In the absence of ligand, GRα resides primarily in the cytoplasm of cells as part of a large multiprotein complex, which consists of the receptor polypeptide, two molecules of hsp90, and several other proteins [13,21-23]. Following ligand binding, the receptor dissociates from the hsps and translocates into the nucleus. The GRα contains two nuclear translocation signals (NL), NL1 and NL2 (Figure 2): NL1 contains a classic basic-type nuclear localization signal (NLS) structure that overlaps with and extends C-terminally from the DBD of GRα [24]. The function of NL1 is dependent on importin α, a protein component of the nuclear translocation system, which is energy-dependent and facilitates the translocation of the activated receptor into the nucleus through the nuclear pore. NL2 spans over almost the entire LBD. In the absence of ligand, binding of hsps with the LBD of GRα masks/inactivates NL1 and NL2, thereby maintaining GRα in the cytoplasm. Inside the nucleus, GRα binds to GREs in the promoter regions of target genes. The interaction of GRα with GREs is dynamic, with the GRα binding to and dissociating from GREs in the order of seconds [25]. GRα also modulates transcriptional activity of other transcription factors by physically interacting with them. After modulating the transcription of its responsive genes, GRα dissociates from the ligand and slowly returns to the cytoplasm as a component of heterocomplexes with hsps [26-28]. Several mechanisms regulate the nuclear export of GRα. The CRM1/exportin and the classic nuclear export signal (NES)-mediated nuclear export machineries were postulated to be involved in GRα nuclear export, based on evidence that leptomycin B, an inhibitor of these systems, abrogated the rapid nuclear to cytoplasmic translocation and cytoplasmic retention of GRα, although no classic NES(s) are evident in the GRα molecule [24,29]. In agreement with the above studies, we have shown that GRα employs 14-3-3σ, a novel molecule that has one NES and regulates numerous signal transduction pathways by changing the intracellular localization of their component proteins [30]. The calcium-calreticulin-mediated, classic NES-independent nuclear export system has also been reported to be involved in the nuclear export and cytoplasmic retention of GRα [31,32].

GRα exerts its classic transcriptional activity on its responsive promoters following binding to GREs [33]. Active endogenous GREs are present in the promoter region of the glucocorticoid-responsive genes. The optimal recognition site is an inverted hexameric palindrome separated by 3 base pairs, PuGNACANNNTGTNCPy, with each GR molecule binding to one of the palindromes [34]. The GRE-bound GRα stimulates the transcription rate of responsive genes by facilitating the formation of the transcription initiation complex, including the RNA polymerase II and its ancillary components via its AF-1 and AF-2 domains [35]. The former is ligand-independent while the latter is ligand-dependent [36].

Research studies aimed to identify molecules that interact with the AF-2 of the GR, have led to several proteins and protein complexes, called coactivators, that form a bridge between the DNA-bound GRα and the transcription initiation complex and assist enzymatically with the transmission of the glucocorticoid signal to the RNA polymerase II [37] (Figure 5). These include: (1) p300 and the homologous cAMP-responsive element-binding protein (CREB)-binding protein (CBP), which also serve as macromolecular docking “platforms” for transcription factors from several signal transduction cascades, including nuclear receptors, CREB, AP-1, NFκB, p53, Ras-dependent growth factor, and STATs [38]. Because of their central position in many signal transduction cascades, the p300/CBP coactivators are also called co-integrators; (2) p300/CBP-associated factor (p/CAF), originally reported as a human homologue of yeast Gcn5, which interacts with p300/CBP and is also a broad transcription coactivator [39,40]; and (3) The p160 family of coactivators, which preferentially interact with the steroid hormone receptors [41]. These include the steroid receptor coactivator-1 (SRC-1), SRC-2, which consists of transcription intermediate factor-II (TIF-II) and the glucocorticoid receptor interacting protein-1 (GRIP-1), and SRC-3, which consists of the p300/CBP/co-integrator-associated protein (p/CIP), activator of thyroid receptor (ACTR) and the receptor-associated coactivator-3 (RAC3) [37,41,42].

The p160 coactivators are the first to be attracted to the DNA-bound steroid hormone receptor and help accumulate p300/CBP and p/CAF proteins to the promoter region, indicating that p160 proteins play an important role in the steroid hormone receptor-mediated transactivation. These coactivators also have intrinsic histone acetyltransferase (HAT) activity through which they loosen the tightly assembled chromatin structure and facilitate access of transcriptional complexes to the promoter regions [37]. HAT activity also modulates the binding of transcription factors to specific elements on their responsive promoters [43,44], as well as the dissociation of coactivators from nuclear receptors or other transcription factors [45]. The p160 family of coactivators and p300/CBP proteins contain one or more copies of the coactivator signature motif sequence LXXLL, where L is leucine and X is any amino acid [41,46]. LXXLL forms an α helical structure and aligns leucine residues form hydrophobic bonds with the AF-2 surface, which is formed by helixes 3, 4 and 12 in the LBD of GRα in response to ligand-binding.

The AF-2 transactivation domain of GRα also attracts several other distinct chromatin modulators such as the mating-type switching/sucrose non-fermenting (SWI/SNF) complex and components of the vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein (DRIP/TRAP) complex [37] (Figure 5). The SWI/SNF complex is an ATP-dependent chromatin remodeling factor with a multi-subunit structure in which an ATPase functions as the catalytic center [47]. Depending on the energy of ATP hydrolysis, it introduces superhelical torsion into DNA. One of its components, SNF2 binds to AF-2 of GRα directly, functioning as an interface between the GR and the SWI/SNF complex [48]. The DRIP/TRAP complex is also a multiprotein conglomerate, which consists of over 10 different proteins, including DRIP205/TRAP220/PBP and components of SMCC [37]. The DRIP/TRAP complex may modulate transcription through interaction and modification of general transcription factors, such as TFIIH or the C-terminal tail of the RNA polymerase II. DRIP205/TRAP220 contains two LXXLL motifs through which it binds to the ligand-activated AF-2 directly [49]. Since the DRIP/TRAP complex and p160 coactivators use the same motif for interaction with the steroid hormone receptors, they may bind to the same site of these receptors and sequentially interact with them for full activation of transcription. Alternatively, they may interact with a particular set of steroid hormone receptors, or have a different effect on different tissues [37,42].

In contrast to the mechanisms of transactivation by AF-2, those of AF-1 are not as well elucidated yet. Using the yeast system, the Ada complex may act on AF-1 transactivation through direct interaction [50]. The SWI/SNF complex and the HAT coactivators, such as p160 and p300/CBP, also physically interact with AF-1 and mediate its transcriptional activity [51-54]. In addition, DRIP150, a component of the DRIP/TRAP complex, and the tumor susceptibility gene 101 (TSG101) interact with the AF-1 of GRα a modified yeast two-hybrid screening [55]. An RNA coactivator, the steroid RNA activator (SRA) also interacts with AF-1 [56]. Given that any of these molecules and complexes interact with both AF-1 and AF-2, it is likely that concurrent activation of AF-1 and AF-2 by their common and/or distinct binding partners may be necessary for optimal activation of GRα-induced transcription [57].

Glucocorticoids exert their diverse effects through its receptor protein module, the GRα. These hormones though, affect other signal transduction cascades through mutual protein-protein interactions with specific transcription factors, by influencing their ability to stimulate or inhibit the transcription rates of the respective target genes. This activity may be more important than the GRE-mediated one, granted that mice harboring a mutant GRα, which is active in terms of protein-protein interactions but inactive in terms of transactivation via DNA, survive and procreate, in contrast to mice with a deletion of the entire GR gene that die immediately after birth from severe respiratory distress syndrome [58,59]. The former mouse model and additional in vitro results indicate that GR interacts with and influences other transcription factors as a monomer [58,60].

The protein-protein interactions of GRα with other transcription factors may take place on the promoters that do not contain GREs (tethering mechanism) as well as on the promoters that have both GRE(s) and responsive element(s) of transcription factors that interact with GRα (“composite promoters”) [61]. Suppression of transactivation of other transcription factors through protein-protein interactions may be particularly important in suppression of immune function and inflammation by glucocorticoids [58,60]. A substantial part of the effects of glucocorticoids on the immune system may be explained by the interaction between GRα and nuclear factor-κB (NF-κB), activator protein-1 (AP-1) and probably signal transducers and activators of transcription (STATs) [62-64]. The following section will discuss further the above interactions and their effects of GRα transcriptional activity.

Glucocorticoids have two major activities on the transcription of glucocorticoid-responsive genes, namely transactivation and transrepression [115]. The former activity is mainly mediated by binding of GRα to its DNA responsive sequences in the promoter regions of genes and stimulating the transcription of downstream sequences. Mechanisms of the latter activity are more complex, mostly mediated by suppression of other transcription factor activities by GRα. At pharmacologic levels, the transactivation activity is well correlated with side effects of glucocorticoids, such as glucocorticoid-associated glucose intolerance and overt diabetes mellitus with insulin resistance, central obesity, osteoporosis and muscle wasting [115]. On the other hand, the transrepressive activity of glucocorticoids is associated mostly with their beneficial therapeutic effects, such as suppression of inflammation and immune activity, and induction of apoptosis of some cancer cells. Thus, significant efforts have been put forth to produce dissociated glucocorticoids with transrepression but no transactivation activity [115].

RU24858, RU40066 and RU24782 were the first steroids reported to have such selectivity, having efficient inhibitory effect on AP-1- and NF-κB-mediated gene induction with reduced transactivation activity in vitro [116]. However, they did not have any therapeutic advantage when they were used in vivo. Compound Abbott-Ligand (AL)-438, a derivative of a synthetic progestin scaffold, binds GRα with similar affinity to that of prednisolone and shows equivalent repression activity in the rat paw-edema inflammatory assay to that of prednisolone [117]. AL-438, however, did not increase circulating glucose levels and bone absorption, in contrast to prednisolone, indicating that this compound is a promising selective glucocorticoid. ZK216348, the (+)-enanitomer of the racemic compound ZK209614, binds GRα and demonstrates anti-inflammatory activity comparable to that of prednisolone for both systemic and topical application with much less unwanted effects on blood glucose and skin atrophy [118]. This compound, however, binds PR and AR, in addition to the GRα, and does not show lear selectivity between transactivation and transrepression in vitro. Compound A (CpdA), a stable analogue of the hydroxyl phenyl aziridine precursor found in the Namibian shrub Salsola tuberculatiformis Botschantzev, exerts anti-inflammatory activity by down-regulating TNFα-induced pro-inflammatory gene expression by inhibiting the transcriptional activity of NF-κB through GRα [119]. CpdA has virtually no stimulatory activity of GR-induced transactivation. Thus CpdA is a fully dissociated compound of plant origin retaining the beneficial anti-inflammatory effect of glucocorticoids.

Another compound, AL082D06 (D06), the tri-aryl methane, specifically binds GRα with a nano-molar affinity and acts as an antagonist for GR but not for other steroid receptors, in contrast to RU 486 [120].

11β-Hydroxysteroid dehydrogenase 1, which catalyzes the conversion of the inactive cortisone to active cortisol, increases intracellular cortisol, potentially contributing to tissue hypersensitivity to glucocorticoids. 11β-HSD1 is widely expressed, particularly in the liver but also in the lung, adipose tissue, blood vessels, ovary and the central nervous system [121]. The transgenic animals over-expressing 11β-HSD1 in adipose tissue, developed significant accumulation of visceral fat, insulin-resistant diabetes mellitus, hyperlipidemia and increased systemic blood pressure, indicating that this enzyme plays a role in the development of visceral obesity-related metabolic syndrome by increasing availability of cortisol in adipose tissue [122,123]. 11β-HSD2, on the other hand, catalyzes the conversion of active cortisol into inactive cortisol, and is expressed in the classic mineralocorticoid-responsive tissues, such as kidney, colon and sweat glands. This enzyme enables these tissues to respond to the circulating mineralocorticoid aldosterone, protecting the mineralocorticoid receptor (MR) from binding to the excess amounts of circulating cortisol, by converting cortisol into inactive cortisone [121].

GR has several phosphorylation sites and all of them are located in the AF-1 subdomain of its N-terminal domain [20,124]. Classically, GRα is phosphorylated after binding to its ligand and this may determine target promoter specificity, cofactor interaction, strength and duration of receptor signaling and receptor stability [124,125]. There are several kinases that phosphorylate GRα in vitro and in vivo; yeast cyclin-dependent kinase p34CDC28 phosphorylates rat GR at serines 224 and 232, which are orthologous to serines 203 and 211 of the human GRα, with the resultant phosphorylation enhancing rat GRα transcriptional activity in the yeast [126]. These residues are also phosphorylated after binding of the GR with agonists or antagonists and the phosphorylated receptor shows reduced translocation into the nucleus and/or altered subcellular localization in mammalian cells [124,127]. The p38 mitogen-activated protein kinase (MAPK) phosphorylates serine 211 of the human GRα, enhances its transcriptional activity and mediates GR-dependent apoptosis [128]. p38 MAPK and JNK also phosphorylate serine 226 of the human GRα and suppress its transcriptional activity by enhancing nuclear export of the receptor [29]. Threonine 171 of the rat GR is also phosphorylated by p38 MAPK and glycogen synthase kinase-3; phosphorylated GR demonstrates reduced transcriptional activity in yeast and human cells, however, the human GRα does not have a threonine residue equivalent to that of the rat GR [129].

The CNS-specific cyclin-dependent kinase 5 (CDK5) physically interacts with the human GRα through its activator component p35, phosphorylates GR at multiple serines including those at 203 and 211, and modulates GR-induced transcriptional activity by changing accumulation of transcriptional cofactors on GRE-bound GR [130]. CDK5 and p35 are expressed mainly in neuronal cells and play important roles in embryonic brain development. Aberrant activation of Cdk5 in the central nervous system also plays a significant role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease and amyotrophic lateral sclerosis [131].

We recently found that adenosine 5’ monophosphate-activated protein kinase (AMPK), a central regulator of energy homeostasis that plays a major role in appetite-modulation and energy expenditure, indirectly phosphorylates human GR at serine 211 and enhances its transcriptional activity through phosphorylation/activation of p38 MAPK [132].

GR forms heterocomplexes with several heat shock proteins, including hsp90, hsp70, hsp56 and possibly hsp23 [33]. Since these proteins bind many proteins helping with the formation of correct assemblies and folding of their partner proteins, they are also called chaperones. Some of these proteins may modulate the transcriptional activity of GRα. One of them, hsp90, regulates GRα-induced transactivation negatively, possibly by affecting recycling of GRα [133]. Receptor-associating protein 46 (RAP46), a cochaperone associated with several heat shock proteins, synergize with hsp70 to regulate GRα transactivation negatively [134].

The ubiquitin/proteasome pathway plays important roles in transcription regulation promoted by numerous trans-acting molecules. Nuclear receptors, including GRα, and the estrogen, progesterone, thyroid hormone, retinoic acid, and peroxisome proliferator-activated receptors, as well as other transcription factors, such as p53, cJun, cMyc and E2F-1, are ubiquitinated and subsequently degraded by the proteasome [135,136]. The transcriptional intermediate molecules, such as nuclear receptor coactivators, chromatin remodeling factors, and some chromatin components, such as histone H1 and HMG proteins, are also ubiquitinated and lysed by the proteasome [135-137]. Moreover, the proteasome interacts with the C-terminal tail of the RNA polymerase II and is directly associated with the promoter regions of several genes, influencing their transcriptional activities [138]. Thus, ubiquitination and subsequent processing of these molecules by the proteasome appear to regulate the transcriptional activity of GRα, possibly by facilitating rapid turnover of promoter-attracted and -associated GRα, finally down-regulating the transcriptional activity of this receptor.

14-3-3 is a family composed of 7 protein isoforms that regulate the activities of many different intracellular proteins in mammalian cells [139,140]. It binds to phosphorylated serine or threonine residues in several specific amino acid sequences and changes the subcellular localization (through its NES) and/or stability of target proteins. 14-3-3η binds to the LBD of GRα and modulates its transcriptional activity through its C-terminal portion that does not contain a binding pocket for phosphorylated amino acids [141,142]. We have recently reported that one of the 14-3-3 family protein members, 14-3-3σ interacts with GRα and regulates GRα-induced transactivation negatively, possibly by shifting the subcellular localization and recycling of GRα from the nucleus towards the cytoplasm thus functioning as an “attached helper” NES [30].

There are several chemical compounds that modulate GR activity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a wide-spread environmental contaminant that produces adverse biologic effects, such as carcinogenesis, reproductive toxicity, immune dysfunction, hepatotoxicity and teratogenesis, suppresses GRα transactivation possibly by reducing the ligand-binding affinity of GRα [143-145].

Geldanamycin, a benzoquinone ansamycin, which specifically binds hsp90 and disrupts its function, suppresses GRα-induced transactivation by inhibiting the translocation of GRα into the nucleus [146,147].

GRα is also regulated by the cellular redox state. Thioredoxin, a compound accumulated during oxidative stress, enhances GRα transactivation, most likely due to functional replenishment of GRα [148].

Ursodeoxycholic acid (UDCA), one of the hydrophilic bile acids, which acts as a bile secretagog, cytoprotective agent and immunomodulator, and is used for the treatment of various liver diseases, including primary biliary cirrhosis, induces translocation of GRα into the nucleus and causes GRα-mediated inhibition of NF-κB transactivation [149]. Cortivazol, a pyrazolosteroid, also induces nuclear translocation of GRα, thereby stimulating GRα-induced transcription [150].

Mizoribine (4-carbamyl-1-β-D-ribofurano-sylimidazolium-5-olate), an imidazole nucleotide with immunosuppressive activity binds to 14-3-3 and enhances 14-3-3/GR interaction, which may further potentiate 14-3-3’s effect on GRα transactivation [151].

Polymorphisms of the GR gene have also been reported. A heterozygous polymorphism replacing aspartic acid to serine at amino acid 363 that mildly increases transcriptional activity of the affected receptor in vitro is associated with increased sensitivity to glucocorticoids, weakly correlating with the development of central obesity and, thus, influencing the metabolic profile and the longevity of humans in a negative fashion [170-172].

The polymorphism in the GR gene that causes arginine to lysine replacement at amino acid 23 (ER22/23EK: GAG AGG to GAA AAG) is associated with relative glucocorticoid resistance by altering the expression levels of GRα translational isoforms [173]. This polymorphism increases muscle mass in males and reduces waist to hip ratio in females, and is associated with greater insulin sensitivity, and lower total and low-density lipoprotein cholesterol levels, indicating that this polymorphism causes beneficial effect on longevity by reducing glucocorticoid action [174,175].

A single nucleotide polymorphism that replaces A with G at the nucleoside 3669 (A3669G) located in the 3’ end of exon 9β has been described in a European population [176]. This polymorphism does not change the amino acid sequence but increases the stability of GRβ mRNA and increases GRβ protein expression, leading to greater inhibition of GRα-induced transcriptional activity and causing glucocorticoid resistance in tissues. The presence of the A3669G allele is associated with reduced central obesity and a more favorable lipid profile in affected subjects [176].

GRβ is a splicing variant of the classic receptor GRα, which does not bind glucocorticoids but functions as a natural dominant negative isoform for GRα transactivation in vitro [7,177]. It is classically reported to localize constitutively in the nucleus and interacts loosely with several heat shock proteins [163,177]. It is likely that the polypeptide encoded by exon 9β , which replaces helix 12 of GRα LBD changes the tertiary structure of LBD, leading to disruption of the ligand-binding pocket and possibly the AF-2 surface in GRβ [178].

Since GRβ shares the same DBD with GRα and binds GREs, competition at the level of GREs was initially thought to be the primary mechanism underlying the dominant negative effect of GRβ on the transcriptional activity of GRα [163]. Given that GRβ and GRα share the entire same N-terminal domain and DBD, and most of the LBD, GRβ may compete with GRα for many other actions of GR, such as dimerization and attraction of transcriptional coactivators [179].

Several studies have demonstrated that increased GRβ expression is associated with reduced sensitivity to glucocorticoids, such as glucocorticoid-resistant asthma, ulcerative colitis, and rheumatoid arthritis [180-183]. GRβ overexpression may also contribute to the pathogenesis of hematologic malignancies, nasal polyps and possibly insulin resistance [182,184-186].