acts as a specific coactivator for the glucocorticoid, androgen and thyroid hormone receptors. This is the first case demonstrating resistance to multiple steroid hormones with identification of the causative gene. We have published these results in this fiscal year. The liver X receptors (LXRs), which belong to the nuclear receptor superfamily, mediate the biologic actions of various lipids, such as the cholesterol metabolites oxysterols, prostanoids and some fatty acids, by directly binding to these molecules. LXRs exist as two subtypes, LXRαand LXRβ, which display distinct patterns of tissue expression. Once LXRs bind their lipid ligands, they form a heterodimer with the retinoid X receptor (RXR), and stimulate the transcription of an array of genes involved in the absorption, efflux, transport, and excretion of cholesterol and other lipids. LXRs also regulate glucose metabolism by decreasing the expression of its rate-limiting enzymes G6Pase and PEPCK, and have anti-inflammatory activity by repressing a set of inflammatory genes in macrophages and other immune cells. We found that activation of LXRs/RXRs repressed GR-stimulated transactivation of glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion: It attenuated dexamethasone-stimulated elevation of circulating glucose in rats and suppressed dexamethasone-induced mRNA expression of G6Pase in rats, mice and human hepatoma HepG2 cells. Mechanistically, we found that LXRα/RXRαbound GREs and inhibited GR binding to these DNA sequences in a gene-specific fashion. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly attenuating the transcriptional activity of GR on glucose and/or lipid metabolism. Based on these findings, we have published one manuscript in this fiscal year Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery. We previously reported that CLOCK/BMAL1 repressed GR-induced transcriptional activity by acetylating GR at several lysine residues located in its hinge region and by suppressing binding of GR to promoter GREs. These findings indicate that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing to the biologic actions of diurnally fluctuating circulating glucocorticoids. We thus performed a human study in which we sampled peripheral blood in the morning and evening from normal subjects, and measured mRNA expression of known glucocorticoid-responsive genes and GR acetylation in circulating lymphocytes. We found that GR was acetylated higher in the morning than in the evening, positively correlating with mRNA expression of CLOCK and BMAL1, while circulating glucocorticoid-stimulated mRNA expression of glucocorticoid responsive genes were repressed by CLOCK/BMAL1 in a gene-specific fashion. These results indicate that the peripheral CLOCK system negatively regulates GR transcriptional activity through acetylation of GR not only in cultured cells but also in humans. We published one manuscript based on these findings in this fiscal year. In another clinically oriented study in which we measured mRNA expression of 190 GR action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsied from 25 obese subjects, we found that levels of evening cortisol is much more important than those in the morning to regulate mRNA expression of glucocorticoid responsive genes. Ratios of morning/evening serum cortisol levels also have a unique effect. It appears that higher sensitivity of tissues to circulating glucocorticoids in the evening due to reduced GR acetylation by CLOCK underlies stronger impact of evening serum cortisol levels to glucocorticoid-regulated gene expression compared to its morning levels. The manuscript based on these findings is now under review. In connection with our study on circadian rhythms, we found one interesting family with seasonal alteration of circadian rhythmicity in collaboration with Dr. F. Halberg, the Univ. of Minnesota. The proband, 61-year old female, has suffered from an annual cycle of severe fatigue including inability to get out of bed, which lasts 2-3 months in the summer and the winter. She, however, is free from symptoms with high achievement during the unaffected months between these bad periods. The proposita demonstrated elongation of circadian rhythmicity (24.84 hours) in blood pressure and vigor rates during the affected periods, while she was in normal 24-hour rhythms in the unaffected months. The cycle of 24.84 hours is exactly the double of the tidal cycle, which is hypothesized to come about gravity-changes caused by movement of the moon around the earth. To identify genetic cause(s) of these manifestations, we have just completed the whole exome analysis that reveals sequence of the entire coding region of genome in 3 affected subjects and 5 unaffected members. We will complete analysis on the obtained results soon.
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