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Recent advances in the molecular mechanisms determining tissue sensitivity to glucocorticoids: novel mutations, circadian rhythm and ligand-induced repression of the human glucocorticoid receptor

Glucocorticoids are pleiotropic hormones, which are involved in almost every cellular, molecular and physiologic network of the organism, and regulate a broad spectrum of physiologic functions essential for life. The cellular response to glucocorticoids displays profound variability both in magnitude and in specificity of action. Tissue sensitivity to glucocorticoids differs among individuals, within tissues of the same individual and within the same cell. The actions of glucocorticoids are mediated by the glucocorticoid receptor, a ubiquitously expressed intracellular, ligand-dependent transcription factor. Multiple mechanisms, such as pre-receptor ligand metabolism, receptor isoform expression, and receptor-, tissue-, and cell type-specific factors, exist to generate diversity as well as specificity in the response to glucocorticoids. Alterations in the molecular mechanisms of glucocorticoid receptor action impair glucocorticoid signal transduction and alter tissue sensitivity to glucocorticoids. This review summarizes the recent advances in our understanding of the molecular mechanisms determining tissue sensitivity to glucocorticoids with particular emphasis on novel mutations and new information on the circadian rhythm and ligand-induced repression of the glucocorticoid receptor.


Background
In humans, glucocorticoids are synthesized by the adrenal cortex and released following activation of the hypothalamic-pituitary-adrenal (HPA) axis, and play an important role in the maintenance of resting and stressrelated homeostasis. Named for their effects on glucose metabolism, glucocorticoids are involved in almost every cellular, molecular and physiologic network of the organism, and regulate a broad spectrum of physiologic functions essential for life, including growth, reproduction, cognition, behavior, cell proliferation and survival, as well as immune, central nervous system (CNS) and cardiovascular functions [1][2][3]. Given their powerful antiinflammatory and immunosuppressive actions, synthetic glucocorticoids represent one of the most widely used therapeutic compounds employed in the treatment of acute and chronic inflammatory/autoimmune and lymphoproliferative disorders [1,3]. However, chronic exposure to glucocorticoids in patients with such disorders leads to multiple adverse effects. Sometimes glucocorticoid resistance of the affected organ or tissue may develop, representing a major challenge for the treatment of these conditions [4].
At the cellular level, the actions of glucocorticoids are mediated by the human glucocorticoid receptor (hGR, NR3C1), which belongs to the steroid/thyroid/ retinoic acid nuclear receptor superfamily of transcription factors [2,5]. Consistent with the pleiotropic effects of glucocorticoids, the hGR is ubiquitously expressed in all human tissues and cells, and is necessary for life after birth [6]. The hGR protein is encoded by exons 2-9 of the hGR gene (located on chromosome 5) and is composed of four distinct regions: the amino-terminal domain (NTD), the DNA-binding domain (DBD), the hinge region and the ligand-binding domain (LBD) [2,5]. Alternative splicing of hGR precursor mRNA gives rise to 5 hGR protein subtypes that have been termed hGRα, hGRβ, hGRγ, hGR-A and hGR-P. An additional cohort of eight receptor proteins (hGRα-A, hGRα-B, hGRα-C1, hGRα-C2, hGRα-C3, hGRα-D1, hGRα-D2, and hGRα-D3) is produced by alternative translation initiation from hGR mRNA [2,5,7]. hGRα-A is the classic full-length 777-amino acid receptor that is generated from the first translation initiation codon [2,5,7]. The other hGRα isoforms have progressively shorter NTDs, possess both common and unique properties and may differentially transduce the glucocorticoid signal to target tissues depending on their selective relative expression and inherent activities. All translational isoforms have similar affinity for the ligand and ability to bind to DNA, consistent with the presence of a common LBD. However, they display differences in their subcellular distribution, with hGRα-D residing constitutively in the nucleus of cells, a fact that indicates that sequences in the NTD of the hGR may play an important role in nuclear translocation, nuclear export and/or cytoplasmic retention of the receptor. In addition, these translational isoforms display significant differences in their ability to regulate gene expression, with the hGRα-C isoforms being the most active, and the hGRα-D subtypes being the most "deficient" in their ability to transactivate glucocorticoid-responsive genes [7]. Recent evidence suggests that hGRα-C3 displays higher transcriptional activity than the other hGRα isoforms, owing to increased recruitment of coactivators at the promoter regions of target genes [8]. In addition to the hGR isoforms generated by alternative splicing or alternative initiation of translation, four novel receptor variants with multiple amino acid replacements/truncation have been described [hGR NS-1, hGR DL-1, hGR-S1 and hGR-S1 (−349A)] [9,10]. Their functional role remains to be elucidated.
The hGR regulates gene expression by either transcriptional activation (transactivation) or transcriptional repression (transrepression). Prior to binding to glucocorticoids, the hGR resides mostly in the cytoplasm of cells as part of a large multiprotein complex [2,11]. Upon ligand-induced activation, the receptor undergoes conformational changes that result in dissociation from this multiprotein complex and translocation into the nucleus, where it binds to glucocorticoid-response elements (GREs) in the promoter region of target genes [2,11,12]. The latter contain hexamer domains in an inverted palindrome arrangement separated by 3 base pairs in the regulatory regions of target genes and regulate their expression positively or negatively through interaction with coactivators [12] or corepressors [2,11], respectively. Glucocorticoids may mediate anti-inflammatory effects via direct binding of hGR to evolutionarily conserved negative GREs (nGREs), which contain an inverted tetrameric palindrome separated by 0-2 base pairs that is distinct from the classic GREs [13]. The ligand-activated hGR can also modulate gene expression independently of DNA-binding, by interacting with other transcription factors, such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), p53 and signal transducers and activators of transcription (STATs). The interaction of hGR with the pro-inflammatory transcription factors NF-κB and AP-1 inhibits their activity and accounts for the major anti-inflammatory and immunosuppressive effects of glucocorticoids [3]. Although the transcriptional activity of hGR is primarily governed by ligand binding, post-translational modifications also play important roles. These covalent changes include methylation, acetylation, nitrosylation, sumoylation, ubiquitination and phosphorylation, and may affect receptor stability, subcellular localization, as well as the interaction of hGR with other proteins [11].
In addition to the above-described genomic actions, glucocorticoids can induce some effects via the GR within seconds or minutes. The non-genomic rapid glucocorticoid actions appear to be mediated by membrane bound GRs, which trigger the activation of kinase signaling pathways, thus influencing many CNS and other tissue functions [14]. On the other hand, the MR functions as the 2nd glucocorticoid receptor in some tissues (e.g. limbic structure of the brain and adipose tissue), which do not express the glucocorticoid-inactivating 11β-hydroxysteroid dehydrogenase 2, and cooperates with the classic GR to regulate expression of common and/or distinct target genes [15].
The cellular response to glucocorticoids displays profound variability both in magnitude and in specificity of action [2,11]. Multiple mechanisms exist to generate diversity, as well as specificity in the response to glucocorticoids, such as pre-receptor ligand metabolism, receptor isoform expression, and receptor-, tissue-, and cell type-specific factors. Furthermore, recent findings from in vitro and in vivo studies have demonstrated the important new role of old molecules, such as the serumand glucocorticoid-inducible kinase 1 (SGK1) [16,17] and FK506 -binding protein 51 (FKBP5) [18,19], in tissue sensitivity to glucocorticoids and associated pathologic conditions. In addition to protein-protein interactions, tissue responsiveness to glucocorticoids has become more complicated since the identification and functional characterization of hGR polymorphisms [20][21][22][23][24]. Interestingly, MR polymorphisms may also play some roles in tissue glucocorticoid sensitivity [25]. Any of the above-described molecular mechanisms may lead to alterations in tissue sensitivity to glucocorticoids, which may take the form of glucocorticoid resistance or glucocorticoid hypersensitivity and may be associated with significant morbidity (Table 1) [26]. This review summarizes the recent advances in the molecular mechanisms underlying tissue sensitivity to glucocorticoids, with particular emphasis on novel mutations and new information on the circadian rhythm of tissue sensitivity to glucocorticoids, and ligand-induced repression of the glucocorticoid receptor.
We have recently described a new case of Chrousos syndrome caused by a novel, heterozygous, point mutation (V423A) in the DBD of the hGRα, and investigated the molecular mechanisms through which the hGRαV423A affects glucocorticoid signal transduction. Compared with the wild-type receptor, the mutant receptor hGRαV423A demonstrated a significant reduction (72%) in its ability to transactivate glucocorticoidresponsive genes, impaired ability to bind to GREs and a marked delay (2.6-fold) in the time required to translocate into the nucleus. However, the hGRαV423A did not exert a dominant negative effect upon the wild-type hGRα, had a similar affinity for the ligand, and displayed a normal interaction with the glucocorticoid receptor-interacting protein-1 (GRIP1) coactivator ( Figure 2) [47].
Structural biology studies using computer-based creation of the 3-dimentional structure of the hGR DBD harboring the V423A mutation showed that the V423A substitution alters the hydrophobic nature of the first zinc finger of the hGRα DBD, and permits water to diffuse into the nearby zinc-binding site. The hydrophobic valine (V) at position 423 in the hGRα DBD shields the four zinc-binding cysteines (C421, C424, C438, and C441) from water, thereby creating a hydrophobic environment. Replacement of valine (V) by alanine (A) at this position destroys the hydrophobic environment and permits water to diffuse into the ion-binding region of the protein, where it is captured by hydrogen bonds to C424 and C441, and reduces their affinity for binding to GREs [47]. These findings expand our knowledge on the previously described molecular mechanisms of action of natural hGR mutants, and may explain the differences observed in the clinical phenotype of subjects with Chrousos syndrome [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][47][48][49].

CLOCK-mediated acetylation of hGR: Implications for circadian rhythm-mediated regulation of glucocorticoid action in target tissues
In humans, circulating cortisol concentrations are tightly regulated by the central components of the HPA axis and fluctuate naturally in an ultradian and circadian fashion, reaching their zenith in the early morning and their nadir in the late evening [1]. Stavrera et al. showed that ultradian glucocorticoid pulses resulted in dynamic association and dissociation of GR with the promoters of target genes leading to cyclic GR-mediated transcriptional regulation [50]. Interestingly, this coupling was not observed upon exposure of cells to synthetic glucocorticoids, since these were shown to prevent the dissociation of GR from the target promoter [50]. In addition to the above findings at the level of target tissues, recent studies have investigated the molecular mechanisms underlying glucocorticoid oscillations [51][52][53]. Walker et al. employed a mathematical model to study how the HPA axis supports ultradian glucocorticoid fluctuations [51]. They demonstrated that the pituitary-adrenal axis can produce ACTH and glucocorticoid ultradian oscillations in the presence of constant levels of CRH [51]. Their results were further confirmed by in vivo studies [53]. Indeed, a constant CRH input in the pituitary-adrenal axis resulted in physiological ultradian fluctuations of ACTH and glucocorticoids, whereas higher levels of CRH led to the disruption of the oscillating patterns [53]. On the other hand, the circadian secretion of cortisol is generated by an evolutionary conserved molecular 'clock', the circadian clock system, which consists of central and peripheral components [54,55]. The central clock system, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, acts as the 'master' oscillator and generator of the body's circadian rhythm under the strong influence of the light/dark input from the eyes [50,54,55]. Interestingly, the neurons of SCN do not express GR; therefore glucocorticoid feedback does not directly affect the activity of the master SCN clock.

Adrenal rests
Modified from References [28] and [29]. *This is the only case of ambiguous genitalia documented in a child with 46,XX karyotype who also harbored a heterozygous mutation of the 21-hydroxylase gene. The peripheral clock system, which is distributed in all organs and tissues, including the central nervous system outside the SCN, acts generally as a 'slave' clock under the regulation of the central SCN clock, by as yet unknown mechanism(s) [54][55][56]. Both clocks communicate with each other and generate circadian rhythmicity by the coordinated activation/inactivation of self-oscillating transcription factors. Central among them are the circadian locomotor output cycle kaput (CLOCK) and its heterodimer partner brain-muscle-arnt-like protein 1 (BMAL1) [54][55][56].
We have recently demonstrated that CLOCK physically interacts with the LBD of the hGR and suppresses the hGR-induced transcriptional activity by acetylating multiple lysine residues (480, 492, 494, and 495) in the hinge region of the receptor. This post-translational modification attenuates the binding of hGR to GREs and its ability to influence glucocorticoid-responsive gene expression. Furthermore, the expression of glucocorticoid-responsive genes fluctuated in a circadian fashion, mirroring in reverse phase the Clock/Bmal1 expression [57]. These findings indicate that CLOCK/BMAL1 is a reversephase negative regulator of glucocorticoid action in target tissues, antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids and providing a local target tissue counter-regulatory feedback loop to the central clock influence on the HPA axis [57]. As a result, tissue sensitivity to glucocorticoids is decreased in the morning (when circulating cortisol concentrations are elevated) and increased in the evening and early night (when cortisol concentrations reach their nadir) (Figure 3).
In addition to these in vitro findings, we examined the acetylation status of hGR and the expression of clockrelated and glucocorticoid-responsive genes in vivo and ex vivo, using peripheral blood mononuclear cells from healthy adult volunteers [58]. The levels of acetylated hGR were higher in the morning and lower in the evening, mirroring the fluctuations of circulating cortisol concentrations Nuclear translocation ↓

Abnormal interaction with GRIP1
Modified from References [29] and [30]. Numbers in the parentheses following authors' names indicate the corresponding references.
in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone, however, some of these genes did not show the expected diurnal mRNA fluctuations in vivo. Instead, their mRNA did not oscillate in the absence of endogenous glucocorticoid, indicating that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. These findings suggest that peripheral CLOCK-mediated circadian acetylation of the hGR functions as a target-tissue, gene-specific counter-regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it in the evening and early night [58]. Furthermore, in another recent study that determined the mRNA expression of~190 hGR action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsies obtained from 25 obese subjects, circulating cortisol concentrations in the evening were more important in regulating the mRNA expression of glucocorticoid-responsive genes than those in the morning [59]. Therefore, it appears that the higher tissue sensitivity to glucocorticoids in the evening, owing to reduced hGR acetylation by CLOCK, underlies the stronger effect of elevated circulating cortisol concentrations on the expression of glucocorticoid-regulated genes in the evening and early night.
In line with our in vitro, in vivo and ex vivo results, Lamia et al. recently showed that CRY1 and CRY2 interacted with the GR in a ligand-dependent fashion, leading to direct inhibition of GR-mediated transactivation of glucocorticoid-responsive genes [60]. Interestingly, this CRY-GR interaction did not affect the transrepressing actions of GR on many inflammatory genes, indicating that cryptochromes, when interact with the GR, could influence the expression of a separate group of GR target genes. This dissociation of the transactivating and transrepressing activities of the GR might reduce the frequency of the undesirable metabolic effects during long-term Figure 2 Μolecular mechanisms through which the natural mutant receptor hGRαV423A causes Chrousos syndrome. Both the wild-type hGRα and the mutant hGRαV423A reside in the cytoplasm in the absence of ligand by forming a heterocomplex with heat shock proteins (HSP) and FKBP51 (FKBP). Upon binding to ligand, the wild-type hGRα dissociates from the heterocomplex partners and translocates into the nucleus, while this process of the mutant hGRαV423A is significantly delayed. The wild-type hGRα stimulates or represses the transcriptional activity of glucocorticoid-responsive genes by attracting to GRE DNA several coactivators including the glucocorticoid receptor-interacting protein 1 (GRIP-1), or by interacting with other transcription factors, such as NF-κB and activator protein-1 (AP-1). The hGRαV423A demonstrates reduced transactivation activity due to decreased ability to interact with GREs, while its activity to repress the transcriptional activity of other transcription factors is preserved. The hGRαV423A does not exert a dominant negative effect upon the wild type-induced transactivation of glucocorticoid-responsive genes. WT: wild-type human glucocorticoid receptor; V423A: human glucocorticoid receptor V423A; HSP: heat shock proteins; FKBP: immunophilins; GRIP-1: glucocorticoid receptor-interacting protein 1; p65: transcription factor p65; p50: transcription factor p50.
administration of synthetic glucocorticoids [60]. Moreover, a very recent study demonstrated that CLOCK/ BMAL1 reduced both maximal GR transactivation of target genes and the efficacy of the receptor to increase the transcription rate of glucocorticoid-responsive genes. On the other hand, the PER1/CRY1 complex reduced the maximal GR transactivation, but not the efficacy of the receptor, suggesting that both elements of the positive and negative arm of the clock system repress GR transcriptional activity [61]. The circadian clock system and the HPA axis regulate the activity of one another through multilevel interactions to ultimately coordinate homeostasis against the day/night change and various unforeseen random internal and external stressors. Uncoupling of or dysfunction in either system alters internal homeostasis and causes pathologic changes virtually in all organs and tissues, including those responsible for intermediary metabolism and immunity [62]. Disrupted coupling of cortisol secretion and target tissue sensitivity to glucocorticoids may account for i) the development of central obesity and the metabolic syndrome in chronically stressed individuals, whose HPA axis circadian rhythm is characterized by blunting of the evening decreases of circulating glucocorticoids, as a result of enhanced input of higher centers upon the hypothalamic paraventricular nucleus secretion of CRH and AVP; and ii) the increased cardiometabolic risk and increased mortality of rotating shift workers or subjects exposed to frequent jet lag because of traveling across time zones [56,63]. In addition, given that tissue sensitivity to glucocorticoids is increased in the evening, clinicians should avoid the administration of high doses of glucocorticoids for the treatment of adrenal insufficiency or congenital adrenal hyperplasia at night, because they increase the possibility of glucocorticoid-related side effects.
At pharmacological concentrations, the transactivational activity of glucocorticoids is correlated with the side effects of these steroids, while their transrepressive activity is associated mostly with their beneficial anti- inflammatory activity. Since CLOCK may differentially regulate these two major class actions of glucocorticoids, administration of these steroids at a specific period of the circadian cycle might increase their pharmacological efficacy, while at the same time reduce their unwanted side effects [62].
Ligand-induced down-regulation (repression) of hGR gene expression Glucocorticoids are among the most potent and effective agents for treating inflammatory diseases and hematologic malignancies. However, a number of patients are often resistant to treatment with glucocorticoids [1][2][3][4]. Therefore, it is important to determine the molecular mechanisms underlying this phenomenon in order to adequately treat patients with these disorders.
Recent studies investigated the molecular mechanisms responsible for repression of hGR gene transcription by glucocorticoids. They demonstrated that glucocorticoid treatment resulted in a rapid decrease in nascent hGR RNA, with maximal repression being sustained for 6 to 8 h. They also identified a functional nGRE in exon 6 of the hGR gene, which played an important role in the repression of the hGR gene. Upon ligand-induced activation, the hGR bound to the nGRE on exon 6 and recruited NCoR1 (nuclear receptor corepressor) and HDAC3 (histone deacetylase 3) to the promoter-proximal region, thereby forming a repression complex and inhibiting hGR transcription [64]. These findings indicate that the hGR concentration can be down-regulated coordinately with excess ligand, regardless of the combinatorial associations of tissue-specific transcription factors. Therefore, although glucocorticoid-induced down-regulation of hGR represents a mechanism for maintaining glucocorticoid homeostasis in normal cells, it appears to have the potential to limit the therapeutic response to glucocorticoids in inflammatory and malignant conditions. As a result, longterm glucocorticoid treatment in patients with the above disorders may lead to constitutive hGR transrepression and glucocorticoid resistance.
In addition to glucocorticoid-induced down-regulation of hGR, the expression of the receptor has been shown to be influenced by epigenetic modifications which occurred in the context of gene-environment interactions. In their pioneering study, Weaver and collaborators demonstrated that maternal behavior had a pivotal role in stress responses of offspring. Indeed, rats exposed to low levels of pup licking and grooming (LG) and archedback nursing (ABN) by their mothers, had increased methylation of the 1 7 promoter of the GR gene, leading to decreased levels of GR in the hippocampus and altered response of the HPA axis to stressors [65]. This epigenetic alteration of GR expression could also be involved in drug-induced glucocorticoid resistance. Moreover, GR protein levels were found to be reduced by micro-RNAs (miRs), such as miR-18a [66], −18 and -124a [67] in neuronal tissues. Uchida et al. used Fischer 344 (F344) rats, a well-known stress-hyperresponsive model, and showed that this strain, upon a 14-day repeat restrain stress, had increased levels of miR-18a and decreased GR protein expression in the PVN, compared with control Sprague-Dawley (SD) rats [66]. Further studies have supported the implication of microRNAs, specifically miR-18 and -124a, in brain responsiveness to glucocorticoids. Both miR-18 and -124a down-regulated the GR, establishing their critical role in the regulation of glucocorticoid responsiveness of the brain [67]. On the other hand, the specific downregulation of the GR in newborn hippocampal cells, using viral-mediated RNA-interference in vivo, increased their neuronal differentiation and migration, as well as their basal excitability. These morphological and functional alterations of hippocampal cells resulted in impairments in memory consolidation [68]. Therefore, blockade of selective GR-dependent processes in brain would be therapeutically beneficial. Zalachoras et al. demonstrated that the non-steroidal GR ligand 1H-pyrazolo[3,4-g]hexahydroisoquinoline or C108297 selectively modulated GR activity in the brain by suppressing CRH gene expression while enhancing GR-dependent memory consolidation by acting as a unique GR modulator [69]. In contradistinction, C108297 antagonized GR-mediated reduction in neurogenesis in the hippocampus following long-term administration of glucocorticoids [69]. Thus, C108297 has a potential to selectively abrogate pathogenic processes associated with GR in the brain, while retaining some beneficial actions of this receptor.

Conclusions
The glucocorticoid receptor is a ubiquitously expressed intracellular, ligand-dependent transcription factor, which mediates the action of glucocorticoids and influences physiologic functions essential for life. The traditional view that glucocorticoids exert their diverse effects through one receptor protein has changed dramatically over the last two decades with the discovery of multiple hGR isoforms arising from the single hGR gene. hGR subtypes with unique expression and gene regulatory profiles are generated by alternative splicing of the nascent transcript, alternative translation initiation of the mature mRNA, and post-translational modifications of the receptor protein.
The capacity of a cell to generate dozens of hGR isoforms that control specific sets of genes and/or differentially regulate common sets provides enormous potential for signaling diversity. Further contributing to the tissue-and cell-specific effects of glucocorticoids is the potential for these isoforms to heterodimerize with each other and cross-talk with other signaling molecules. The combinatorial nature of glucocorticoid signaling pathways indicates that alterations in hGR action may have important implications for many critical biological processes and may account for the alterations in tissue sensitivity to glucocorticoids, as well as the variations in response to glucocorticoid treatment documented in clinical practice. A greater understanding of the role that hGR heterogeneity plays in the cellular response to glucocorticoids should aid in the development of new glucocorticoid compounds with selective activities that may offer the prospect of an improved outcome with lesser side effects.