Chapter 6 – Glucocorticoid Receptor

Updated August 2010

INTRODUCTION

Glucocorticoids are steroid hormones secreted by the adrenal glands, important for maintenance of basal and stress-related homeostasis. They regulate a variety of biologic processes and exert profound influences on many physiologic functions [1, 2]. In pharmacologic doses, glucocorticoids are used as potent immunosuppressive agents in the management of many inflammatory, autoimmune and proliferative diseases [3]. At the cellular level, the actions of glucocorticoids are mediated by an intracellular receptor protein, the glucocorticoid receptor (GR), which functions as a hormone-activated transcription factor that regulates the expression of glucocorticoid-responsive genes. These genes probably represent 5-20% of the human genome and can be influenced by the ligand-activated GR directly or indirectly [4]. The GR is ubiquitously expressed in almost all human tissues and organs. The human (h) GR, a single polypeptide chain of 777 amino acid residues, belongs to the steroid/sterol/thyroid/retinoid/orphan receptor superfamily of nuclear transactivating factors, with over 150 members currently cloned and characterized across species [5].

 

EVOLUTION OF THE GLUCOCORTICOID RECEPTOR

The glucocorticoid receptor (GR) is a member of the steroid hormone receptor subfamily of nuclear receptors (Figure 1). This receptor family of vertebrates consists of six evolutionarily related steroid hormone receptors: two for estrogens (ERα and ERβ) and one each for androgens (AR), progestins (PR), glucocorticoids (GR), and mineralocorticoids (MR) (Figure 1). Steroid receptors evolved in the chordate lineage after the separation of deuterostomes and protostomes, prior to or at the base of the Cambrian explosion about 540 million years ago [6, 7]. The receptor phylogeny suggests that two serial gene duplications of an ancestral steroid receptor gene occurred before the divergence of lamprey and jawed vertebrates. The first gene duplication created an estrogen receptor (ER) and a 3-ketosteroid receptor, whereas the second duplication of the latter gene produced a corticoid receptor and a receptor for 3-ketogonadal steroids (androgens, progestins, or both). Therefore, the ancestral vertebrates had three steroid receptors: an estrogen receptor (ER), a receptor for corticoids and a receptor that bound androgens, progestins or both. At some later time within the gnathostome lineage, each of these three receptor genes were duplicated yet again to yield the six steroid receptors currently found in jawed vertebrates: the ER creating ERα and ERβ, the corticosteroid receptor yielding the GR and MR, and the 3-ketogonadal steroid receptor producing the PR and AR. Therefore, the genome of ‘higher’ vertebrates is thought to be the result of two genome duplication events that occurred early in chordate evolution [6, 8]. Although the timing of these events is not entirely clear, it is most likely that one duplication occurred before the lamprey-gnathostome divergence and one after [6, 9].

Figure 1: Steroid hormone receptors and their homologies expressed as percent identity with the primary sequence of the human GRα. GRα; glucocorticoid receptor α, MR; mineralocorticoid receptor, PR-A; progesterone receptor-A, AR; androgen receptor, ERα: estrogen receptor α, ERß: estrogen receptor ß Modified from [261].

The GR and its closest family member, the MR, descend from duplication of the ancestral corticoid receptor (AncCR) gene deep in the vertebrate lineage, approximately 450 million years ago [10]. The GR is activated by cortisol, while the MR is activated by aldosterone in tetrapods and by deoxycorticosterone (DOC) in teleosts. The MR is also sensitive to cortisol, though considerably less so than to aldosterone and DOC [10]. Like the MR, the AncCR is sensitive to aldosterone, DOC and cortisol, indicating that the specificity of the GR for cortisol is evolutionarily derived [10].

To determine how the preference of the GR for cortisol evolved, Ortlund et al. identified substitutions that occurred during the same period as the shift in GR function [11]. Using maximum likelihood phylogenetics, it was shown that from the common ancestor of all jawed vertebrates (AncGR1), the GR retained AncCR’s sensitivity to aldosterone, DOC, and cortisol, whereas at the next node, the GR from the common ancestor of bony vertebrates (AncGR2) had a phenotype like that of modern GRs, responding only to cortisol. These findings indicate that the specificity of the GR for cortisol evolved during the interval between these two speciation events, approximately 420 to 440 million years ago [11].

 

STRUCTURE OF THE HUMAN GR GENE AND PROTEIN

All steroid hormone receptors including GR display a modular structure comprised of five to six regions (A-F): the amino-terminal A/B region, also called immunogenic or N-terminal domain, and the C and E regions, which correspond to the DNA- and ligand-binding domains, respectively (Figure 2). The GR cDNA was isolated by expression cloning in 1985 [12]. The human GR gene consists of 9 exons and is located on chromosome 5. Alternative splicing of the GR gene in exon 9 generates two highly homologous receptor isoforms, termed α and ß. These are identical through amino acid 727, but then diverge, with GRα having an additional 50 amino acids and GRß having an additional, nonhomologous 15 amino acids. The molecular weights of these receptor isoforms are 97 and 94 kilo-Dalton, respectively. GRα is expressed virtually all organs and tissues and resides primarily in the cytoplasm, and represents the classic glucocorticoid receptor that functions as ligand-dependent transcription factor. GRß, also expressed ubiquitously, does not bind glucocorticoid agonists and functions as a dominant negative receptor for GRα-induced transcriptional activity [13].

The N-terminal domain (NTD) of GRα contains a major transactivation domain, termed activation function (AF)-1, which is located between amino acids 77 and 262 of the GRα [14, 15]. AF-1 belongs to a group of acidic activators, such as VP16, NF-kB, p65, p53 and hepatocyte nuclear factor (HNF)-4, contains four α-helices, and plays an important role in the communication between this domain and molecules necessary for the initiation of transcription, such as coactivators, chromatin modulators and basal transcription factors, including RNA polymerase II, TATA-binding protein (TBP) and a host of TBP-associated proteins (TAF_IIs) [16]. GRα AF-1 is relatively unfolded at the basal state, while it forms a significantly complex helical structure in response to binding to cofactors, such as TBP [17].

The DNA-binding domain (DBD) of GRα corresponds to amino acids 420-480 and contains two zinc finger motifs through which GRα binds to specific DNA sequences, the glucocorticoid-responsive elements (GREs) [18, 19]. The DBD is the most highly conserved domain throughout the steroid receptor family. It has two similar zinc finger modules, each nucleated by a Zn ion coordination center held by four cysteine residues and followed by α-helix (Figure 3 A). The N-terminal’s first α-helix lies in the major groove of the double-stranded DNA, while the C-terminal part of each module is positioned over the minor groove (Figure 3 B). Recent research indicated that GREs with different sequences influence the 3-dimensional structure of the DBD and induce transcriptional activity differentially [20]. This suggest that DNA is a sequence-specific allosteric modulator of GR-induced transcriptional activity that may explain in part the gene-specific transcriptional effects of this receptor.

The ligand-binding domain (LBD) of the human GRα corresponds to amino acids 481-777, binds to glucocorticoids and plays a critical role in the ligand-induced activation of GRα. The crystal structure of the GRα LBD was successfully analyzed by using a point mutant containing a single replacement of phenylalanine at amino acid 602 by serine, and is comprised of 12 α-helices and 4 small β-strands that fold into a three-layer helical domain [21] (Figure 4). Helices 1 and 3 form one side of a helical sandwich, while helices 7 and 10 form the other side. The middle layer of the helices (helices 4, 5, 8, and 9) is present in the top but not the bottom half of the protein. This arrangement of helices creates a cavity in the bottom half of the LBD, which is surrounded by helices 3, 4, 11 and 12, and functions as a ligand-binding pocket [21-23]. Interaction of the LBD with the heat shock protein (hsp) 90 contributes to the maintenance of the protein structure that allows LBD to associate with ligand. Ligand-binding induces a conformational change in the LBD and allows GRα to communicate with several molecules, such as importin α of the nuclear import system, components of transcription initiation complexes and other transcription factors that mediate the ligand-dependent actions of GRα. The LBD also contains another transactivation domain, termed AF-2. The activity of AF-2 is ligand-dependent.

 

TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF GR ISOFORMS

As described above, the human GR gene expresses two mRNAs through alternative use of exon 9α and ß, and produces two splice variants. The GRα mRNA further expresses multiple isoforms by using at least 8 alternative translation initiation sites [24] (Figure 5). Since GRß shares a common mRNA domain that contains the same translation initiation sites with the GRα [12], the GRß variant mRNA seems also to be translated through the same initiation sites to a similar host of ß isoforms. Translational GRα isoforms were differentially expressed in various cell lines [24]. They were produced by ribosomal leaky scanning and/or ribosomal shunting from their alternative translation initiation sites located at amino acids 27 (GRα-B), 86 (GRα-C1), 90 (GRα-C2), 98 (GRα-C3), 316 (GRα-D1), 331 (GRα-D2) and 336 (GRα-D3), C-terminally from the classic translation start site (1: for the GRα-A) [24]. Thus, they had different lengths of their NTDs but the same DBDs and LBDs. Compared to GRα-A, the -C2 and -C3 isoforms had stronger transcriptional activities on a synthetic GRE-driven promoter, while GRα-D1, -D2 and -D3 demonstrated weaker activities [24]. GRα-B and -C1, however, possessed transcriptional activities similar to that of GRα-A [24]. All GRα isoforms translocated into the nucleus in response to ligand, while they were differentially distributed in the cytoplasm and/or the nucleus in the absence of ligand and displayed distinct transactivation or transrepression patterns on global gene expression examined by cDNA microarray analyses [24]. Thus, these N-terminal GR isoforms may differentially transduce glucocorticoid hormone signals to tissues, depending on their selective relative expression and inherent activities.

It is likely that differential cell-specific production and functional differences similar to those of the GRα translational isoforms might be present between the putative GRß translational isoforms as well.

The human GR gene has eleven different promoters with their alternative first exons (1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J) [25, 26] (Figure 6). Therefore, the human GR gene can produce eleven different transcripts from different promoters that encode the same GR proteins sharing a common exon 2, which contains the translating ATG codon. 1A1, 1A2, 1A3 and 1I are located in the distal promoter region spanning ~32,000-36,000 bps upstream of the translation initiation site, while 1B, 1C, 1D, 1E, 1F, 1H and 1J position in the proximal promoter region located up to ~5,000 bps upstream of such a site [25]. Through differential use of these promoters, the levels of GR protein isoforms can vary considerably among tissues [25]. Thus, splice and translational GR isoforms expressed from different promoters appear to form up to 256 different combinations of homo- and hetero-dimers with varying expression levels and transcriptional activities. The marked complexity in the transcription/translation of the GR gene allows cells/tissues to respond appropriately to the circulating concentrations of glucocorticoids depending on their needs [27].

 

ACTION OF GR

1. Nucleocytoplasmic Shuttling of GRα

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 protein [18, 28-30] (Figure 7). 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 [31]. 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 [32]. 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 [33-35].

Several mechanisms have been postulated for the regulation of GRα nuclear export [27]. The CRM1/exportin and the classic nuclear export signal (NES)-mediated nuclear export machinery does not appear to be functional in GRα, based on evidence that GRα is insensitive to leptomycin B, an inhibitor of this export system, and does not contain  classic NES(s) [31, 36, 37]. Rather, the Ca²⁺-binding protein calreticulin plays a role in the nuclear export of GRα, directly binding to the DBD of this receptor [37-39].

2. Mechanisms of Transcriptional Activation by GRα 

GRα exerts its classic transcriptional activity on its responsive promoters following binding to GREs [40]. 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 [41]. 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 [42] (Figure 2). The former is ligand-independent while the latter is ligand-dependent [43].

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 [44] (Figure 8). 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, cAMP response element-binding protein (CREB), activator protein-1 (AP-1), nuclear factor of kB (NF-kB), p53, Ras-dependent growth factor, and signal transducers and stimulators of transcription (STATs) [45]. 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 [46, 47]; and (3) The p160 family of coactivators, which preferentially interact with the steroid hormone receptors [48]. 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) [44, 48, 49].

The p160 coactivators are the first to be attracted to the DNA-bound steroid hormone receptor and help accumulating 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 [44]. HAT activity also modulates the binding of transcription factors to specific elements on their responsive promoters [50, 51], as well as the dissociation of coactivators from nuclear receptors or other transcription factors [52]. 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 [48, 53]. 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 [44]. 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 [54]. 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 [55]. The DRIP/TRAP complex is also a multiprotein conglomerate, which consists of over 10 different proteins, including DRIP205/TRAP220/PBP and components of SMCC [44]. 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 [56]. 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 [44, 49].

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 [57]. 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 [58-61]. In addition, DRIP150, a component of the DRIP/TRAP complex, and the tumor susceptibility gene 101 (TSG101) interact with the AF-1 of GRα in  a modified yeast two-hybrid screening [62]. An RNA coactivator, the steroid RNA activator (SRA) also interacts with AF-1 [63]. 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 [64].

3. Interaction of GR with Other Transcription Factors

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 [65, 66]. The former mouse model and additional in vitro results indicate that GR interacts with and influences other transcription factors as a monomer [65, 67].

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  promoters that have both GRE(s) and responsive element(s) of transcription factors that interact with GRα (“composite promoters”) [68] (Figure 9). Repression of transactivation of other transcription factors through protein-protein interactions may be particularly important in suppression of immune function and inflammation by glucocorticoids [65, 67]. A substantial part of the effects of glucocorticoids on the immune system may be explained by the interaction between GRα and NF-kB, AP-1 and probably STATs [69-71]. Recent reports also indicate that GR directly interacts with  transcription factors “T-box expressed in T-cells” (T-bet) and GATA-3, which play key roles respectively in the differentiation of T helper-1 and T helper-2 lymphocytes [72, 73]. GR also influences indirectly the actions of the interferon regulatory factor-3 (IRF-3) through the p160 nuclear receptor GRIP, by competing with this factor for binding to the coactivator [74]. These transcription factors are important for the regulation of immune function and the above interactions may explain some GR actions on the immune system. The following section will discuss further the above interactions and their effects on GRα-induced transcriptional activity.

A. Nuclear Factor-kB (NF-kB)

NF-kB is one of the most important transcription factors that regulate inflammation and immune function. NF-kB is stimulated by many inflammatory signals and cytokines [70, 75]. It is a dimer of various members of the NF-kB/Rel family, including p50 (and its precursor p105), p52 (and its precursor p100), c-Rel, ReA and RelB in mammalian organisms. The heterodimer p65/p50 is a major and the most abundant form of NF-kB. In its inactive form, NF-kB forms a trimer with an additional regulatory protein, IkB in the cytoplasm. A variety of extracellular signals, such as bacterial and viral products like lipopolysaccharide (LPS), released intracellular components, such as heat shock proteins after physical or chemical stress, and several proinflammatory cytokines induce phosphorylation of IkB by activating a cascade of kinases. The phosphorylated IkB then dissociates from NF-kB and is catabolized, while the liberated NF-kB enters into the nucleus, where it binds to the kB-responsive elements in the promoter regions of its responsive genes. Ligand-activated GRα directly binds NF-kB p65 at its Rel homology domain through its DBD and suppresses the transcriptional activity of NF-kB, while NF-kB suppresses GRα-induced transactivation through GREs. Interaction with GRα inhibits binding of NF-kB to its responsive elements or neutralizes its ability to transmit an effective signal [76-79]. The LBD of GRα is necessary for this suppressive action [80]. GRα also suppresses NF-kB-induced transactivation by an additional mechanism, in which GRα tethered to the kB-responsive promoters attracts histone deacetylase and/or modulates the phosphorylation of the RNA polymerase II C-terminal tail [81, 82]. In addition, the ligand-activated GRα increases the synthesis of IkB by stimulating its promoter activity through classic GREs, thus segregating active NF-kB from the nucleus by forming inactive heterocomplexes with IkB in the cytoplasm [83].

B. Activator Protein-1 (AP-1)

AP-1 is a transcription factor, which regulates diverse gene expression involved in cell proliferation and differentiation [69, 84, 85]. It acts as a dimer of members of the bZip protein family, in which c-Fos and c-Jun heterodimers are most abundant. AP-1 transduces the signal of phorbol esters, growth factors and pro-inflammatory cytokines, such as IL-1 and TNFα. These molecules stimulate different members of the mitogen-activated protein kinase family, e.g., extracellular signal-regulated kinase p38, and Jun N-terminal kinase (JNK). Once these kinases are activated, they stimulate the synthesis of specific transcription factors involved in the induction of fos and jun gene transcription, as well as enhance the transcriptional activity of both pre-existing and newly synthesized c-Fos/c-Jun proteins. AP-1 and GRα mutually interact and repress each other’s transcriptional activities on their respective responsive promoters. The LBD and DBD of GRα and the leucine zipper domain of c-Jun are necessary for this interaction [19]. Inhibition of binding of AP-1 to DNA may be one of the underlying mechanisms of GRα-induced suppression of AP-1-mediated transactivation. Furthermore, GRα competes with AP-1 for the p300/CBP coactivator, which has a limited reserve, therefore, AP-1 may not have access to adequate amounts of this coactivator to exert its transcriptional activity fully [86].

C. cAMP-Responsive Element-Binding Protein (CREB)

CREB functions downstream of many hormones and peptides that bind to the cell surface G-protein-coupled receptors, which employ cAMP as their second messenger. CREB is also a member of bZip transcription factors [87].  It forms homo- and hetero-dimers with other proteins of the same family and binds to the cAMP-responsive element (CRE). Stimulation of the above receptors induces the accumulation of cAMP that leads to activation of cAMP-dependent protein kinase A (PKA). This kinase then phosphorylates CREB at a specific serine residue and promotes recruitment of the transcriptional co-activator CBP to stimulate transcription. GRα and CREB mutually repress the transcription from their simple responsive promoters [88, 89]. Although direct association of GRα and CREB has been reported in vitro, their direct physical interaction is still unclear [88, 90]. On the other hand, they synergistically activate the transcription of composite promoters, such as that of phosphoenolpyruvate carboxykinase (PEPCK) and somatostatin, which contain both GRE and CRE sequences [90, 91].

D. Transforming Growth Factor (TGF) ß Downstream Smad6

Members of the Smad family of proteins transduce signals of transforming growth factor (TGF) ß superfamily members, such as TGFß, activin and bone morphogenetic proteins (BMPs), by associating with the cytoplasmic side of the type I cell surface receptors of these hormones [92]. Nine distinct vertebrate Smad family members have been identified, which are classified into three groups: receptor-regulated Smads (R-Smads), such as Smad1, 2, 3, 5 and 8, a common-partner Smad (Co-Smad), Smad4, and inhibitory Smads (I-Smads) like Smad6 and Smad7 [92].

Upon binding of TGFß, activin or BMP to their receptors, cytoplasmic R-Smads are phosphorylated by the receptor kinases, translocate into the nucleus and stimulate the transcriptional activity of TFGß-, activin- or BMP-responsive genes by binding to their response elements located in their promoter regions as a hetero-trimer with Co-Smad [92]. I-Smads, such as Smad6 and Smad7, act as inhibitory molecules in the TGFß family signaling, by forming stable associations with activated type I receptors, which prevent the phosphorylation of R-Smads [92]. Smad6 also competes with Smad4 in the heteromeric complex formation induced by activated Smad1 [93]. In addition, I-Smads directly suppress the transcriptional activity of TGFß family signaling by binding to promoter DNA and attracting histone deacetylases and/or the C-terminal binding protein (CtBP) [94-96]. Since I-Smads are produced in response to activation of TGFß family signaling [97], they literally function in the negative feedback regulation of the Smad signaling pathways. Smad6 preferably inhibits BMP signaling, while Smad7 is a more general inhibitor, repressing TGFß and activin signaling, in addition to that of BMP [98].

We found that Smad6 physically interacts with the N-terminal domain of the GRα through its Mad-homology 2 domain and suppresses GR-mediated transcriptional activity in vitro [99]. Adenovirus-mediated Smad6 overexpression also inhibits glucocorticoid action in rat liver in vivo, preventing dexamethasone-induced elevation of blood glucose levels and hepatic mRNA expression of the phosphoenolpyruvate carboxykinase, a well-known rate-limiting enzyme of hepatic gluconeogenesis [99]. Smad6 suppresses GR-induced transactivation by attracting histone deacetylase 3 (HDAC3) to DNA-bound GR and by antagonizing acetylation of histones H3 and H4 induced by the p160 histone acetyltransferase [99]. Thus, Smad6 regulates glucocorticoid actions as a corepressor of the GR. It appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anti-catabolic and pro-wound healing properties of the TGFß family of proteins through cross-talk between TGFß family members and glucocorticoids [99].

E. CAAT/Enhancer-binding Protein (C/EBP)

C/EBP is also one of the bZip family transcription factors, which has diverse effects on proliferation, development and differentiation in the fetus, influencing the liver, adipose, immune and hematopoietic tissues. It is present and functions mainly as a homodimer of proteins in a family that is composed of 6 different members [100]. C/EBPß, which is also known as the nuclear factor IL-6 (NF-IL6), synergistically stimulates transcription of GRα on the composite promoter that contains both C/EBPß- and GRα-binding sites [101]. GRα, on the other hand, enhances C/EBPß activity on its simple responsive promoter [101, 102]. Direct in vitro binding of these proteins has been reported.

F. Other Transcription Factors

Functional interaction of GRα has also been reported with other transcription factors, including the chicken ovalbumin promoter-upstream transcription factor II (COUP-TFII), HNF-6, NR4A orphan receptors, such as neuron-derived orphan receptor-1 (NOR-1), nuclear receptor-related 1 (NURR1) and Nur77, p53, GATA-1, Oct-1 and -2, NF-1 and Sp-1. COUP-TFII is an orphan nuclear receptor, which plays important roles in neurogenesis as well as glucose,  lipid and xenobiotics metabolism.  It physically interacts with the hinge region of GRα and suppresses GR-induced transcriptional activity by attracting a corepressor, the silencing mediator for retinoid and thyroid hormone receptors (SMRT) [103]. Mutual protein-protein interaction of GRα and COUP-TFII was necessary for glucocorticoid-induced enhancement of the promoter activity and the endogenous mRNA expression of the COUP-TFII-responsive phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of hepatic gluconeogenesis, suggesting that COUP-TFII may participate in some of the metabolic effects of glucocorticoids through direct interactions with GRα [103]. HNF6 is also an orphan nuclear receptor that plays an important role in the hepatic metabolism of glucose. It represses GRα-induced transactivation by direct binding to the DBD of GRα through its N-terminal domain [104]. Interaction of another orphan nuclear receptor Nur77 and GRα family members is critical for the regulation of proopiomelanocortin (POMC) gene expression [105]. p53, a transcription factor functioning as a tumor suppressor, physically interacts with GRα in the cytoplasm along with an additional protein Hdm2. GRα and p53 mutually repress each other’s transcriptional activity by increasing their degradation rates [106, 107]. GRα also interacts with Oct-1 and -2 on the mouse mammary tumor virus (MMTV) promoter and the gonadotropin-releasing hormone promoter [108-112]. The POU domain of Oct-1 and the DBD of GRα interact with each other in vitro. NF-1, which also stimulates the MMTV promoter, interacts with GRα and modulates this promoter’s activity [112, 113]. GATA-1, a transcription factor that plays an essential role in erythroid differentiation is repressed by GRα at the experimental cellular levels. The N-terminal domain of GRα is necessary for the interaction with GATA-1 [114].

FACTORS THAT MODULATE GR ACTION

1. New Ligands with Selective Activities

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-kB-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 clear 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-kB 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].

2. Epigenetic modulation of the GR

a. Acetylation and CLOCK-mediated counter regulation of target tissue glucocorticoid action against diurnally fluctuating circulating glucocorticoids

All steroid hormone receptors including the GR are acetylated by several acetyltransferases, such as p300, p/CAF and Tip60,  and have common acetylation sites in a consensus amino acid motif, KXKK, located in their hinge region [121-123]. The human GR is acetylated at lysine 494 and 495 of such an acetylation motif also located in its hinge region, and was reported to be deacetylated by HDAC2, an effect that is required for suppression of NF-kB-induced transcriptional activity by the activated GR [124] (Figure 10). This finding indicates that aceylation of GR at these lysine residues attenuates the repressive effect of the GR on this transcription factor. In agreement with these results, we recently found that the Clock transcription factor acetylates GR at the multiple lysine cluster that includes lysines 494 and 495, and represses GR-induced transcription of several glucocorticoid-responsive genes [125]. Clock, the “circadian locomotor output cycle kaput”, and its heterodimer partner “brain-muscle-arnt-like protein 1” (Bmal1), belong to the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) superfamily of transcription factor, and play an essential role in the formation of the circadian oscillation rhythm of both hypothalamic suprachiasmatic nucleus (SCN) and peripheral CLOCK systems that function as internal circadian time keepers [126]. The CLOCK system located in the SCN of the brain hypothalamus, acts as the “master” oscillator and generator of the body’s circadian rhythm, while the peripheral CLOCK system virtually distributed in all organs and tissues, including the CNS outside the SCN, acts generally as a “slave” CLOCK under the influence of the central SCN CLOCK. The Clock transcription factor shares high amino acid and structural similarity with the activator of thyroid receptor (ACTR), a member of the p160-type nuclear receptor coactivator family with inherent histone acetyltransferase (HAT) activity, and thus, has such enzymatic function [127].

Clock physically interacts with GR LBD at its nuclear receptor interacting domain (NRID) in its middle portion, and acetylates human GR at amino acid 480, 492, 494 and 495. Acetylation of GR attenuates binding of the receptor to GREs, and hence, represses GR-induced transactivation of GRE-driven promoters [125] (Figure 11). Since the lysine residues acetylated by Clock are located in the C-terminal extension (CTE), which is known to play a role in DNA recognition by steroid hormone receptors [128], it is likely that acetylation of these residues reduces binding of GR to GREs by altering the action of the CTE. The part of the hinge region acetylated by Clock is also overlapping with the nuclear localization signal (NLS) [31, 125], thus it is also possible that acetylation of GR alters nuclear translocation of this receptor. It is well known that the central master CLOCK located in SCN creates diurnal fluctuation of circulating cortisol, thus peripheral CLOCK-mediated repression of GR transcriptional activity in glucocorticoid target tissues functions as a local counter regulatory mechanism for oscillating circulating cortisol [129]. Loss of synchronization between the circadian rhythm of circulating cortisol produced by the central CLOCK and peripheral CLOCK-mediated repression of GR transcriptional activity that is observed in subjects with frequent trans-time zone travel and night shift work may develop functional hypercortisolism in target tissues, which could underlie multiple components of the metabolic syndrome with the resultant cardiovascular complications that are commonly seen in these subjects.

b. Phosphorylation

GR has several phosphorylation sites and all of them are located in the NTD [27, 130] (Figure 10). 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 [130, 131]. 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 [132]. 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 [130, 133]. The p38 mitogen-activated protein kinase (MAPK) phosphorylates serine 211 of the human GRα, enhances its transcriptional activity and mediates GR-dependent apoptosis [134]. p38 MAPK and JNK also phosphorylate serine 226 of the human GRα and suppress its transcriptional activity by enhancing nuclear export of the receptor [36]. 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 [135].

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 [136]. 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 [137].

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 through activation of p38 mitogen-activated protein kinase. Through phospohrylation of GRα, AMPK regulates glucocorticoid actions on carbohydrate metabolism, modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion [138]. Indeed, activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. These findings indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues, and activation of AMPK could be a promising target for developing pharmacologic interventions in metabolic disorders in which glucocorticoids play major pathogenetic roles.

c. Ubiquitination

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 [139, 140]. 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 [139-141]. 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 [142]. 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. Indeed, mouse GR contains a PEST motif at amino acids 407-426 (399-419 in human GRα) through which the ubiqunine-conjugating enzyme E2 and the ubiquitine-ligase enzyme E3 recognize their substrates [143]. The lysine residue of the mouse GR located at amino acid 426 (419 in human GRα) appears to be ubiquitinated, as inhibition of ubiquitination by compound MG-132 enhanced the transcriptional activity of the wild type GR, while the mutant receptor with lysine to alanine replacement at amino acid 426 demonstrated elevated transcriptional activity and was insensitive to MG-132 [143] (Figure 10). Ubiquitination of GR also influences motility of the receptor inside the nucleus, which was evaluated with the fluorescence recovery after photobleaching (FRAP) technique, possibly by changing association of the receptor to the nuclear matrix through ubiquitination [144-146].

d. SUMOylation

GR is also SUMOylated. SUMOylation is the reaction of conjugating the small ubiquitin-related modifier (SUMO) peptide (~100 amino acid peptide with molecular mass of ~11 kDa) to substrate proteins conducted by an enzymatic cascade similar to those of ubiquitination but specific to SUMOylation [147]. The human GRα has three SUMOylation sites, at lysines 277, 293 and 703 [148] (Figure 10). The first 2 sites are located in the NTD and act as major SUMOylation sites, while the last site is positioned in the LBD. SUMOylation of GR suppresses GR-induced transcriptional activity of  a promoter containing multiple GREs, possibly by influencing the synergistic effect of multiple GRs bound on this promoter [149-151]. DAXX, which binds SUMOylated promyelocytic leukemia protein (PML) and other transcription factors, was postulated to mediate SUMOylation-induced repression of GR transcriptional activity [152]. Other molecules, such as histone deacetylases (HDACs) and the protein inhibitors of activated STAT (PIAS) family, which interact with SUMOylated proteins and GR as well [99, 153, 154], might also participate in SUMO-mediated repression of GR transcriptional activity, as the DAXX effect appears to be cell type- and/or cellular context-specific [155]. It is known that phosphorylation of rat GR at amino acid position 246 (226 in the human GRα) by JNK facilitates SUMOylation of the receptor and regulates GR-induced transcriptional activity in a target gene-specific fashion [149].

3. 11 ß-Hydroxysteroid Dehydrogenases (11ß-HSDs)

11ß-Hydroxysteroid dehydrogenase 1, which catalyzes the conversion of the inactive cortisone to active cortisol, increases intracellular cortisol, potentially contributing to tissue hypersensitivity to glucocorticoids. 11ß-HSD1 is widely expressed, particularly in the liver but also in the lung, adipose tissue, blood vessels, ovary and the central nervous system [156]. The transgenic animals over-expressing 11ß-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 [157, 158]. 11ß-HSD2, on the other hand, catalyzes the conversion of active cortisol into inactive cortisone, 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 [156]. 

4. Chaperones and Cochaperones

            GR forms heterocomplexes with several heat shock proteins, including hsp90, hsp70, hsp56 and possibly hsp23 [40]. 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 [159]. Receptor-associating protein 46 (RAP46),  a cochaperone associated with several heat shock proteins, synergize with hsp70 to regulate GRα transactivation negatively [160].

6. Chemicals and Other Compounds

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 [161-163].

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 [164, 165].

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 [166].

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-kB transactivation [167]. Cortivazol, a pyrazolosteroid, also induces nuclear translocation of GRα, thereby stimulating GRα-induced  transcription [168].

Mizoribine (4-carbamyl-1-ß-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 [169].

7. Non-coding RNA Growth Arrest-specific 5 (Gas5)

The availability of nutrients influences cellular growth and survival by affecting gene transcription, while glucocorticoids also influence gene transcription and have diverse activities on cell growth, energy expenditure, and survival. It was recently reported that the growth arrest-specific 5 (Gas5), which is a non-protein coding RNA expressed from the exonic sequence of the Gas5 gene and is accumulated in cells whose growth has been arrested due to lack of nutrients or growth factors, functions as an RNA repressor of the GR and some other steroid hormone receptors [170]. Indeed, Gas5 sensitized cells to apoptosis by suppressing glucocorticoid-mediated induction of several responsive genes, including those encoding the cellular inhibitor of apoptosis 2 and the serum/glucocorticoid-responsive kinase (SGK). Gas5 bound to the DNA-binding domain of the glucocorticoid receptor (GR) by acting as a decoy “glucocorticoid response element (GRE)”, thus, competing with DNA GREs for binding to the GR (Figure 12). Thus, Gas5 is a ribo-repressor of the GR, influencing cell survival and metabolic activities during starvation by modulating the transcriptional activity of the GR. Detailed physiologic as well as pathologic roles of Gas5 in the regulation of local glucocorticoid actions has not been elucidated as yet.

THE SPLICING VARIANT GRß ISOFORM

The GRß isoform, which is expressed from the GR gene through alternative use of its specific exon 9ß, is known to have a dominant negative activity on classic GRα-induced transcriptional activity [13, 171]. This isoform was identified in both the zebrafish and humans, and was recently reported in mice [12, 172, 173]. Since human (h) GRß shares the first 727 amino acids from the N-terminus with hGRα [12, 174] (Figure 2), hGRß shares the same NTD and DBD with hGRα, but has a unique “LBD”. Since the divergence point (amino acid 727) is located at the C-terminal end of helix 10 in the hGRα LBD, the hGRß “LBD” does not have the helices 11 and 12 of  hGRα. As these helices are important for forming the ligand-binding pocket and for the creation of the AF-2 surface upon ligand binding [21], GRß cannot form an active ligand-binding pocket, does not bind glucocorticoids, and so, does not directly regulate GRE-containing, glucocorticoid-responsive gene promoters. In the absence of the hGRß “LBD”, the truncated hGR consisting of the NTD and DBD is transcriptionally active on GRE-containing promoters [175], thus the hGRß “LBD” somehow attenuates the transcriptional activity of the other subdomains of the molecule on GRE-driven promoters.

The dominant negative activity of GRß was first demonstrated in transient transfection-based reporter assays using GRE-driven reporter genes [13, 176], but was subsequently confirmed on endogenous, glucocorticoid-responsive genes, such as the mitogen-activated protein kinase phosphatase-1 (MPK-1), myocilin and fibronectin [177, 178]. Further, GRß was shown to attenuate glucocorticoid-induced repression of the tumor necrosis (TNF) α and interleukin (IL)-6 genes [177]. We also confirmed this negative effect of GRß on GRα-mediated transrepression using microarray analyses [179]. Several mechanisms explaining this GRß function have been reported, including (1) competition for GRE binding through their shared DBD, (2) heterodimerization with GRα and (3) coactivator squelching through the preserved AF-1 domain [13, 175, 176]. All these different mechanisms of action appear to be functional, depending on the promoters and tissues affected by this GR isoform.  Recently, the human GRß was shown to possess  intrinsic transcriptional activity independent of its dominant negative effect on GRα-induced transcriptional activity, while the physiologic role(s) of this activity remain(s) to be examined [171, 179, 180]. Inside the cells, hGRß can localize both in the cytoplasm and the nucleus [181, 182].

Similar to the human GR gene, the zebrafish (z) GR gene consists of 9 exons and produces the zGRα and zGRß proteins, which contain 746 and 737 amino acids, respectively [172] (Figure 13). zGRα and zGRß share the N-terminal 697 amino acids, whereas they have specific C-terminal portions, which contain 47 and 40 amino acids, respectively. In contrast to hGRα and hGRß, which are produced through alternative use of specific exon 9α and 9ß, zGRα and zGRß are formed as a result of intron retention [172]. zGRα and zGRß use exon 1 to exon 8 for their common N-terminal 697 amino acids. zGRα uses exon 9 for its specific C-terminal portion, while zGRß continuously employs the rest of exon 8 and uses a stop codon located at the 3’ portion of this exon to express its specific C-terminal peptide [172]. Protein alignment comparison of hGRß and zGRß indicated that these two molecules employ exactly the same divergence point, while their ß isoform-specific C-terminal peptides show little sequence homology [172]. These pieces of molecular information indicate that hGRß and zGRß evolved independently. Mouse (m) GRß is also produced in the same fashion as zGRß, indicating that intron retention may be a general mechanism for expressing this receptor isoform in organisms, while splicing-mediated expression employed by the human GRß is rather unique [183]. Nevertheless, zebrafish and mouse GRß demonstrated the same functional properties as those of hGRß, namely, inability to bind glucocorticoids, a dominant negative activity on  respectively zGRα- and mGRα-induced transactivation  of GRE-drive promoters, and a strikingly similar tissue distribution as hGRß [172, 183]. Thus, hGRß, mGRß and zGRß were produced through convergent evolution, most likely developed through a strong requirement of this type of GR isoform in the survival of these species.

The presence of nonligand-binding C-terminal variants is not unique to the GR. Similar to the human, mouse and zebrafish GR, several other human steroid and nuclear receptors, e.g. ERß, thyroid hormone receptor α (TRα), vitamin D receptor, constitutive androstane receptor (CAR), dosage-sensitive sex reversal-1 (DAX-1), NURR-2, NOR-2, peroxisome proliferators-activated receptor α (PPARα), and PPARγ, all have C-terminally truncated receptor isoforms that are defective in binding to cognate ligands and have a dominant negative activity on their corresponding classic receptors [184-193]. This suggests that evolution has allowed the development and retention of such alternative nuclear receptors, probably because they play useful biologic roles.

Several clinically oriented investigations suggest that GRß is responsible for the development of tissue-specific insensitivity to glucocorticoids in various disorders, most of them associated with dysregulation of immune function. They include glucocorticoid-resistant asthma, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), ankylosing spondylitis, chronic lymphocytic leukemia and nasal polyps [194-200]. In these studies, various immune cells expressed elevated levels of GRß, which correlated with reduced sensitivity to glucocorticoids. Elevated levels of pro-inflammatory cytokines, such as IL-1, -2, -4, -7, -8 and -18, TNFα, and interferons α and g, might have been responsible for increased GRß expression in cells from patients with these pathologic conditions, as these cytokines experimentally stimulated the expression of GRß in lymphocytes, neutrophils or airway smooth muscle cells [201-206]. Further, presence of a single nucleotide polymorphism in the 3’ untranslated region of the hGRß mRNA (rs6198G allele), which increases the stability of the mRNA, and thus, causes elevated expression of the GRß protein, was associated with increased incidence of RA, SLE, high blood pressure, ischemic heart disease and nasal carriage of Staphylococcus aureus [195, 207-209], possibly through inhibition of glucocorticoid actions by the  increased concentrations of GRß. These pieces of clinical evidence further support that GRß has dominant negative activity on GRα-induced transcription inside the human body, functioning as a negative regulator of glucocorticoid actions in local tissues.

PATHOLOGIC MODULATION OF GR ACTIVITY

1. Natural Pathologic GR Mutations that Cause Familial/Sporadic Generalized Glucocorticoid Resistance or Chrousos Syndrome

Mutations in the glucocorticoid receptor gene result in familial/sporadic generalized glucocorticoid resistance syndrome or Chrousos syndrome (see recent review by Charmandari and Kino) [210]. The condition is characterized by hypercorticosolism without Cushingoid features [211, 212]. To overcome reduced sensitivity to glucocorticoids in tissues, affected subjects have compensatory elevations in circulating cortisol and adrenocorticotropic hormone (ACTH) concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, and resistance of the hypothalamic-pituitary-adrenal (HPA) axis to dexamethasone suppression, but no clinical evidence of hypercortisolism. The excess ACTH secretion additionally causes increased production of adrenal steroids with mineralocorticoid activity, such as deoxycorticosterone (DOC) and corticosterone and/or androgenic activity, such as androstenedione, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS); The former accounts for symptoms and signs of mineralocorticoid excess, such as hypertension and hypokalemic alkalosis. The latter accounts for manifestations of androgen excess, such as ambiguous genitalia and precocious puberty in children, acne, hirsutism and infertility in both sexes, male-pattern hair-loss, menstrual irregularities and oligo-anovulation in females, and adrenal rests in the testes and oligospermia in males. The clinical spectrum of the condition is broad and a large number of subjects may be asymptomatic, displaying biochemical alterations only.

More than 10 kindreds and sporadic cases with abnormalities in the GR number, affinity for glucocorticoid, stability, and translocation into the nucleus have been reported [213-221]. The molecular defects have been elucidated in six kindreds and seven sporadic cases (Figure 14 and Table 1). The propositus of the original kindred was a homozygote for a single nonconservative point mutation, replacing aspartic acid with valine at amino acid 641 in the LBD of GRα; this mutation reduced the binding affinity of the affected receptor for dexamethasone by three-fold and caused loss of transactivation activity [218]. The proposita of the second family had a 4-base deletion at the 3’-boundary of exon 6, removing a donor splice site. This resulted in complete ablation of one of the GR alleles in affected members of the family [219]. Recent research employing mice with GR haploinsufficiency confirmed that ablation of one GR allele is sufficient to develop generalized glucocorticoid resistance [222]. The propositus of the third kindred had a single homozygotic point mutation at amino acid 729 (valine to isoleucine: V729I) in the LBD, which reduced both the affinity and transactivation activity of GRα [221]. Several pathologic heterozygotic mutations located in LBD of the GRα have been recently found in patients listed in Table 1. They include point mutations replacing isoleucine at amino acid 559 to asparagine (I559N) [223], valine at 571 to alanine (V571A) [224], glutamic acid at 679 to serine (G679S) [225, 226], isoleucine at 747 to methionine (I747M) [227], leucine at 773 to proline (L773P) [228] and arginine at 714 to glutamine (R714Q) [229]. A homozygous, frameshift mutation with a 2 bp deletion at amino acid position 773 has also been reported [230]. These mutations cause variety of molecular defects in the mutated GRα, such as reduced affinity to ligand dexamethasone, reduced or no interaction with p160 type nuclear receptor coactivators, and slower translocation into the nucleus [231]. So far only one mutation has been reported in the DBD of GRα that replaces arginine at amino acid 477 by histidine (R477H) [225]. This mutant receptor does not bind to GREs, but preserves normal affinity for dexamethasone [225, 226].

Table 1: Mutations in the human glucocorticoid receptor gene causing  Chrousos syndrome Mutation Position
Authors	cDNA*	Amino acid	Molecular Defects	Genotype	Phenotype	References
Chrousos et al. Hurley et al.	1922A>T	D641V	Transactivation¯ Affinity to ligand¯ (x3) Nuclear translocation: 22 min Abnormal interaction with GRIP1	Homozygous	Hypertension Hypokalemic alkalosis	[211, 218]
Karl et al.	4bp deletion in exon-intron 6		GRα number: 50% reduction Inactivation of affected allele	Heterozygous	Hirsutism Male-pattern hair-loss Menstrual irregularities	[219]
Malchoff et al.	2185G>A	V729I	Transactivation¯ Affinity to ligand¯ (x4) Nuclear translocation: 120 min Abnormal interaction with GRIP1	Homozygous	Precocious puberty Hyperandrogenism	[221]
Karl et al. Kino et al.	1676T>A	I559N	Transactivation¯ Transdominance (+) Decrease in GR binding sites Nuclear translocation: 180< min Abnormal interaction with GRIP1	Heterozygous	Hypertension Oligospermia Infertility	[220, 223]
Ruiz et al. Charmandari et al.	1430G>A	R477H	Transactivation¯ No GREs binding Decrease in GR binding sites Nuclear translocation: 20 min	Heterozygous	Hirsutism Fatigue Hypertension	[225, 226]
Ruiz et al. Charmandari et al.	2035G>A	G679S	Transactivation¯ Affinity to ligand¯ (x2) Nuclear translocation: 30 min Abnormal interaction with GRIP1	Heterozygous	Hirsutism Fatigue Hypertension	[225, 226]
Mendonca et al.	1712T>C	V571A	Transactivation¯ Affinity to ligand¯ (x6) Nuclear translocation: 25 min Abnormal interaction with GRIP1	Homozygous	Ambiguous genitalia Hypertension Hypokalemia Oligo-amenorrhea	[224]
Vottero et al.	2241T>G	I747M	Transactivation¯ Transdominance (+) Affinity to ligand¯ (x2) Nuclear translocation¯ Abnormal interaction with GRIP1	Heterozygous	Cystic acme Hirsutism Oligo-amenorrhea	[227]
Charmandari et al.	2318T>C	L773P	Transactivation¯ Transdominance (+) Affinity to ligand¯ (x2.6) Nuclear translocation: 30 min Abnormal interaction with GRIP1	Heterozygous	Fatigue Anxiety Acne Hirsutism Hypertension	[228]
Charmandari et al.	2209T>G	F737L	Transactivation¯ Transdominance (time-dependent) (+) Affinity to ligand¯ (x1.5) Nuclear translocation :180 min	Heterozygous	Hypertension Hypokalemia	[267]
Charmandari et al.	1201G>C	D401H	Transactivation­ Transdominance (-) Affinity to ligand: no chnage Nuclear translocation: Normal	Heterozygous	Hypertenstion Diabetes mellitus Accumulation of visceral fat	[232]
McMahon et al.	2bp (TG) deletion at 2318 and 2319	F774SfsX24	No transactivation activity No ligand-binding activity	Homozygous	Severe hypoglycemia developed 1 day after birth Hypertension Fatigues with feeding	[230]
Nader et al.	2141G>A	R714Q	Transactivation¯ Transdominance (+) Affinity to ligand¯ (x2.0) Nuclear translocation: 20 min Abnormal interaction with GRIP1	Heterozygous	Hypoglycemia developed at age 2 years and 10 months Hypertension Accelerated bone age Mild clitoromegaly	[229]

 

 

2. GR Mutation-mediated  Hypersensitivity Syndrome

Only one mutation has been also reported in the GRα NTD that replaces aspartic acid at amino acid 401 by histidine (D401H) [232]. The patient harboring this heterozygous mutation demonstrated manifestations consistent with glucocorticoid hypersensitivity, in accordance with the in vitro results that the mutant receptor hGRαD401H demonstrated a 2.4-fold increase in its ability to transactivate the glucocorticoid-responsive promoters. This condition represents the mirror image of the Chrousos syndrome.

3. GR Polymorphisms

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 [233-235]. This polymorphism found at amino acid 363 was first described by Karl et al. [219].

            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 [236].  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 [237, 238].

            A single nucleotide polymorphism that replaces A with G at the nucleoside 3669 (A3669G) located in the 3’ end of exon 9ß has been described in a European population [239]. This polymorphism does not change the amino acid sequence but increases the stability of GRß mRNA and increases GRß 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 [239].

 

3. Viral Infection

A. Human Immunodeficiency Virus Type-1

Patients with the Acquired Immunodeficiency Syndrome (AIDS), which is caused by the infection of the Human Immunodeficiency Virus type-1 (HIV-1), have several manifestations compatible with increased activity of GRα. They develop reduction of innate and T helper 1-directed cellular immunity, which is also seen in conditions of glucocorticoid excess. Patients with AIDS often develop symptoms and signs that manifest in hypercortisolemic states, such as muscle wasting, myopathy, dyslipidemia and visceral obesity-related insulin resistance [240-244]. Therefore, it is possible that some HIV-1-related factor(s) may modulate the function of GRα in patients with AIDS.

We have shown that one of the HIV-1 accessory proteins, Vpr, a 96-amino acid virion-associated protein with multiple functions [245, 246] enhances GRα transactivation by functioning as a coactivator [247] (Figure 15). Indeed, Vpr contains a nuclear receptor signature motif LXXLL at amino acids 64-68. This motif is used by host nuclear receptor coactivators to bind nuclear receptors [48]. Similarly, through this motif, Vpr directly binds to GRα and cooperatively enhances its activity on its responsive promoters along with host nuclear receptor coactivators SRC-1 and p300/CBP [247]. Vpr directly binds p300 at its C-terminal amino acids 2045-2191, where the p160 coactivators also bind [248]. Since Vpr circulates at detectable levels in HIV-1-infected individuals and is able to penetrate the cell membrane, its effects may be extended to cells not infected by HIV-1 [249, 250]. Indeed, extracellularly administered Vpr polypeptide regulates glucocorticoid-responsive genes, such as IL-12, in the same way as the potent glucocorticoid, dexamethasone [251].

Another HIV-1 accessory protein, Tat, which functions as a major transactivator of the HIV-1 long terminal repeat promoter [252] also potentiates GRα activity moderately, possibly by increasing the accumulation of the positive transcription elongation factor b (pTEFb) [253-255] (Figure 15).  Like Vpr, Tat readily penetrates the cell membranes [256] and may, therefore, modulate the transcriptional activity of GR in cells/tissues not yet infected by HIV-1.

Through Vpr and Tat, HIV-1 may facilitate the transcription of genes encoding its own proteins by directly stimulating viral proliferation. On the other hand, by enhancing transactivation of GR, these proteins may contribute to the proliferation of the virus indirectly, possibly by suppressing the host immune system [253, 257]. Extensive further clinical and basic investigations are crucial to address the relevance of the above in vitro evidence.    

B. Adenovirus

Adenoviruses cause illness of the respiratory system, such as common cold syndrome, pneumonia, croup and bronchitis, as well as illnesses of other organs, such as gastroenteritis, conjunctivitis and cystitis. They encode the E1A protein, which is expressed just after the infection and is necessary for the transcriptional regulation of the adenovirus-encoded genes [258]. In addition to the viral genes, E1A regulates the transcriptional activity of a variety of host genes through interaction with the host transcriptional integrator p300 and its homologous molecule CBP [45, 259] (Figure 15).  In an in vitro system, E1A, in contrast to Vpr, blocks the actions of glucocorticoids on the transcriptional activity of genes, producing resistance to glucocorticoids [248].

E1A also interacts with the C-terminal tail-binding protein (CtBP), which functions as a transcriptional repressor for numerous transcription factors, by communicating with the class II histone deacetylases and other inhibitory molecules like the retinoblastoma protein (Rb) [260] (Figure 15). E1A suppresses functions of p300/CBP and CtBP by binding to their functionally critical domains [45, 260]. Although there is no supportive clinical evidence, it is highly possible that adenovirus changes the peripheral action of glucocorticoids as well as of other bioactive molecules that activate nuclear hormone receptors and directly regulates the transcriptional activity of their target genes, ultimately contributing to the pathologic states observed in adenoviral infection. 

ACKNOWLEDGEMENTS

This literary work was funded in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, and the Biomedical Research Foundation of the Academy of Athens and the University of Athens, Athens, Greece.