Glucocorticoids, the end-products of the hypothalamic-pituitary-adrenal (HPA) axis, have a broad array of life-sustaining functions, and play an important role in the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Thus, changes of tissue sensitivity to glucocorticoids may develop pathologic states and influence their disease course. We investigated the pathophysiologic mechanism of the familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations in the glucocorticoid receptor (GR) gene. We found in a Colombian patient a heterozygotic mutation that replaced aspartic acid to histidine at amino acid 401 (GRD401H). Using panels of assay methods, we have characterized details of molecular defects of GRD401H and found that this mutant receptor demonstrated 2-3 times stronger transcriptional activity than the wild type receptor, while the patient showed a mixed phenotype characteristic to both glucocorticoid resistant and hypersentivitvity states. Thus, this mutant receptor causes tissue-specific alterations of glucocorticoid activity. We published in this year a landmark review manuscript in the Journal of Clinical Endocrinology & Metabolism on the familial/sporadic glucocorticoid resistance syndrome and molecular defects of the pathologic mutant GRs reported to date.? ? 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 on more than 90 percent of the endogenous glucocorticoid-responsive genes tested. CDK5 phosphorylated GR at multiple serines, including those located at 203, 211 and 226 in its N-terminal domain. 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 MR and modulated MR-induced transcriptional activity similarly to GR. ? ? To find more intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screening assays using the GR DBD, and found that SET/TAF-Ibeta and the noncoding (nc) Gas5 interacted with this portion of the GR. The former molecule is a part of the SET-CAN oncogene product, as well as a component of the inhibitor of acetyltransferases (INHAT) complex. We found that SET/TAF-Ibeta acts as a negative regulator of GR transcriptional activity and that ligand-activated GR stimulates transcription by displacing the INHAT complex from histones via physical interaction through its DBD. In contrast to Set/TAF-Ibeta, the Set-Can fusion protein bound GREs regardless of ligand availability and strongly suppressed GR-induced transcriptional activity/histone acetylation, possibly participating in the development of glucocorticoid insensitivity in acute undifferentiated leukemia with Set-Can translocation. Gas5 also interacted with the GR DBD. 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 as a single sense RNA in a yeast two-hybrid screening and in a RNA-protein co-immunoprecipitation assay. Gas5 RNA prevented the association of the GR with its regulatory DNA elements in a chromatin immunoprecipitation assay and suppressed its transcriptional activity on a glucocorticoid-responsive endogenous promoter. Serum starvation-induced Gas5 suppressed glucocorticoid-mediated cellular inhibitor of apoptosis 2 (cIAP2) mRNA expression, while Gas5 siRNA completely abolished this suppression. Furthermore, serum starvation-induced Gas5 prevented apoptosis of growth-arrested cells through cIAP2 protein expression. Gas5 has one GRE-like sequence in its 3 portion in its 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-related co-repressor of the GR harboring an RNA mock 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 will also indicate that Gas5 may contribute to the cellular adaptive reaction to starvation, preventing apoptosis and saving energy resources. ? ? Glucocorticoids play central roles in the regulation of basal and stress-related energy metabolism. Adenosine 5 monophosphate-activated protein kinase (AMPK), on the other hand, is the master regulator of energy homeostasis, sensing energy depletion inside the body and stimulating pathways that increase fuel uptake and save on peripheral supplies. We hypothesized that these major regulatory systems communicate with each other to maintain proper metabolic activity in the body. We found that AMPK regulates the actions of glucocorticoids at the cellular level by targeting their intracellular glucocorticoid receptor (GR) and altering its ability to modify transcription of target genes in a tissue- and promoter-specific fashion. AMPK does this by phosphorylating serine 211 of the GR indirectly through phosphorylation and subsequent activation of p38 mitogen-activated protein kinase (MAPK). At the animal level, activation of AMPK in rats completely reversed glucocorticoid-induced hepatic steatosis and strongly suppressed glucocorticoid-mediated stimulation of glucose and fatty acid metabolism. Transcriptomic analysis of liver tissue from these rats using microarrays suggested mutual regulation between the AMPK and glucocorticoid signaling pathways directed mostly from the former to moderate the activities of the latter. Thus, the AMPK-mediated energy control system governs also glucocorticoid action at target tissues. Our results also indicate that activation of AMPK could be a promising target for the development of pharmacologic interventions to metabolic disorders caused by excess amounts of glucocorticoids.? ? Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion as a result of the control exerted by central components of the HPA axis, 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 found that CLOCK/BMAL1 repressed GR-induced transcriptional activity in a HAT activity-dependent fashion. In serum shock-synchronized cells, transactivational activity of GR, accessed longitudinally by mRNA expression of an endogenous responsive gene, fluctuated spontaneously in a circadian fashion, in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically and CLOCK suppressed binding of GR to promoter GREs by acetylating a cluster of lysine residues located in its hinge region. 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. Further, these results suggest that a peripheral target tissue circadian rhythm indirectly influences functions of every organ/tissue inside the body through modulation of the ubiquitous and diverse actions of glucocorticoids.
Showing the most recent 10 out of 57 publications
