We investigated the pathophysiologic mechanism of familial/sporadic glucocorticoid resistance syndrome, a prototype of glucocorticoid resistance caused by mutations in the glucocorticoid receptor (GR) gene. We found a 2-year old girl with a heterozygotic mutation replacing arginine to glutamine at amino acid 714 (GR R714Q). The patient showed severe hypertension and hypoglycemia as well as accelerated bone age, while the mutant receptor demonstrated reduced transcriptional activity, reduced affinity to dexamethasone and defective formation of the activation function (AF) 2. Computer-based structural analysis on the ligand-binding domain (LBD) of GR R714Q revealed that the mutation breaks the salt bridge between original R714 and E662, while negative charge of E662 is instead balanced by the formation of a stable new salt bridge with R704. Impact of this conformational change is transmitted to distant LBD portion and eventually destroys the ligand-binding pocket and the AF2 surface. Glucocorticoids play an essential role in the homeostasis of the central nervous system (CNS) and influence diverse functions of neuronal cells. We previously reported that the cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of CNS, and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with GR through its activators p35/p25 and differentially regulated the transcriptional activity of the GR by phosphorylating its multiple serines. Since glucocorticoids employ the mineralocorticoid receptor (MR) as a functional receptor in the brain in addition to GR, we examined the effects of CDK5 on MR and found that this kinase phosphorylated serines 129 and 158, and threonine 250 of the human MR and modulated MR-induced transcriptional activity similarly to GR. We then focused on the brain-derived neurotrophic factor (BDNF), which plays critical roles in the neurobiability, synaptic plasticity, consolidation of memory and emotional changes. MR-specific aldosterone and GR-specific dexamethasone respectively increased and reduced BDNF mRNA/protein expression in rat cortical neuronal cells, while CDK5 enhanced their effects on the BDNF expression. These results suggest that aberrant activation of CDK5 may regulate neuronal activities through corticosteroid receptors/BDNF, further contributing to the development of neurodegenerative diseases, and possibly, mood disorders. We further examined implication of GR/CDK5 interaction in the pathogenesis of mood disorders, by measuring CDK5/p35/p25 expression, CDK5 kinase activity and GR phophorylation in brain hippocampal samples obtained from deceased patients with major depression. We have also started in vivo study examining the effect of CDK5-mediated phosphorylation of GR and MR in animal behavior by collaborating with Dr. Osborne Almedia, the Max Planck Institute of Psychiatry, Munich, Germany. To find intracellular molecules potentially influencing tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screening assays using the GR DBD, and found that the noncoding (nc) RNA Gas5 interacted with this portion of the GR. This ncRNA accumulates in growth-arrested cells, but its physiologic roles are not known as yet. We found that Gas5 bound specifically to GR at its DBD, prevented the association of the GR with its regulatory DNA elements and suppressed its transcriptional activity. Serum starvation-induced Gas5 suppressed glucocorticoid-mediated mRNA expression of the cellular inhibitor of apoptosis 2 and the glucocorticoid/serum-inducible kinase genes, and prevented apoptosis through these molecules. Gas5 has one GRE-like sequence in its 3'portion in the intra-molecular double helical structure, through which this ncRNA interacts competitively with the GR DBD by mimicking DNA GREs. Thus, Gas5 is a growth arrest/starvation-related co-repressor of the GR with a decoy RNA GRE, restricting the expression of steroid-responsive genes. This is a novel concept suggesting competition between ncRNA and genomic DNA for the DBD of steroid receptors. This work also indicates that Gas5 may contribute to the cellular adaptive response to starvation. To elucidate in vivo actions of Gas5, we have started developing mice over-expressing Gas5 RNA under the control of the teracyclin-responsive promoter. Changes in energy supply act as major stressors and glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, thus we examined influence of the adenosine 5'monophosphate-activated protein kinase (AMPK) on glucocorticoid action, as AMPK is an evolutionarily well-conserved serine/threonine kinase and plays central roles as a cellular sensor of metabolic needs and regulator of the body's energy balance. We found that AMPK regulated glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive GILZ gene, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Since increased action of glucocorticoids is associated with development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacologic interventions to these pathologies. Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of the 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 in both 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 the biologic actions of diurnally fluctuating circulating glucocorticoids. We 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.
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