Selective transrepression versus transactivation mechanisms by glucocorticoid receptor modulators in stress and immune systems

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European Journal of Pharmacology 583 (2008) 290 – 302 www.elsevier.com/locate/ejphar

Review

Selective transrepression versus transactivation mechanisms by glucocorticoid receptor modulators in stress and immune systems Karolien De Bosscher a,⁎, Kathleen Van Craenenbroeck a , Onno C. Meijer b , Guy Haegeman a a

Laboratory of Eukaryotic Gene Expression & Signal Transduction (LEGEST), Department of Molecular Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium b Div Medical Pharmacology, Leiden/Amsterdam Center for Drug Research & Leiden University Medical Center, PO Box 9502, 2300 RA Leiden, The Netherlands Accepted 12 November 2007 Available online 31 January 2008

Abstract Glucocorticoids control immune homeostasis and regulate stress responses in the human body to a large extent via the glucocorticoid receptor. This transcription factor can modulate gene expression either through direct DNA binding (mainly resulting in transactivation) or independent of DNA binding (in the majority of cases resulting in transrepression). The aim of this review is to discuss the mechanistic basis and applicability of different glucocorticoid receptor modulators in various affections, ranging from immune disorders to mental dysfunctions. © 2008 Elsevier B.V. All rights reserved. Keywords: Glucocorticoid receptor; Transrepression; Stress; HPA; Inflammation; NF-κB

Contents 1. 2. 3. 4. 5. 6.

Glucocorticoids and mineralocorticoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The glucocorticoid receptor and the mineralocorticoid receptor . . . . . . . . . . . . . . . . . . . . . . . . Functionality of the glucocorticoid receptor and the mineralocorticoid receptor as transcription factors . . . Cross talk between the glucocorticoid receptor and pro-inflammatory transcription factors in the nucleus . . Transactivation and transrepression mechanisms on hypothalamus–pituitary–adrenal (HPA) axis regulation . Glucocorticoid receptor-based therapy in brain disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Current glucocorticoid receptor-based therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Glucocorticoid receptor modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Dissociated glucocorticoid receptor modulators in immune-related disorders . . . . . . . . . . . . . . . 7.2. Combination therapies in immune disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Role for selective glucocorticoid receptor modulators in brain disorders? . . . . . . . . . . . . . . . . . 8. Future perspectives for glucocorticoid receptor modulation strategies in brain disorders . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. LEGEST, Department of Molecular Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium. Tel.: +32 92645147; fax: +32 92645304. E-mail address: [email protected] (K. De Bosscher). 0014-2999/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.11.076

K. De Bosscher et al. / European Journal of Pharmacology 583 (2008) 290–302

1. Glucocorticoids and mineralocorticoids In higher organisms, the adrenal cortex synthesizes two classes of steroidal hormones that fulfill an important role in the maintenance of basal and stress-related homeostasis, namely glucocorticoids and mineralocorticoids. Besides their clinically well appreciated anti-inflammatory and immunosuppressive actions, glucocorticoids (i.e. corticosterone in rodents and cortisol in humans) are also involved in the regulation of sugar, fat and protein metabolism, in body growth, and reproductive processes (Chrousos and Kino, 2007; Tronche et al., 2004). From different pathologies, such as Addison's disease and Cushing's syndrome, characterized by hypo- and hyperphysiological concentrations of glucocorticoids respectively, it has been recognized that glucocorticoids also have an impact on mood and cognitive functions (Chrousos and Gold, 1998; de Kloet et al., 2007; Gold et al., 1995). Due to its pervasiveness in the human body and its wide range of functions, it is perfectly understandable that a chronic imbalance in the levels of glucocorticoids, inevitably leads to an equally broad range of pathophysiological effects. Indeed, increased systemic levels of glucocorticoids are notorious for a load of unwanted side effects, including diabetes, hypertension, cataract, osteoporosis, growth retardation, skin thinning, muscle weakening, etc. (Schäcke et al., 2002). Furthermore, the interference with processes that regulate fat distribution and water household equilibria contributes to the development of a hunch-back and the so-called moon face. These features are, in comparison to diabetes and osteoporosis, perhaps not the clinically most important or costly side effects, but for the patients themselves they are very distressing and undoubtedly have an impact on the morale of the patient. Endogenous glucocorticoids are released in the blood upon stressful situations (ranging from emotional stress to infectious insults). Their role is to facilitate a plenitude of processes all aimed at coping with, recovery from, and adaptation to the stressor. Secretion is regulated through the activity of an upstream neuroendocrine cascade. Stressful sensory and/or visceral stimuli are integrated at the level of the hypothalamus, from which corticotropin-releasing hormone (CRH) and arginine vasopressine (AVP) are secreted, followed by the synthesis and release of adrenocorticotrophic hormones (ACTH) from the pituitary gland and ending with glucocorticoid synthesis by the adrenal glands (Reichardt and Schütz, 1998; Turner-Cobb, 2005). After the coordinated regulation of immune, endocrine and neurological responses, glucocorticoids inhibit the synthesis and secretion of CRH and ACTH, effectively inhibiting their own synthesis and thereby adequately restoring homeostasis. An additional level of control is imposed by cortisol-binding globulin, a protein that checks ligand availability of glucocorticoids in the blood and that has a high affinity but a limited capacity for glucocorticoid binding (Hammond et al., 1990), as well as by enzymes that interconvert glucocorticoids and their inactive metabolites (Holmes and Seckl, 2006), and by membrane pumps that may limit access to tissues (Karssen et al., 2001). In the brain, glucocorticoids influence the activity of neurons. Excitability of neurons, and therefore the responsiveness of nerve cells to incoming stimuli are regulated by glucocorticoids, but so

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are neuronal morphology and survival (Joëls, 1997; McEwen and Sapolsky, 1995). Glucocorticoids have clear effects on memory functions, but also influence other functions of the central nervous system, including normalization of the cardiovascular tone, arousal and sleep (Chrousos and Kino, 2007). Chronically elevated levels of glucocorticoids have been linked to hippocampal degeneration in man, possibly resulting in memory impairments (Lupien et al., 1998). They have also been associated with higher risk for psychosis, as well as stress-related affective disorders such as depression (de Kloet et al., 2007). It is interesting to note that both glucocorticoid excess and glucocorticoid deficiency can boil down to cognitive disorders. In our contemporary western society, the glucocorticoid-mediated adaptive responses that serve to anticipate on adverse events and danger and which should at due time be translated into a necessary state of fear and the ability to withdraw from danger, have for some individuals turned into maladapted pathological sequelae of anxiety, insomnia and depression (Chrousos and Kino, 2007). Compared to glucocorticoids, mineralocorticoids are far less multi-tasking. They are mainly involved in controlling the water household system, which is indisputably important as well. A large body of evidence further suggests that the mineralocorticoid aldosterone signals in vascular cells, and in this way can contribute to vascular remodeling and target organ damage, independent of its salt balancing and blood pressure regulatory effects (Duprez, 2007; Fiebeler et al., 2007). Remarkably, a potential therapeutic role for mineralocorticoid receptor antagonists, as vascular protectants across a spectrum of cardiovascular disease, has been suggested, even under situations where circulating levels of aldosterone are low (Funder, 2007). 2. The glucocorticoid receptor and the mineralocorticoid receptor Both steroids exert their function by binding to their respective intracellular receptors, the glucocorticoid receptor and the mineralocorticoid receptor, which are very closely related. The glucocorticoid receptor displays a widespread tissue distribution whilst the mineralocorticoid receptor is more restricted to kidney (collecting ducts), heart, intestine, limbic neurons (Funder, 1992) and circumventricular tissues in brain (reviewed in GómezSánchez, 1997). These expression patterns reflect their respective physiological roles. Intriguingly, whereas mineralocorticoids (typically aldosterone) only bind to the mineralocorticoid receptor, glucocorticoids can bind to both receptors and even with a higher affinity to the mineralocorticoid receptor than to the glucocorticoid receptor (de Kloet, 2004). In certain brain regions and in kidney epithelial cells, that show co-expression of both receptors, the enzyme 11-β-HSD type II inactivates glucocorticoids, allowing an exclusive activation of the mineralocorticoid receptor by mineralocorticoids and helping to generate the necessary specificity (Funder et al., 1988; Robson et al., 1998). In other tissues cortisol is the main ligand for the mineralocorticoid receptor, due to its approximately 1000× excess over aldosterone in the plasma. When coexpressed, as is the case in neuronal layers of the hippocampus, the glucocorticoid receptor

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and the mineralocorticoid receptor may form heterodimers that can bind DNA, adding another level of complexity for fine-tuning learning processes, memory and mood (Trapp and Holsboer, 1996). Notably, the expression of the glucocorticoid receptor itself is also controlled by glucocorticoids, via a process that is called homologous downregulation, further reflecting a tight control in the glucocorticoid receptor-dependent stress system (Lowy, 1991). To illustrate, it has been shown in rats that an increase in basal and stress-coupled corticosterone secretion due to prenatal immune challenges was correlated with a diminished expression of hippocampal glucocorticoid receptors (Reul et al., 1994). In the hippocampus, both the mineralocorticoid receptor and the glucocorticoid receptor play a role in the control of neurotransmission and plasticity, but they mediate different, and sometimes opposite effects upon glucocorticoids. In general, mineralocorticoid receptor-mediated effects are associated with an increased excitability of the neuronal network, whereas acute glucocorticoid receptor activation sensitizes the neurons for stimuli that decrease neuronal activity (de Kloet et al., 1999; Joëls and Van Riel, 2004). Chronic exposure to glucocorticoids may have rather dramatic effects on hippocampal function. For example, exposure to chronic stress leads to impaired plasticity in CA1 pyramidal neurons, which is quickly restored upon blocking the glucocorticoid receptor (Krugers et al., 2006). Many of these effects are mediated by genomic mechanisms (Meijer, 2006), but some effects, e.g. hippocampal glutamate transmission, are mediated through non-genomic pathways (Karst et al., 2005). Both the mineralocorticoid receptor and the glucocorticoid receptor can act as transcription factors (see further below). In most systems tested, with the notable exception of models of renal aldosterone-mediated action, the glucocorticoid receptor is much stronger both as an inducer and a repressor of transcriptional activity. Upon investigating acute explant hippocampal slices, certain genes involved in hippocampal neurotransmission and plasticity were found to be downregulated 1 h after glucocorticoid receptor activation while virtually none were upregulated (Morsink et al., 2007). 3. Functionality of the glucocorticoid receptor and the mineralocorticoid receptor as transcription factors The above described observations bring us to the role of the glucocorticoid receptor as a transcription factor, able to regulate various gene expression programs. Fig. 1 summarizes the multiple levels at which the functionality of the glucocorticoid receptor is controlled. Just as most other nuclear receptor family members, including the mineralocorticoid receptor, the glucocorticoid receptor is build-up in a tripartite modular fashion, typically composed of an N-terminal transactivation domain, a central DNA-binding domain and a C-terminal ligand-binding domain. The DNA-binding domain is closely intertwined with a glucocorticoid receptor dimerization interface, localized around the Zinc finger regions of this domain. Ligand binding converts the inactive, predominantly cytosolic receptor into an active transcription factor residing mainly in the nucleus. Without taking exceptions into account, two main groups of glucocorticoid receptor-regulated target genes can be distin-

Fig. 1. This picture represents the multiple levels at which the functionality of the glucocorticoid receptor (denoted as GR) is modulated. Signalling through the glucocorticoid receptor is controlled at the cellular level by heat shock proteins and immunophilins, ensuring optimal ligand loading, by the type of ligand (agonists, antagonists or selective modulators) and by the availability of ligand (through the enzymatic activity of 11-β-hydroxysteroid dehydrogenases (light green triad)). At the systemic level signalling through the glucocorticoid receptor is regulated through tissue access-controlling factors (such as cortisolbinding globulin, membrane pumps, such as pgp, or through the activity of antagonizing hormones, such as e.g. insulin and glucagon). More simple, the glucocorticoid receptor's activity is also determined by its tissue distribution, availability of the receptor (feedback control by homologous downregulation) and by diverse modifications (phosphorylation, acetylation, nitrosylation, methylation, ubiquitinylation, sumoylation). Nucleocytoplasmic shuttling also regulates glucocorticoid receptor-mediated responses at the cellular level (dark green triad). The activity of the glucocorticoid receptor is influenced by the occurrence of different glucocorticoid receptor variants, including splice variants (GRβ molecules), isoforms and polymorphisms (blue triad). The transcriptional outcome by the glucocorticoid receptor as a transcription factor is influenced through interactions with corepressors (e.g. NcoR, SMRT, HDACs), coactivators (e.g. SRC, GRIP, CBP) and chromatin-modulating factors (e.g. Brg1, Brm) (red triad). The activity of the glucocorticoid receptor is also influenced by interactions with other transcription factors (cross talk, e.g. with the mineralocorticoid receptor), by regulation at the level of RNA (RNA cofactors) and by interactions with the factors that affect the basal transcription machinery (orange triad).

guished. One set of genes contains in their promoter DNA a glucocorticoid-responsive element (GRE), typically a palindromic sequence onto which activated glucocorticoid receptor can directly bind as a homodimer and drive gene expression (e.g. glucose-6-phosphatase, tyrosine aminotransferase, glutamine synthetase, mouse mammary tumour virus, GlucocorticoidInduced Leucine Zipper (GILZ), human placental alkaline phosphatase) (Adom et al., 1991; Beato, 1991; Cannarile et al., 2001; De Bosscher et al., 2005; Hasselgren, 1999; van de Werve et al., 2000). On synthetic promoters, the mineralocorticoid receptor will recognize the same response elements, but tends to be a weaker transcription factor, which may act as a partial agonist at the genomic level (Meijer, 2006). The mineralocorticoid receptor and the glucocorticoid receptor also share in vivo target genes such as Serum and glucocorticoid-inducible kinase 1 (Sgk1) and Glucocorticoid-Induced Leucine Zipper (Bhalla et al., 2006). However, in brain there are distinct sets of genes regulated by low and high levels of corticosterone, presumably reflecting largely distinct targets of the mineralocorticoid receptor and the glucocorticoid receptor (Datson et al., 2001). The other group of

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genes is regulated by the glucocorticoid receptor, believed to be acting as a monomer, without the need for a direct DNA contact (Reichardt et al., 1998). Hereby, the glucocorticoid receptor influences the activation steps and/or activity of other transcription factors, including NF-κB, AP-1, CREB, GATA-1, GATA-3, t-Bet, Oct1, C/EBPβ, STAT5 (Caldenhoven et al., 1995; Chang et al., 1993; De Bosscher et al., 2006; Imai et al., 1993; Jonat et al., 1990; Liberman et al., 2007a,b; Lidén et al., 2000; Rogatsky and Ivashkiv, 2006; Schüle et al., 1990; Stöcklin et al., 1997; YangYen et al., 1990). The transcriptional outcome is then mainly of an inhibitory nature (especially for inflammatory gene expression), but under certain conditions (depending on the transcription factor composition or cellular context), it can also further enhance gene expression. The mineralocorticoid receptor is much weaker at transrepression (Pearce and Yamamoto, 1993), but may be able to exert transrepressive effects in some settings (Meijer et al., 2000b). Although direct DNA binding is dispensable for this type of regulation, mutational studies have not only pointed to the importance of the ligand-binding domain, but also to a need for the DNA-binding domain in protein–protein cross talk interactions (Evans, 1989; Heck et al., 1994; Lidén et al., 1997). 4. Cross talk between between the the glucocorticoid glucocorticoid receptor receptor and and proinflammatory transcription factors in the pro-inflammatory transcription factors in nucleus the nucleus Over the past years it has become increasingly clear that the signalling components of different signal transduction pathways, originating from different extracellular stimuli, interconnect and communicate with each other, a process known as ‘cross talk’. One well known example of this, with a widespread clinical application, is the cross talk between NF-κB and the glucocorticoid receptor. NF-κB is a crucial engine that drives many proinflammatory cytokines, enzymes and adhesion molecules. For this reason, it constitutes an important pharmacological target in the fight against chronic inflammatory disorders and cancer (Gilmore and Herscovitch, 2006). Unravelling the mechanism(s) whereby glucocorticoids inhibit NF-κB, as well as AP-1, another pro-inflammatory transcription factor, is still actively ongoing and has been extensively reviewed during the past years (Adcock et al., 2006; De Bosscher et al., 2003; Ito et al., 2006a; Necela and Cidlowski, 2004). The cross talk phenomenon not only increases the number of possible cellular responses but also adds on a level of complexity by making the predictions of a certain transcriptional outcome (gene activation or gene repression) for a given gene family rather difficult. Indeed, it has already been shown that the direction of gene regulation (stimulatory or inhibitory or status quo) is highly context-dependent (signal-dependent, gene-dependent and cell type-dependent) (De Bosscher et al., 2000; Glass and Ogawa, 2006; Ogawa et al., 2005; Rogatsky et al., 2002, 2001). Coactivators, in their most strict definition [of which CREBbinding protein (CBP), p300, Glucocorticoid Receptor Interacting Protein 1 (GRIP1/TIF2) and Steroid Receptor Coactivator-1 (SRC-1) were the first ones characterized], play a role in transcription either through their intrinsic histone acetyltransferase (HAT) activity or through the recruitment of other HAT-containing

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enzymes, leading to a relaxed chromatin environment. Corepressors, such as Nuclear corepressor (NcoR), Silencing Mediator of Retinoic acid and Thyroid hormone receptor (SMRT) and histone deacetylases (HDACs) mediate gene repression via deacetylating histone tails, thereby compacting the chromatin. Also methyltransferases and demethylases have been implicated in gene regulatory processes. Intriguingly, recent observations have revealed a high degree of promiscuity in cofactor functioning. For example, even within the same cell line, the GRIP1 cofactor can change its role from being a coactivator to a corepressor in glucocorticoid receptor-dependent gene regulation of AP-1-driven gene expression (Rogatsky et al., 2001). Similarly, rather than acting as a corepressor, HDAC1 can act as a coactivator for the glucocorticoid receptor at the MMTV promoter (Qiu et al., 2006). Not only histones, but also transcription factors themselves are subject to modifications that modulate their transcriptional activities. In this respect, it was recently suggested that deacetylation of the glucocorticoid receptor by HDAC2 seems a prerequisite for association with activated p65 and subsequent inflammatory gene repression (Ito et al., 2006b). Increasing evidence further suggests that the constellation of unique glucocorticoid receptor isoforms within certain cell types could co-determine cell-specific responses to glucocorticoids (Lu and Cidlowski, 2006). Finally, in one single cell type, different surfaces of the receptor regulate glucocorticoid receptor-target genes, in a gene-specific manner (Rogatsky et al., 2003). Taken together, it is therefore of no surprise that also cell-specific glucocorticoid receptor-dependent cross talk effects have been observed, as illustrated by the following examples in the brain. Glucocorticoids mediate downregulation of the serotonin 5-HT1A receptor gene in granule cells of the hippocampal dentate gyrus but not in the adjacent pyramidal neurons of the CA1 area (Meijer and de Kloet, 1995), although the latter do abundantly express glucocorticoid receptor and 5-HT1A receptors (Meijer and de Kloet, 1998; Meijer et al., 2003). The mineralocorticoid receptor can transrepress 5-HT1 receptor expression, and similarly as for the glucocorticoid receptor, very much in a cell context-dependent manner (Meijer et al., 2000b). Similarly, the expression of IκBα is lower in the neuronal cortex upon glucocorticoid treatment versus the untreated set-up, whilst in the periphery of the same animal the expression of IκBα is enhanced by glucocorticoids (Unlap and Jope, 1997). Activated glucocorticoid receptor not only interferes with the activation and activity of NF-κB, it can also modulate the activation and activities of AP-1 and CREB transcription factors. Regulatory elements for either one or both of these transcription factors are not only present in cytokine promoters, but also in promoters of genes coding for dopamine receptors, e.g. AP-1 in the dopamine receptor D5 promoter (Beischlag et al., 1995), or for enzymes involved in catecholamine biosynthesis, e.g. AP-1 and CRE in tyrosine hydroxylase (Sabban and Kvetnansky, 2001). Depending on the content of the dimeric AP-1 (c-Jun/c-Jun as opposed to c-Jun/c-Fos), the cell type and the cofactor context, it has been observed that activated glucocorticoid receptor can either activate or repress AP-1-driven gene expression (Diamond et al., 1990). Therefore, in order to know the outcome of AP-1activating stressors combined with glucocorticoid signalling on

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the abovementioned factors in certain areas of the brain, it will be crucial to study the gene and protein expression profile in these specialized tissues, in a time-dependent manner. Consistent herewith, the stress-induced gene expression patterns of catecholamine biosynthetic enzymes have been found to be highly cell type-dependent and differing between the sympathetic ganglia as compared to the adrenal medulla (Sabban et al., 2004a,b). Clearly, additional cellular components can contribute to a specific and very different transcriptional outcome. The fact that there is a highly variable stoichiometry of specific coregulators (e.g. SRC1 splice variants, SRC2, SRC3) in different brain regions already provides one of many possible explanations for the observed differences in steroid responses (Meijer et al., 2005, 2000a). These coregulators are themselves subject to hormonal regulation and interestingly, their expression is more frequently regulated at the level of degradation than at the transcriptional level (Bousios et al., 2001; Lonard and O'Malley, 2006; Misiti et al., 1998). The above described insights imply that, in order to design effective therapeutic strategies, we must find the key how to reliably predict the transcriptional outcome with a given set of parameters to our availability (DNA-regulatory elements, transcription factor composition and modification, promoter organization, indirect protein–protein interactions, cofactor constitution and modifications, chromatin build-up). We must also look beyond the single gene level; we need to be able to have a bird's eye view of the overall transcriptional outcome, bearing in mind the benefit for the organism as a whole. Recent studies have already attempted to gain this insight (Glass and Ogawa, 2006; Pascual and Glass, 2006), and have tackled the problem accordingly, with the goal of obtaining an integrated view of gene regulatory processes via multiple signalling pathways. 5. Transactivation and transrepression mechanisms on hypothalamus–pituitary–adrenal (HPA) axis regulation As discussed above, not only the synthesis of glucocorticoids and their supply from the bloodstream to the periphery are tightly regulated and subject to feedback control, changes in glucocorticoid receptor levels themselves also impact on physiological and pathophysiological conditions. The importance of glucocorticoid receptor activity for the stress system is clear, from both pharmacological studies as well as genetic models. Findings with the transgenic glucocorticoid receptor over- and underexpressing mice, as well as brain-specific glucocorticoid receptor knockout animals (Kellendonk et al., 2002; Reichardt et al., 2000, 2001; Tronche et al., 1999, 1998; Urani and Gass, 2003), highlight the existence of a direct connection between HPA axis regulation, stress responses and central glucocorticoid receptor expression levels. Natural fluctuations of glucocorticoid receptor levels in control regions of the brain, which can arise from prenatal and postnatal experiences, may influence HPA axis regulation and may thus contribute to the way adults can deal with stressful situations (Liu et al., 1997; Reul et al., 1994). Both transactivation and transrepression mechanisms play a role in these effects. Mouse models that bear a point mutation in the glucocorticoid receptor at the dimerization interface (A458T in

mice, in the D-loop of the second Zinc finger in the DNA-binding domain) have been developed and also do survive (Reichardt et al., 1998). Their great advantage is that they allow discriminating between different activities of the receptor. In cell culture overexpression studies, glucocorticoid receptor A458T failed to bind onto glucocorticoid-responsive elements and to transactivate glucocorticoid-responsive element-dependent reporters, whilst its transrepressive capacity on AP-1-dependent gene expression remained unaffected (Heck et al., 1994). Consequently, by studying mice of which the glucocorticoid receptor has a point mutation in the dimerization domain, the group of Schütz aimed to distinguish between processes that are either dependent of glucocorticoid receptor dimerization (direct DNA binding of glucocorticoid receptor as a homodimer onto promoters of target genes, typically those required for energy homeostasis e.g. the gluconeogenic TAT-enzyme in the liver), or else independent of glucocorticoid receptor dimerization in vivo (typically transcription factor cross talk in the frame of antiinflammatory gene expression mechanisms e.g. repression of AP1 activity in gelatinase B or collagenase-3) (Reichardt et al., 2001; Tronche et al., 1998). Remarkably enough and important for drug development strategies, a part of the HPA axis control mechanism seems dependent on glucocorticoid receptor dimerization, whilst a part seems independent on glucocorticoid receptor dimerization. Indeed, pro-opiomelanocortin (POMC) gene expression (the gene coding for ACTH), as well as prolactin gene expression in the anterior pituitary were upregulated in glucocorticoid receptor dim mice (derepression), whereas CRH gene expression remained unchanged. So, only the repression of POMC (and subsequently ACTH) in the anterior pituitary seems dependent on the glucocorticoid receptor dimerization interface. Repression of CRH in the median eminence by monomeric glucocorticoid receptor may be effected through protein–protein interaction with CREB, AP-1 and Nurr77, three transcription factors that have been identified in the promoter control region of CRH and that can serve as glucocorticoid receptor targets (Malkoski and Dorin, 1999; Reichardt et al., 1998), although a negative glucocorticoidresponsive element that is substantially different from the canonical palindrome has also been proposed to play a role (Malkoski and Dorin, 1999). As a comparison, both CRH and POMC were found to be upregulated in the glucocorticoid receptor null mice. Notably, the upregulation of ACTH in the anterior pituitary of glucocorticoid receptor dim mice was not translated into an increase in ACTH serum levels, demonstrating an additional control level by monomeric glucocorticoid receptor, namely ACTH secretion (Reichardt and Schütz, 1998). Thus, even in the same physiological system, glucocorticoid receptor can clearly operate via different mechanisms, depending on the gene and even for the same gene, depending on the target tissue. Of note, not all unaffected aspects of glucocorticoid receptor function in the dim mouse necessarily depend on transrepression mechanisms. In the adrenal medulla, glucocorticoids control catecholamine biosynthesis through a glucocorticoid receptor-induced transcriptional upregulation of the PNMT gene (phenylethanolamine-N-methyl-transferase), which codes for an enzyme that converts noradrenaline to adrenaline. Interestingly, in adrenal cells derived from glucocorticoid receptor null mice, the

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transcription of PNMT is abolished, whereas in adrenals isolated from glucocorticoid receptor dim mice, mRNA expression was unaffected (Reichardt et al., 1998). Following a detailed analysis of the PNMT promoter region using glucocorticoid receptor wild type and glucocorticoid receptor dimer mutants, it was concluded that on certain promoters containing multiple glucocorticoid responsive elements, such as of the PNMT gene, glucocorticoid receptor molecules are able to form concerted multimers, independently of the DNA-binding domain dimer interface (Adams et al., 2003). 6. Glucocorticoid receptor-based therapy in brain disorders 6.1. Rationale An abnormal glucocorticoid secretion and/or changed gene expression levels of glucocorticoid receptor in brain regions have been recognized in dopamine-coupled brain disorders, such as schizophrenia and depression, which include cognitive deficits such as working-memory disturbances among their characteristics (Mizoguchi et al., 2004; Young, 2006). Increased cortisol levels have also been connected to other psychiatric disorders including autism, Alzheimer's disease and Parkinson's disease (DeBattista et al., 2006; Peeters et al., 2004), making a strong case for an association between hypercortisolism and neurotoxic effects (Young, 2006). The observation that a prolonged exposure to increased amounts of corticosterone dampens 5-HT responses in the hippocampus, further supports the notion that hypercortisolism constitutes a real risk factor for the development of major depression in genetically predisposed individuals (Joëls and Van Riel, 2004). Although the molecular evidence steadily increases, a definitive role for the HPA axis and/or a direct involvement of glucocorticoid receptor signalling in the pathogenesis of these diseases remains a debated matter (reviewed in Van Craenenbroeck et al., 2005). Anyhow, it is clear that a detailed study on the underlying molecular mechanisms of glucocorticoid receptor in the brain is of paramount importance to stimulate the development of novel therapeutics acting in a more specific manner.

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receptor are minor and there is no known affinity for monoamine, histamine or cholinergic receptors (DeBattista et al., 2006). The observation that transgenic mice expressing glucocorticoid receptor specifically in the forebrain are more likely to develop a depressive behaviour (Wei et al., 2004), anyhow confirms the hypothesis that a close connection exists between affective diseases and HPA axis hyperactivity (Belanoff et al., 2001; Chu et al., 2001; Mayer et al., 2006). Rapid effects of RU486 on neurogenesis are furthermore suggested to be an important contributing factor for their mechanism of action (Mayer et al., 2006). In patients suffering from a bipolar disorder, RU486 administered at 600 mg once a day for 7 days improved both the neurocognitive functioning and mood (Young et al., 2004). The same treatment regimen in patients with psychotic major depression was found to be consistently and robustly effective in reducing delusions, hallucinations and paranoia but did not affect the depressed state (DeBattista et al., 2006; Flores et al., 2006). Safety and efficacy profiles for the usage of RU486 in psychotic major depression patients are now being evaluated in a Phase III clinical trial, and will show to what extent the published beneficial effects of glucocorticoid receptor blockade (van der Lely et al., 1991) hold for larger groups of patients. The adverse effects upon RU486 treatment range from rash, fatigue and loss of appetite to agitation and constipation (Flores et al., 2006). It seems paradoxical but also glucocorticoid receptor agonists, such as dexamethasone, have been investigated for their benefit as a short treatment in patients with psychotic major depression (Arana et al., 1995). The rationale for this is that the low-dosed agonists cannot pass the blood-brain barrier, making it functionally equivalent to centrally working glucocorticoid receptor antagonists (Karssen et al., 2005). Anyhow, only a short-term usage would be advisable, so to help avoid many of the classical side effects. 7. Glucocorticoid receptor modulators 7.1. Dissociated glucocorticoid receptor modulators in immunerelated disorders

6.2. Current glucocorticoid receptor-based therapies A therapeutic benefit of cortisol synthesis inhibitors, such as ketoconazole and metyrapone, has been reported for some depressed patients. However, a variety of potential serious side effects hampers their usage and the concern that their clinical effects can remain up to eight months after stopping the treatment makes it far from an ideal therapy (reviewed in DeBattista et al., 2006). Despite some controversies, more and more evidence supports a central role of hypercortisolaemia in the pathogenesis of psychotic major depression. Consequently, exploring the applicability of mifepristone (or RU486), a potent antagonist of glucocorticoid receptor, as an antidepressant was a logic step. RU486 is not only an antagonist of glucocorticoid receptor, at low concentrations it also antagonizes progesterone receptors and is therefore mostly known and applied as an anti-gestagenic agent (Ulmann et al., 1990). Effects of RU486 on the mineralocorticoid

Since the 1940s glucocorticoids have been used in the clinic, on an empirical basis, without any knowledge on their molecular target(s). Although they belong to the most proficient anti-inflammatory class of drugs, even today, serious side effects restrict their applicability, especially upon prolonged usage. In fact, if glucocorticoids were just being discovered today, it is quite unlikely they would know such a widespread usage in the clinic as they do now, as their side effect profile is of an amazingly broad spectre. Alas, for quite a number of patients suffering from chronic ailments including nasal polyposis, rheumatoid arthritis, inflammatory bowel syndrome etc., there are little worthy alternatives. As soon as it was realized that a differential molecular basis exists for the transactivation versus the transrepression potential of glucocorticoid receptor, more or less mirroring the side effect profile versus the anti-inflammatory action mechanism, a quest has been ongoing to design the ideal glucocorticoid receptor

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agonist, i.e. one that retains the beneficial anti-inflammatory activities without the unwanted side effects (Vanden Berghe et al., 1999; Vayssière et al., 1997). The term ‘dissociated’ ligand refers to a ligand that can discriminate between gene transactivation and gene transrepression mechanisms. Fig. 2 depicts this working hypothesis, but also puts into perspective that the mechanisms underlying beneficial effects and side effects are not to be considered as pure black and white phenomena. The transgenic mice expressing dimerization-deficient glucocorticoid receptor, in which the glucocorticoid receptor-mediated transactivation of genes with simple glucocorticoid responsive elements is hampered, but where the tethering function of the glucocorticoid receptor is intact, are still able to tackle inflammatory insults (Reichardt et al., 2001). This observation confirmed the hypothesis that protein–protein interactions between monomeric glucocorticoid receptor and pro-inflammatory transcription factors such as NF-κB, AP-1, IRF-3, NF-AT and CREB, without the need for direct DNA binding by the glucocorticoid receptor, is probably the main contributing mechanism explaining its immune-modulating capacities. However, as one could have expected from the complexity of nature itself, the work model based on the assumption of discrete positive and negative regulatory actions of the glucocorticoid receptor in generating adverse effects versus beneficial effects, respectively, is not exactly watertight (Coghlan et al., 2003). As already explained above, the PNMT gene constitutes one example of a gene promoter onto which glucocorticoid receptor molecules that are defective in their dimerization domain

Fig. 2. This picture displays the different mechanisms that are held accountable for the beneficial versus the detrimental effects upon long-term glucocorticoid usage. The left side of the figure represents the adverse effects, which can be established not only by transactivation (red circle), which is well recognized, but also by transrepression mechanisms (orange circle). Some side effects manifest themselves through a balance of both transrepression and transactivation mechanisms (purple square). The right side of the figure represents the beneficial effects of glucocorticoids, which include cytokine gene repression through transrepression (also well characterized) (green circle), the inhibition of immune cell migration as well as anti-proliferative effects. A broadening class of genes that contributes to the beneficial effects of glucocorticoid-induced glucocorticoid receptor activation is transcribed through transactivation mechanisms (e.g. IκB-α, glucocorticoid-Induced Leucine Zipper, Mitogenactivated protein kinase phosphatise 1, lipocortin 1 and secretory leukocyte peptidase inhibitor) (yellow circle).

(and also display defects of DNA binding on some glucocorticoid responsive element-containing genes) can still bind and transactivate (Adams et al., 2003; Reichardt et al., 1998). Similarly, parts of the glucocorticoid-dependent feedback regulation in the HPA axis depend on glucocorticoid receptor monomers whilst other parts depend on an intact glucocorticoid receptor dimer interface. Moreover, there are genes dependent on the transactivation by the glucocorticoid receptor that have been described to also exert antiinflammatory actions, e.g. lipocortin, Secretory Leukocyte Peptidase Inhibitor (SLPI), GILZ, MAP Kinase Phosphatase-1 (MKP-1), IκB-α, although they seem to represent a minority when compared to the whole family of glucocorticoid-repressed cytokines, chemokines and inflammatory enzymes (Adcock et al., 2006; Barnes, 2005, 2006; De Bosscher et al., 2003; Rhen and Cidlowski, 2005). However, dexamethasone-induced thymus involution is hampered in glucocorticoid receptor dim mice (Reichardt et al., 1998). To reconcile the conflicting pieces of evidence, an alternative model in which glucocorticoids exert anti-inflammatory effects at both transcriptional and posttranscriptional levels, both by activating and inhibiting expression of target genes, has been proposed (Clark, 2007). Consequently, looking for a glucocorticoid receptor agonist with at least a well improved therapeutic index may most probably be a more realistic and rewarding goal for both the pharmaceutical industry and the patient. A number of steroidal and non-steroidal glucocorticoid receptor modulators with these properties have recently been characterized; however, the molecular basis for the observed in vivo selectivity is not always understood. In the past, candidates with a good dissociated profile in vitro have been quite disappointing upon investigating their in vivo-profile (Belvisi et al., 2001a,b). It seems that in vitro-screening procedures, preferentially used by the pharmaceutical industry to select drug candidates because of the high-throughput applicability, do not necessarily reflect or efficiently predict characteristics that occur in vivo. This approach may therefore not always yield the best candidates or may even miss interesting candidates e.g. because of too stringent criteria for the effective dose range. A classical example is the comparison of non-steroidal compounds, usually acting at the micromolar range, like the natural NF-κB inhibitors genestein, humulone, or the non-steroidal glucocorticoid receptor modulator CpdA (De Bosscher et al., 2005; Dijsselbloem et al., 2007; Lee et al., 2007) with steroidal compounds, acting in the nanomolar range. The lower the effective dose the better, therefore it is quite a challenge to modify promising new drug targets in such a way that the initial characteristics are kept but their efficacy is improved. A recent paper by the group of Yamamoto demonstrates that a family of structurally similar, non-steroidal glucocorticoid receptor modulators displays surprisingly differing properties with regard to their action spectrum and functionality (Wang et al., 2006). Recently developed, non-steroidal compounds that do not completely dissociate transactivation from transrepression, but nevertheless show a markedly improved therapeutic potential over prednisolone, include AL-438 and ZK 216348 (Coghlan et al., 2003; Schäcke et al., 2004). One non-steroidal plant-derived compound, CpdA, proved to be a glucocorticoid receptor modulator

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with a more sharply dissociated profile not only in vitro but also in vivo. The effectiveness of CpdA was demonstrated further in the collagen-induced rheumatoid arthritis mouse model and confirmed by gene expression analysis in human synovial fibroblasts (De Bosscher et al., 2005; Dewint et al., 2008; Gossye et al., in preparation). It must be said that, no matter how promising certain target molecules may seem in a laboratory setting, only a very little fraction makes it to the pharmacist's shelves. One of the classical, and understandable, reasons for this is that structurally different and therefore atypical ‘druggable’ candidates are generally considered as a too highrisk business for the pharmaceutical industry. 7.2. Combination therapies in immune disorders The observation that the inhibition of NF-κB, either pharmacologically or genetically, can reduce various chronic inflammatory diseases, makes it an interesting potential therapeutic target. However, upon tackling NF-κB, one must proceed with caution, as this transcription factor is also an important regulator of the immune system and its uncontrolled complete blockage will definitely introduce a plethora of side effects to the organism. Glucocorticoids are not the only drugs that are able to modulate the activity of NF-κB. As a consequence, a strategy gaining more and more interest is to investigate the therapeutic potential and effects at the gene and protein expression level of a well balanced combination therapy of glucocorticoids with other NF-κB inhibitors. Recent data from our group show promising perspectives upon combining glucocorticoids with fibrates (agonists for the PPARα receptor, which upon activation can also block NF-κB in the context of vascular inflammation) (Bougarne et al., in preparation), or upon combining glucocorticoids with inhibitors of specific kinases that modulate the activity of NF-κB (Beck et al., in preparation) or upon combining glucocorticoids with a range of natural compounds that inhibit NF-κB (Van Cleemput et al., in preparation) in the context of anti-inflammatory strategies. Overall, the strong point in using combinations of NF-κB inhibitors resides in the fact that (although NF-κB is indeed the main downstream target for both some of these compounds and glucocorticoids clearly target more than one level) the way through which they exert this inhibition may vary. Consequently, the net beneficial effect may be stronger, if needed, or complementary or may display a better side effect profile. Combination strategies and intermittent strategies are in fact already used in the clinic, for example in rheumatoid arthritis, following the credo: ‘as much as necessary but as little as possible’ (Buttgereit et al., 2005). The aim is not only to impact on the specific glucocorticoid-induced side effect profile, but often also to lower the occurrence of therapy resistance (Chikanza, 2002). A variant of the combination strategy is the development of so-called nitrosteroids, e.g. NO-prednisolone. The additionally released NO not only synergizes with the glucocorticoid moiety in antiinflammatory potency, but seems to induce less osteoporosis in animal models than conventional prednisolone (Paul-Clark et al., 2002, 2003). To avoid all undesirable side effects of a certain therapy may be utopia, but the goal is to keep working towards the development of

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target gene-selective and at the same time tissue-selective drugs. This represents a real challenge for the years to come. One step in this direction is already being made by the development of dissociated compounds. Another step constitutes the promising effects of liposomal glucocorticoids, in a model of rheumatoid arthritis and in a model of multiple sclerosis (Metselaar et al., 2004; Schmidt et al., 2003). These liposomes accumulate specifically at sites of inflammation and because of the encapsulation, off-target side effects may less likely occur. However, as the polyethylene glycol (PEG) coating on the liposomes can cause hypersensitivity reactions in up to 10% of the patients, efforts now focus on the development of new long-circulating glucocorticoid liposomes without the PEG. If this would prove a successful strategy and even more so when applied with the socalled SEGRA's (Selective Glucocorticoid Receptor Agonists), the era of cortisone phobia may perhaps finally come to an end. 7.3. Role for selective glucocorticoid receptor modulators in brain disorders? The brain is another major target for glucocorticoids, both with respect to feedback regulation of the HPA axis, as well as in relation to stress-induced changes in metabolic, cognitive and affective functions. Therefore, there might be a role for selective glucocorticoid receptor modulators as regulators of disturbed HPA axis signaling, or in relation to other aspects of brain functioning. The latter option is exemplified by the successful use of the glucocorticoid receptor antagonist RU486 in at least some patients suffering from psychotic major depression (Belanoff et al., 2001; van der Lely et al., 1991). Recently, there has been a new wave of interest in finding more selective glucocorticoid antagonists, steroidal or non-steroidal, with improved selectivity profiles for the glucocorticoid receptor over other steroid receptors, comparing to RU486 (Akritopoulou-Zanze et al., 2004; Einstein et al., 2004; Jiang et al., 2007; Miner et al., 2005; Morgan et al., 2002; Peeters et al., 2004; Rosen and Miner, 2005). With the aim of potentially applying these selective glucocorticoid receptor antagonists in metabolic disorders, such as type II diabetes, the influence on gene expression has been investigated mainly in liver and muscle cell lines. So far, more advanced studies on the in vivo potency, efficacy and side effect profile of any of these novel selective antagonists in relationship to psychiatric affections have not yet been conducted. It will be interesting to find out what the impact is of these improved glucocorticoid receptor antagonists in affective disorders, as compared to current therapies. The genomic effects of glucocorticoid on brain tissue clearly involve both transrepressive as well as transactivating aspects (Karst et al., 2000; Morsink et al., 2006). However, it is very unclear which mechanisms are responsible for which effects, although we know that glucocorticoid receptor-mediated facilitation of spatial learning in mice depends on the glucocorticoid receptor dimer interface (Oitzl et al., 2001). Therefore, one definite use of dissociated ligands is the possibility to use these as a research tool to unravel the central mechanisms of glucocorticoid receptor action. As an example, the effects of the nonsteroidal selective glucocorticoid receptor modulator CpdA may

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be used to probe glucocorticoid receptor-NF-κB interactions in the brain. NF-κB is involved in important aspects of neuronal functioning that are distinct from its involvement in (neuro) inflammatory processes and this is particularly well established for hippocampal plasticity (Kaltschmidt et al., 2005; Meffert and Baltimore, 2005). As mentioned, the 5-HT1A receptor promoter is an example of a gene on which glucocorticoid receptor (as well as mineralocorticoid receptor) cross talk with NF-κB is relevant in that brain area. Given the role of NF-κB on hippocampal functioning, and the well established role of the hippocampus on the processing of stressors and the activation of the HPA axis, we assume that glucocorticoid receptor-NF-κB cross talk is probably relevant for regulations of the HPA axis. Dissociated glucocorticoid receptor modulators such as CpdA (De Bosscher et al., 2005) will form an appropriate tool to test such hypotheses. Promoters of both the CRH and the POMC gene contain putative negative glucocorticoid responsive elements, nGREs (of which not much is known in terms of structural demands for the glucocorticoid receptor) and several binding sites for cross talk partners (Drouin et al., 1998; Malkoski and Dorin, 1999). Louw et al. already showed that peripheral HPA axis parameters change upon long-term treatment of rats with CpdA (Louw and Swart, 1999). It will be particularly interesting to see how dissociated glucocorticoid receptor modulators act on the core of the HPA axis, and compare this to the genomic model of the glucocorticoid receptor dim mice. 8. Future perspectives for glucocorticoid receptor modulation strategies in brain disorders It is clear that some recently discovered aspects of glucocorticoid receptor-coupled mechanisms have to be borne in mind when it comes to the search for new targets or to novel drug design. Firstly, from a therapeutical perspective, an important barrier to overcome, when using glucocorticoid receptor modulators to treat brain affections, is the activity of Pgp (P-glycoprotein) at the blood-brain intersection. This protein, encoded by mdr genes (multidrug resistance genes) limits the access of many compounds to the brain, including synthetic and natural glucocorticoids (Karssen et al., 2002, 2001; Schinkel et al., 1995). Pgp inhibitors are therefore already used to increase glucocorticoid uptake in the brain with respect to depression (Pariante et al., 2003). In this respect, it is of note that we have observed that CpdA readily activates brain steroid receptors (Sarabdjitsinh, OCM, GH and KDB, unpublished observations). Secondly, target tissue responsiveness co-determines the precise impact of glucocorticoid hormones. A big issue in the clinic is the development of a so-called glucocorticoid resistance (Schaaf and Cidlowski, 2002). It is quite hard to dissect the underlying mechanistic basis for this observation, as more than one mechanism may account for this phenomenon, let alone finding the key how to prevent or treat this adverse event. Equally important to realize is the fact that glucocorticoid sensitivity in the HPA axis can be independently regulated from glucocorticoid sensitivity in peripheral tissues (Chrousos and Kino, 2007). Hyperactivity of the glucocorticoid receptor is not

only brought about by increased levels of glucocorticoids, it can also be mediated by an increase in receptor sensitivity. In this respect, the glucocorticoid receptor polymorphism BclI has recently been associated with a higher predisposition to develop a major depressive illness (van Rossum et al., 2006). The same was found for the ER22/23EK polymorphism, a bit surprising, as this glucocorticoid receptor polymorphism has rather been linked to glucocorticoid resistance. Glucocorticoid receptor polymorphisms exist that can distinguish between transactivation and transrepression mechanisms. In this respect the glucocorticoid receptor-9β has been described to preferentially affect glucocorticoid receptor-mediated transrepression (van den Akker et al., 2006). Overall, variants of the glucocorticoid receptor gene do seem to play a role in the pathophysiology of depression. Glucocorticoid receptor polymorphisms have in this respect convincingly been linked to adrenocortical responses followed by psychosocial stress (Wust et al., 2004). Importantly, glucocorticoid receptor polymorphisms may also contribute to an observed variability in treatment responses (Brouwer et al., 2006; van Rossum et al., 2006). Interestingly, not only polymorphisms in a particular gene itself but also in its promoter region may be an important determining factor explaining an increased susceptibility to stress-related disorders. A recently characterized polymorphism in the 5′ region of the NR3C1 gene (coding for the glucocorticoid receptor) is suggested to play a role in the genetic vulnerability for major depression (van West et al., 2006). Up to now, not only a series of different glucocorticoid receptor polymorphisms (reviewed in DeRijk et al. in this issue and DeRijk and de Kloet, 2005), but also an increasing number of receptor variants arising from alternative splicing, have been described (Yudt and Cidlowski, 2002). Thus an even higher variability in responses is to be expected, when also the recently characterized isoforms of the glucocorticoid receptor are taken into consideration. As their functionality can differ from the classical glucocorticoid receptor to quite some extent (Lu and Cidlowski, 2006), it is most conceivable that different glucocorticoid receptor modulators can impact differently on these receptor variants, making it hard to make predictions about the expected therapeutic outcome. Thirdly, newly discovered physiological regulators represent popular therapeutic targets. Hereto the cofactors or coregulators form no exception. More than 270 coregulators have been identified to date. In cancer, coregulators have been shown to serve as either oncogenes or tumour suppressors, depending upon signalling and cell context, and genes coding for coregulators are more and more appreciated in a role of ‘master genes’ (O'Malley, 2007). Examining cofactor recruitment together with gene expression profiling should further contribute to our knowledge on the basis of selectivity. This recent insight will undoubtedly bring about a novel wave in drug development research, where the focus will shift from selective ligand design for nuclear receptors to selective modulation of coregulator activities. To illustrate, a significant association has been made between single-nucleotide polymorphisms in FKBP5 (a glucocorticoid receptor-regulating cytoplasmic cochaperone of Hsp90) and both the response to antidepressants and the recurrence of depressive episodes (Binder et al., 2004).

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In conclusion, a continuation of detailed molecular studies on the underlying mechanisms of the glucocorticoid receptor in the brain by the use of in vivo models, ex vivo tissue and brain cell lines will be most helpful in order to put the pieces of this puzzle, one by one, together. Of note, it may need to be re-emphasized that corticosteroids act through two receptor types; both glucocorticoid and mineralocorticoid receptors. These can work in synergy, or antagonize each other's actions. The glucocorticoid receptor and its ligands have received much more attention, because of their important use as anti-inflammatory drugs, and in relation to brain because of the straightforwardness of targetting this lowest affinity stress-related hormone receptor. However, the two receptor types do interact, both within the cell, and through their mutual influence on the amount of circulating ligand. Affecting the functionality of one receptor may have a serious impact on signalling through the other receptor, and this impact should always be realized and adequately addressed in the quest for novel therapeutics. Excitingly, it seems that for the coming years there will at least be no shortage in glucocorticoid receptor-associated targets, of which the potential as therapeutic agents still needs to be explored. It will nevertheless be a challenge to find out which respective strategy will yield the most advantageous improved therapeutic index for the different brain and immune disorders. Acknowledgements KDB and KVC are postdoctoral fellows at the FWOVlaanderen. Financial support for OCM was provided by NWO (016.036.381) and for two members from UGent (KDB and GH) by the IAP/6 program. References Adams, M., Meijer, O.C., Wang, J., Bhargava, A., Pearce, D., 2003. Homodimerization of the glucocorticoid receptor is not essential for response element binding: activation of the phenylethanolamine N-methyltransferase gene by dimerization-defective mutants. Mol. Endocrinol. 17, 2583–2592. Adcock, I.M., Caramori, G., Ito, K., 2006. New insights into the molecular mechanisms of corticosteroids actions. Curr. Drug Targets 7, 649–660. Adom, J., Carr, K.D., Gouilleux, F., Marsaud, V., Richard-Foy, H., 1991. Chromatin structure of hormono-dependent promoters. J. Steroid Biochem. Mol. Biol. 40, 325–332. Akritopoulou-Zanze, I., Patel, J.R., Hartandi, K., Brenneman, J., Winn, M., Pratt, J.K., Grynfarb, M., Goos-Nisson, A., Von Geldern, T.W., Kym, P.R., 2004. Synthesis and biological evaluation of novel, selective, nonsteroidal glucocorticoid receptor antagonists. Bioorg. Med. Chem. Lett. 14, 2079–2082. Arana, G.W., Santos, A.B., Laraia, M.T., McLeod-Bryant, S., Beale, M.D., Rames, L.J., Roberts, J.M., Dias, J.K., Molloy, M., 1995. Dexamethasone for the treatment of depression: a randomized, placebo-controlled, doubleblind trial. Am. J. Psychiatry 152, 265–267. Barnes, P.J., 2005. Molecular mechanisms and cellular effects of glucocorticosteroids. Immunol. Allergy Clin. North Am. 25, 451–468. Barnes, P.J., 2006. Corticosteroids: the drugs to beat. Eur. J. Pharmacol. 533, 2–14. Beato, M., 1991. Transcriptional control by nuclear receptors. FASEB J. 5, 2044–2051. Beischlag, T.V., Marchese, A., Meador-Woodruff, J.H., Damask, S.P., O'Dowd, B.F., Tyndale, R.F., van Tol, H.H., Seeman, P., Niznik, H.B., 1995. The human dopamine D5 receptor gene: cloning and characterization of the 5′flanking and promoter region. Biochemistry 34, 5960–5970.

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