Steroids 78 (2013) 59–68

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Steroids

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Review

It takes two to tango: Dimerisation of glucocorticoid receptorand its anti-inflammatory functions

Mark Nixon, Ruth Andrew ⇑, Karen E. ChapmanEndocrinology, University/British Heart Foundation Centre for Cardiovascular Science, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ,United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 May 2012Received in revised form 28 August 2012Accepted 7 September 2012Available online 2 November 2012

Keywords:GlucocorticoidsSignallingTranscriptionInflammation

0039-128X/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.steroids.2012.09.013

⇑ Corresponding author. Tel.: +44 (0) 131 242 6763E-mail address: Ruth.Andrew@ed.ac.uk (R. Andrew

For a number of years, there has been a widespread view that the adverse side-effects of prolonged glu-cocorticoid (GC) treatment are a result of glucocorticoid receptor (GR)-mediated gene activation, whilstthe beneficial anti-inflammatory effects result from GR-mediated ‘transrepression’. Since the introduc-tion of the dimerisation-deficient GR mutant, GRdim, was apparently unable to activate gene transcription,yet still able to repress pro-inflammatory gene transcription, the search for novel GR modulators has cen-tred on the separation of gene activation from repression by prevention of GR dimerisation. However,recent work has questioned the conclusions drawn from these early GRdim studies, with evidence thatGRdim mutants not only activate gene transcription, but that, in direct contradiction to the initial GRdim

work, are also capable of forming dimers. This review of the current literature highlights the versatilityof the GR in forming homodimer interactions, as well as the ability to bind to alternate nuclear receptors,and investigates the potential implications such varying GR dimer conformations may have for the designof GR ligands with a safer side effect profile.

� 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592. GC action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593. GR-mediated effects on transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604. GR dimerisation; identification of distinct signalling pathways? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615. Is blocking GR homodimerisation favourable to inducing an ‘anti-inflammatory profile’? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636. The future: selective glucocorticoid receptor modulators (SGRMs) that induce ‘favorable’ conformations in GR structure. . . . . . . . . . . . . . . . . . 647. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

1. Introduction

The anti-inflammatory properties of the human glucocorticoidcortisol were first reported over half a century ago [1]. Since then,glucocorticoids (GCs) have remained the leading treatment forinflammatory conditions, particularly with the development ofsynthetic GCs, including dexamethasone, betamethasone, triam-cinolone, prednisone, prednisolone and methylprednisolone. Inthe UK alone, long-term (>3 years) prescription of GCs has in-creased 34% over the past two decades [2], whilst in the US over44 million prescriptions for GCs are written annually [3]. Yet de-

ll rights reserved.

; fax: +44 (0) 131 242 6779.).

spite the vast usage of these compounds and extensive researchover the past 50 years, much remains to be elucidated concerningthe mechanisms behind their beneficial anti-inflammatory actionsand their detrimental side effects. However, we are a great dealcloser to uncovering the diverse signalling pathways behind theiractions. In this review, we discuss recent advances in GC receptor(GR) dimer- and ‘monomer’-mediated action and how this researchis advancing development of new therapies for inflammation.

2. GC action

GCs are steroid hormones synthesised from the precursor cho-lesterol within the zona fasciculata of the adrenal cortex that exerttranscriptional effects principally through GR, a member of the

Fig. 1. Glucocorticoid receptor (GR) structure. Representation of the three main regions within GR, the activation function-1 (AF-1) transactivation domain, the DNA-bindingdomain (DBD) and the ligand-binding domain (LBD), and the major functions of each of these domains. Within the DBD, the second-zinc is highlighted, demonstrating thesubstitution (A458T) into the D-loop (shown in bold) to generate the dimerisation-deficient GR mutant model (GRdim). Within the LBD, two residues known to form areciprocal hydrophobic interface between GR dimers are highlighted.

60 M. Nixon et al. / Steroids 78 (2013) 59–68

nuclear receptor superfamily that includes all the steroid recep-tors. These receptors share a common domain structure with apoorly conserved amino-terminal domain (NTD), a highly con-served central DNA-binding domain (DBD) and a well-conservedC-terminal ligand-binding domain (LBD) that also contains struc-tures critical for interaction with other transcriptional regulators(Fig. 1) [4–6]. Alternative splicing of the GR gene generates severalsplice variants [7], of which the most widely and highly expressedis GRa, with expression of the minor isoform GRb being muchmore restricted [8]. The differential splicing of GRb alters the C-ter-minus of the protein so that it is unable to bind GCs. However, GRbretains the ability to bind DNA and has been demonstrated to act asa dominant negative inhibitor of GRa [9]. Given that the majorityof GC action is exerted through GRa, here ‘GR’ within the text re-fers to GRa. Of the three major domains, the NTD is the most var-iable, and contains a transactivation domain, activation function-1(AF-1). This AF-1 region plays a key role in transcriptional activa-tion, with reports of AF-1 interacting with several transcriptionfactors, including TATA-binding protein (TBP) and CREB-bindingprotein (CBP) [6,10]. Recent work has also identified a role forphosphorylation of several serine residues within this domain,namely S203, S211 and S226 (human GR), in modulating GR func-tion. Phosphorylation of these residues, particularly S211, alterstranscription [11,12] (reviewed [13]). The LBD consists of 11helices folded into a globular structure that forms the ligandbinding pocket [14]. The importance of the ligand in determiningthe conformation assumed by the receptor after ligand bindinghas been demonstrated in a study investigating the binding ofthe GR agonist dexamethasone and the GR antagonist mifepristone[15]. Dexamethasone binding resulted in a specific conformationalchange within helix 11 of GR (equivalent to helix 12 in mostother members of the family), favoring an interaction with thecoactivator TIF2, whilst mifepristone induced an alternate confor-mation within helix 11, impairing the recruitment of TIF2 andinstead preferentially binding the corepressor NCoR. As such,the structure of the ligand itself is responsible for mediatingdownstream effects.

The small, hydrophobic nature of GCs allows diffusion acrossthe outer membrane of their cellular targets, although they mayalso be actively transported into the cell [16], after which they bindto cytosolic GR. In the resting (ligand-free) state, GR resides in thecytoplasm where its association with a large multi-protein chaper-one complex maintains a conformational state favouring high-affinity ligand binding [17,18]. Following ligand binding the chap-erone complex dissociates, resulting in conformational changesthat unmask nuclear localisation sequences, facilitating transloca-tion to the nucleus in an importin-a and importin-7 dependentmanner [19]. GR export from the nucleus may involve calreticulin-and exportin-1 based mechanisms, as well as potentially being reg-ulated through a nuclear retention signal [20]. As such, the subcel-lular localisation of GR is determined by a number of factorsincluding ligand binding, and the rate of nuclear import/export.

Although GCs act primarily through interactions with the GR,some crucial physiological effects are also mediated by the miner-alocorticoid receptor (MR), which has high affinity for endogenousGCs [21,22]. Whilst not as widely distributed as GR, MR is ex-pressed in a number of tissues [23]. However, GC access to MR isrestricted in most tissues through the action of 11b-hydroxysteroiddehydrogenase type 2 (11b-HSD2), an enzyme that converts activeGC (cortisol in humans, corticosterone in rodents) to intrinsicallyinert forms (cortisone in humans, 11-dehydrocorticosterone inrodents) [24–26]. Thus, when co-expressed with MR, 11b-HSD2prevents its activation by GC, conferring mineralocorticoid-specificity, but in its absence, MR is a high affinity GC receptor[27]. There is a current debate as to whether GCs are able to accessMR in the presence of 11b-HSD2 and how they regulate MR activityonce bound [22,28].

3. GR-mediated effects on transcription

Following translocation to the nucleus, GR activates or re-presses target gene transcription [29–32]. Until recently, the con-ventional view of GR-mediated gene activation has been one inwhich homodimers bind to ‘palindromic’ glucocorticoid response

M. Nixon et al. / Steroids 78 (2013) 59–68 61

elements (GREs) within the regulatory regions of target genes [33].Sequences resembling the consensus GRE (an inverted hexamericrepeat with three base pair separation between half-sites; AGA-ACAnnnTGTTCT) can be found in the GC-responsive mouse mam-mary tumour virus (MMTV) long terminal repeat [34], theglucose-6-phosphatase (G6P) gene promoter [35], the tyrosineamino-transferase (TAT) gene promoter [36] and the phosphoenol-pyruvate carboxykinase (PEPCK) gene promoter [37], as well asseveral other GC-regulated genes, where they are critical for GC-induction. However, recently it has become clear that GR bindingsites can deviate considerably from the consensus, depend signifi-cantly on adjacent transcription factor binding sites (many areembedded in ‘glucocorticoid responsive units’) and do not neces-sarily require ‘conventional’ homodimer binding as describedabove [38–40]. In contrast to classical GR-mediated gene activation(sometimes referred to as ‘transactivation’), GR also represses genetranscription, reportedly through both direct and indirect DNA-binding mechanisms. The latter is sometimes referred to as trans-repression, to distinguish it from the direct binding mode of action(see below), though the terminology as it refers to GR is confused.Here, we use the term ‘transrepression’ to refer to transcriptionalrepression by GR that is mediated through interference with theactivity of another transcription factor and does not involve GRbinding to the repressed gene. Direct DNA-binding occurs throughGR binding to negative GREs (nGREs) within the promoter region ofthese genes [41]. However, the absence of nGREs from multipleGC-responsive genes indicated the presence of alternative modelsof repression. In particular, GR does not appear to bind directlyto inflammatory gene promoters. Instead GR-mediated repressionis through antagonism of pro-inflammatory transcription factoractivity, particularly NF-jB and AP-1. As such, this mode of repres-sion (‘transrepression’) has been primarily attributed to GR-proteininteractions with a variety of transcription factors including NF-jB[42,43], AP-1 [44,45], STAT5 [46] and CREB [47]. Furthermore,transrepression appears to occur at much lower GC concentrationsthan gene activation by GR [48]. Perhaps one of the most interest-ing observations in GR-mediated repression was that these GR-protein interactions were apparently mediated by monomers ofGR rather than homodimers, which had been up that point as-sumed to be the sole mode of DNA-binding by GR, responsiblefor both activation and repression [49–51].

A point of contention in the trafficking of GR from cytoplasm tonucleus has been the subcellular location of GR dimerisation. Formany years, it was believed that GR dimers only formed uponDNA-binding [52]. However, more recent work has shown thatGR is capable of forming dimers in solution (albeit with a relativelyweak affinity compared to other nuclear receptors), independent ofDNA-binding [53]. This was taken a step further using nuclearlocalisation signal (NLS)-deficient GR mutants within cells, whichremain in the cytoplasm even after ligand binding [54]. Whenco-transfected with wild-type GR, it was shown that this NLS-defi-cient GR could translocate into the nucleus as a result of dimerisa-tion with the wild-type receptor. This implies that GR dimerisationoccurs outside of the nucleus, in a DNA-independent manner.Interestingly, the initial report on GR dimerisation in solution alsodescribed a similar result for the formation of GR/MR heterodimers[53], which is discussed further below.

4. GR dimerisation; identification of distinct signallingpathways?

The exciting work on GR-mediated repression of pro-inflamma-tory transcription factor activity appeared to demonstrate that themechanisms behind the beneficial anti-inflammatory effects of GCswere distinct from those responsible for adverse metabolic effects

of GC, these being attributed to transrepression and gene activa-tion, respectively. Even more intriguing was the suggestion thattransrepression effects of GR could be dissociated from gene acti-vation by manipulation of GR dimerisation.

This concept first arose in the 1990s following the introductionof point mutations into GR that affected dimerisation. Of the threemajor domains within the GR structure, the LBD was the first to beshown to play a role in dimer formation, with an initial report inrat GR revealing interactions of GR homodimers through the GRLBD [53]. However, it was not until the determination of the crystalstructure of the human GR LBD that the arrangement of such ahomodimer was defined [14]. It was demonstrated that key hydro-phobic interactions exist reciprocally between residues P625 andI628 of GR LBD homodimers, and that these play a functionally sig-nificant role in GR action (Fig. 1). With comparable expression towild-type GR, mutation of either P625 or I628 to alanine (P625Aand I628A respectively) in the full-length receptor reduced activa-tion of a reporter driven by the MMTV promoter. Interestingly theI628A mutant, which showed a 10-fold decrease in dimerisation,was still capable of repressing NF-jB-dependent gene activationto similar extent to wild-type GR [14].

Despite this demonstration of the importance of LBD residues indimer interactions, the majority of work investigating regions ofGR that mediate homodimerisation involves mutation of aminoacids within the DBD carried out several years earlier (Fig. 2). Hereit was demonstrated that the ability to dimerise when bound toDNA was dependent upon 5 amino acids in the second zinc fingerof the DBD, termed the D-loop [55]. This work, considered togetherwith the evidence from the LBD mutants, suggests that severalcontacts between GR monomers, both in the ligand binding andDNA-binding domains, stabilise the dimer interface (Fig. 1)[52,56]. Importantly, the work investigating the role of the D-looponly utilised the DBD in isolation and therefore did not test the ef-fect of the mutation of the D-loop upon dimerisation of the intactGR. Initial in vitro work found that a point mutation in the D-loop(A458T) impaired the ability of GR to bind to the palindromic GRE,preventing GC-induced gene transcription, but not GC-mediatedrepression of AP-1 transcription [55]. Shortly after this, a knock-in mouse model was established in which this point mutationwas introduced into the endogenous GR gene, generating micewith ‘dimerisation-deficient GR’, termed GRdim [57]. Unlike GR�/�

mice, GRdim mice are viable and do not display the impaired lungfunction that causes death in GR�/� mice. As was seen in vitro,these GRdim mice, unlike wild-type mice, were apparently unableto activate gene transcription in response to dexamethasone, as as-sessed by liver TAT expression [57]. Supporting previous cell cul-ture work, GRdim mice were able to repress NF-jB- and AP-1-driven inflammatory gene transcription to a similar extent to thatseen in wild-type mice [57,58]. Further work looking specifically atthe in vivo response of GRdim mice to inflammatory insult showedGCs were able to suppress inflammation-induced oedema in GRdim

mice, albeit more weakly than in wild-type mice [59].This work appeared to indicate that dimerisation was critical for

gene activation, whilst gene repression was maintained in mutantGR incapable of dimerisation. Until recently, this has been inter-preted as evidence that GR monomers mediated transrepression,yet a more contemporary interpretation is that some or all of theeffects in GRdim mice may result from the formation of ‘alternate’GR dimer or multimer complexes (Fig. 2). Indeed, growing evi-dence has revealed caveats to the ‘dissociated model’ based onthe GRdim model. Several groups have used GRdim mice to examinethe role of GR dimerisation in the regulation of gene transcription,some of which are cited in Table 1. Supporting a role for apparentGR homodimer-independent gene activation, a recent study profil-ing hepatic gene expression in GC-treated wild-type and GRdim

identified a subset of genes induced by GCs in both [39]. Indeed,

Fig. 2. Theorised glucocorticoid receptor (GR) dimer interactions influencing transactivation. (A) The ‘conventional’ head-to-head model of GR homodimerisation involvinginteractions (shown as a red star) within the D-loop of the DNA-binding domain (DBD) and ligand-binding domain (LBD) of each GR molecule. (B) In the GRdim mutant model,interactions between the LBD of two GR molecules are still possible, however, ablation of GR–DBD interactions prevents stabilisation of head-to-head dimerisation and thusprevents binding to palindromic glucocorticoid response elements (GRE). However, it may be that interactions occur between the LBD and the N-terminal domain thatstabalises an ‘alternate’ head-to-tail homodimer, allowing DNA-binding to GRE half-sites that are direct repeats (C). (D) A ‘multimeric’ configuration not involving directdimerisation, but rather the formation of a DNA-bound complex with other transcription factors. (E) Heterodimer formation with the mineralocorticoid receptor (MR)involves appears to involve interactions in the LBD, although again based on interactions in other receptors may involve the N-terminal domain.

62 M. Nixon et al. / Steroids 78 (2013) 59–68

some genes were more strongly induced in GRdim than in wild-typemice, whilst others were induced to a similar extent in both [39].These findings indicate that GC-induced transactivation can stilltake place in the absence of ‘conventional’ homodimerisation.Interestingly, this had been elegantly demonstrated several yearsbeforehand by Adams et al. in the case of phenylethanolamine N-methyltransferase (PNMT) [60], a classically GC-activated gene[61]. A point mutation (A477T) in the second zinc finger of therat GR that blocked ‘conventional’ GR homodimerisation, analo-gous to A458T used by Heck et al. [55] and Reichardt et al. [57], re-sulted in stronger induction of PNMT promoter activity in reporterassays than wild-type GR. Rather than a single palindromic GRE asoriginally reported [62], the PNMT promoter has a number of GREhalf sites [60]. Whilst mutation of any single one of these half siteshad only a minor effect on PNMT promoter activation by GC, muta-tion of two or more strongly reduced activation [63]. Based on thearrangement of half-sites, it was proposed that at the PNMT gene,GR forms ‘multimers’ through protein–protein interactions distinctfrom the second zinc finger-mediated mechanism used for dimer-isation at other genes such as TAT (Fig. 2D). This is supported bythe description of other GC-responsive genes containing multipleGRE half-sites located throughout the promoter region, includingCytochrome P450 (CYP) 3A5, a-amylase 2 (AMY2) and serum-and glucocorticoid-induced protein kinase (SGK) [64–66]. Possibly,GR monomers are recruited to other DNA-bound transcriptionalfactors, forming large multimeric complexes [67–69]. Further sup-porting evidence for the formation of multimers as oppose to di-mers arises from the fact that genes containing these multiple

GREs, such as AMY2 and SGK, are activated by GRdim mutants[39]. This may also occur for GR regulation of dual specificity phos-phatase-1 (DUSP-1) (also known as MAP kinase phosphatase-1),where four cis-acting elements mediate the transcriptional induc-tion by GCs [70].

A recent study by Meijsing et al. suggests the GRE is an impor-tant allosteric determinant of GR conformation. They used reporterassays based on different endogenous GREs, with three GR vari-ants, each with a unique mutation in either the N-terminal AF-1domain, DBD or AF2/LBD, theorising that different GR surfacesare displayed at distinct GREs [38]. Indeed substitution of a singlebase pair in the GRE had distinct effects on the transcriptionalactivity of GR mutants. Strikingly, mutation in the dimer-interface(A477T) of the DBD prevented GR-mediated induction of the SGKgene (with a non-consensus half-site sequence TGTCCG), yet in-creased induction of the UDP-galactose ceramide galactosyltrans-ferase (CGT) gene (with a non-consensus half site sequenceTGTACG). This suggests that the actual DNA sequence to whichGR binds serves as a ‘ligand’ for GR, determining transcriptionalactivity or dictating mode of GR dimerisation.

Does this mean that GR is capable of gene activation as a mono-mer, or is formation of ‘alternate’ GR homodimers a more likelyscenario? A recent paper by Jewell et al. has shed new light onthe ability of GR to dimerise [71]. Utilising transfected cellsexpressing comparable levels of fluorescently-tagged wild-typeGR (GRWT) and GRdim, dexamethasone treatment induced forma-tion of GRdim/GRWT heterodimers as well as GRdim homodimers.This work provided further evidence that the initial conclusion that

Table 1Glucocorticoid regulation of genes.

Gene name Tissue/cell type Identified GRE (species) Regulation inGRdim models

References

Genes upregulated by GCsInsulin-like growth factor bindingprotein 1 (IGFBP1)

Human ostesosarcoma cells 2 GRE (human), 1 GRE (rat) No [109–111]

Tyrosine amino-transferase (TAT) Mouse liver, Human hepatocyte, Humanostesosarcoma cells

2 GRE (human) No [38,39,112]

Phosphoenolpyruvatecarboxykinase (PEPCK)

Human hepatocyes 2 GRE (rat) No [113,114]

FK506 binding protein 5 (FKBP5) Mouse liver, Human ostesosarcoma cells 2 GRE (human, rat, mouse) No [38,39]Dual specificity proteinphosphatase 1 (DUSP-1)

Mouse liver, Mouse macrophages, Mouseendothelial cellsMouse liver

1 GRE (human) YesNo

[39,70,85,91]

Phenylethanolamine N-methyltransferase (PNMT)

Rat adrenal cells 4 GRE and multiple half-sites(rat)

Yes [63,115]

NF-kB inhibitor alpha (IkBa) Mouse fibroblasts Several half-sites identified(human)

No [59,84,89]

Interleukin 10 (IL-10) Human monocytes 1 GRE (human) Unknown [116,117]Annexin-1 Human monocytes No functional GRE identified

(human)Unknown [118,119]

Glucocorticoid-induced leucinezipper (GILZ)

Human ostesosarcoma cells, Human epithelialcells, Mouse osteoblasts

2 GRE (human) Yes [38,102,120,121]

Serum- and glucocorticoid-induced protein kinase (SGK)

Human ostesosarcoma cells 1 GRE (rat) Yes [109,122]

Dickkopf-1 (Dkk-1) Human osteoblasts 1 GRE (human) Unknown [123,124]Prototypic long pentraxin 3 (PTX3) Human fibroblasts, Mouse fibroblasts None identified No [125]Period circadian protein homolog 1(Per1)

Mouse fibroblasts, Mouse osteoblasts 1 GRE (mouse) No [102,126]

Receptor activator of nuclear factorkappa-B ligand (RANKL)

Human fibroblast-like synoviocytes, Humanosteosarcoma cells, Mouse calvarial bones

1 half-site (mouse) Yes [102,127,128]

Amylase alpha 2 (Amy2) Mouse liver, Mouse pancreas 2 half-sites (mouse) Yes [39,65]

Gene name Tissue/cell type GR binding to nGRE or alternatetranscription factor (TF)

Regulation inGRdim models

References

Genes downregulated by GCsMatrix metalloproteinase 3(MMP3)

Human fibroblast-like synoviocytes, Mouse skin Alternate TF (tethering) Yes [100]

Interleukin 6 (IL-6) Mouse macrophages Alternate TF (tethering) Yes [59]Tumor necrosis factor alpha(TNFa)

Mouse macrophages Alternate TF (tethering) Yes [59]

Interleukin 8 (IL-8) Human ostesosarcoma cells Alternate TF (tethering) Unknown [129]Pro-opiomelanocortin (POMC) Mouse fibroblasts nGRE (human, rat) Yes [130,131]Osteocalcin Human osteoblasts, Mouse osteoblasts nGRE (human, rat) Unknown [102,132,133]Prototypic long pentraxin 3 (PTX3) Human dendritic cells Alternate TF (tethering) Yes [125]Interleukin 11 (IL-11) Mouse osteoblasts Alternate TF (tethering) Yes [102]Collagenase-3 (MMP13) Mouse fibroblasts Alternate TF (tethering) Yes [58]

NB. Column ‘Regulation in GRdim models’: no = not comparable to wild-type, yes = comparable to wild-type.

M. Nixon et al. / Steroids 78 (2013) 59–68 63

GRdim could not dimerise was premature. Despite this, severalquestions remain to be answered. Given the replication of resultsfrom earlier studies in which cells expressing GRdim failed to in-duce MMTV activity, this work by Jewell et al. suggests that the for-mation of ‘conventional’ GR homodimers by GRdim is unlikely(Fig. 2B). However, whilst ‘conventional’ GRdim homodimer forma-tion may not take place, ‘alternate’ homodimer formation is astrong possibility, and this theory gains credence when one consid-ers evidence from related nuclear receptors. Activation of theandrogen receptor (AR) can involve contacts between the NTDand LBD within a single AR molecule/monomer [72]. Although itremains unknown whether or not the NTD of one AR moleculeinteracts with the LBD of a second AR molecule, it is plausible that‘head-to-tail’ GR dimers could form (Fig. 2C) in much the same wayas heterodimers of RXR with its various partners, as well as RXRhomodimers, assemble on DNA [73–76].

Whilst there is only recent evidence of ‘alternate’ GR homodi-mers, there is long-standing evidence for the formation of GR/MRheterodimers (Fig. 2E). Early reports demonstrated that not onlyare GR/MR heterodimers capable of binding DNA, but they alsoregulate gene transcription in a manner distinct from GR and MRhomodimers [77,78]. Co-expression and ligand-activation of bothGR and MR in transfected cells synergistically increased MMTV-

driven luciferase activity [77], though others have found mutualantagonism between GR and MR at a TAT3–TATA-driven reporter.Thus, the effects of GR/MR heterodimers are likely to be cell- andgene-specific [78]. Perhaps a more interesting revelation was thatthe GR/MR interaction was mediated by the GR-LBD and not thecontacts shown to be important for homodimerisation of theDBD [53]. The formation of such heterodimers is likely to haveimplications for an inflammatory setting for two main reasons.Firstly, MR is expressed in macrophages, a major cellular compo-nent of the inflammatory response, whilst 11b-HSD2 is not, en-abling GC activation of the MR [79,80]. Secondly, in contrast tothe anti-inflammatory actions of GR activation, several studieshave indicated a pro-inflammatory role for MR signalling [81–83]. As such, further research into this area is necessary to improvethe understanding of GR/MR regulation of gene transcription.

5. Is blocking GR homodimerisation favourable to inducing an‘anti-inflammatory profile’?

Whilst a great deal of work has focussed on the ability of GR torepress pro-inflammatory gene transcription, it is important toremember that GR also activates genes encoding anti-inflamma-tory mediators including IjBa, DUSP-1, glucocorticoid-induced

64 M. Nixon et al. / Steroids 78 (2013) 59–68

leucine zipper (GILZ) and Annexin-A1 [84–88]. The relevance ofIjBa induction to the anti-inflammatory actions of GCs is obvious,with IjBa retaining NF-jB in the cytoplasm [84,89]. Similarly, An-nexin-A1 has clear anti-inflammatory roles as demonstrated inknockout mice [88]. Recently, DUSP-1, which inactivates MAPKsby catalysing the removal of phosphates at key residues, has beenrevealed as an important anti-inflammatory enzyme [85,90–93].Moreover, the anti-inflammatory effects of GCs are largely attenu-ated in mice lacking DUSP-1 [90], including in zymosan-inducedinflammation [85]. However, GCs retain efficiency in DUSP-1�/�

mice subject to mast-cell dependent anaphylaxis [94], demonstrat-ing other anti-inflammatory effects of GCs are required in thismodel. A further caveat is provided by recent work from Vandevy-ver who showed that in contrast to wild-type mice, TNFa failed toinduce DUSP-1 in GRdim mice, resulting in higher circulating mark-ers of inflammation [95]. This led the authors to suggest that GRdimerisation is required for DUSP-1 expression, which in turn, iscritical for mounting an anti-inflammatory response. This is atodds to previous work demonstrating DUSP-1 induction in variouscells expressing GRdim, including macrophages, by dexamethasone[39,85]. The authors suggested that such differences may be due totissue-specific effects, and as such, the differing ‘models’ used inthese studies yielded different results [95]. However, given recentfindings, it would appear that it is an oversimplification to statethat GR dimerisation is ‘required’ for gene induction in any studyutilising the GRdim model. Rather, one could theorise from suchwork that it requires a specific GR homodimer conformation,namely ‘conventional’ GR homodimerisation, likely dependent onits GRE arrangement. This is a distinction that will need to be madein future studies as the diverse mechanisms of GR-mediated sig-nalling continue to be investigated.

6. The future: selective glucocorticoid receptor modulators(SGRMs) that induce ‘favorable’ conformations in GR structure

The original hypothesis which attributed the beneficial anti-inflammatory effects of GCs to transrepression and proposed thatdetrimental effects stemmed from gene activation stimulated a

Fig. 3. Search for an improved anti-inflammatory profile for glucocorticoid receptorinflammatory effects and adverse side effects mediated through both transrepressionmediated anti-inflammatory response has led to the concept of an ‘idealised GR profile’ ththe desired anti-inflammatory effects. However, a ligand that permits such effects has, tothe theory that transrepression could be separated from transactivation through ablademonstrated that these GR mutants are not as ‘inert’ as initially believed in terms of transtill likely to have a significantly improved side effect profile than currently existing the

major industrial effort to develop ‘dissociated’ GR ligands thatcould distinguish between transrepression and gene activation.Several synthetic GCs, RU24782, RU24858 and RU40066, weredeveloped that appeared to dissociate these pathways, at leastwhen tested on a limited number of promoter constructs in vitro[96]. However, compared to prednisolone, RU40066 was ineffec-tive in vivo in reducing croton oil-induced oedema, and whilst bothRU24782 and RU24858 did reduce inflammation, both were lesseffective than prednisolone [96,97]. Nonetheless, such anti-inflam-matory potential led to further investigation of these compounds.Yet despite this initial promise, these synthetic GCs induced sideeffects similar to those seen with typical GC therapy [98,99],though with severity reflecting their reduced potency with respectto prednisolone. As we further unravel the intricate mechanism ofGR action, it would appear that the ‘holy grail’ of a fully ‘dissoci-ated’ GR ligand may be unattainable (Fig. 3B). Nevertheless, thisdoes not detract from the overwhelming need to develop new GRligands that maximise anti-inflammatory effects, whilst limitingadverse side effects. Although recent studies have questioned theconclusions drawn from the early GRdim studies in terms of inabil-ity to form dimers and complete abolition of transactivation, theyhave also provided intriguing insights into GR signalling, particu-larly the role of ‘conventional’ or ‘alternate’ GR homodimers. Assuch, manipulation of GR homodimers, or even GR/MR heterodi-mers, is likely to provide a possible route for therapeutic advance-ment (Fig. 3C).

Such development has inevitably proved difficult. However,whilst most of the dissociated GR ligands tested to date were spe-cifically designed and then subsequently tested on simple assaysin vitro, Compound A (CpdA), first described by Dewint et al., pro-vides an exception. This plant-derived GR agonist potently sup-pressed inflammation associated with collagen-induced arthritis,yet failed to induce hepatic PEPCK and G6P [100]. This was attrib-uted to impairment of GR dimerisation following CpdA binding,which was supported by immunoprecipitation, along with evidencethat CpdA actively disrupted pre-existing GR homodimers [54,100].However, whilst CpdA does not allow GR to activate simple GREssuch as those in the PEPCK and TAT genes, it might allow GR induc-

(GR) ligands. (A) Glucocorticoid (GC) activation of GR results in beneficial anti-and transactivation. (B) The discovery of multiple pathways in generating a GR-at incorporates selective transactivation and selective transrepression, yielding only

date, remained out of reach. (C) Use of the GR-‘dimerisation-deficient’ mutants led totion of dimerisation, thus preserving anti-inflammatory action. Recent work hassactivation, yet a ligand that inhibits GR dimerisation, ‘conventional’ or otherwise, israpies.

M. Nixon et al. / Steroids 78 (2013) 59–68 65

tion of genes containing alternate GREs similar to those in PNMT.Given that GRdim mice are susceptible to GC-induced osteoporosis,Rauch et al. tested the effects of CpdA on osteoblast differentiation[101]. Osteoblast differentiation is normally inhibited by GCs via aGR-mediated increase in the ratio of receptor activator of nuclearkappa-B ligand (RANKL) to its decoy receptor osteoprotegerin(OPG), as well as repression of osteoblast IL-11 levels and STAT3tyrosine phosphorylation [102]. Unlike dexamethasone, CpdAtreatment of osteosarcoma cells had no effect on RANKL expression,nor did it suppress IL-11 levels in primary osteoblasts as dexameth-asone did [101]. Whilst this supports a lack of effect of CpdA onosteoblast differentiation, it is particularly intriguing given thatCpdA did suppress levels of other cytokines in these cells, namelyIL-6 and CXCL-10. The authors suggest that this is due to IL-11expression being driven by AP-1-mediated transcription [102],whilst CpdA-driven repression targets NF-jB- over AP-1-mediatedtranscription. However, it remains to be seen whether CpdA is sim-ilarly bone-sparing in vivo, whilst the possibility exists that at leastsome effects are ‘off-target’ and not mediated through GR.

A further aspect that must be considered in the search forSGRMs is their regulation of GR levels. It is clearly established thatclassical GR agonists such as dexamethasone and endogenous GCsdown-regulate GR mRNA and protein levels [103–105], the lattervia ligand-dependent targeting of GR to proteasomal degradation[106], thus limiting GR-mediated gene activation and preventingover-stimulation. A study by Avenant investigated the role of li-gand-specific GR degradation and whether this mechanism wasphosphorylation-dependent [107]. In COS-1 cells, the half-life ofGR in the presence of dexamethasone was approximately 10 h(12 h in the presence of cortisol), whereas CpdA extended thehalf-life to approximately 42 h. A role for phosphorylation wasinvestigated, however neither phosphorylation of S226 nor S211determined GR degradation [107]. The authors suggested that li-gand-induced conformational changes are likely to determineinteractions with the proteasome, thus influencing degradation.Furthermore, the varying effects on GR half-life may reflect the rel-ative binding affinity of these ligands to the GR molecule. Althoughligand-free GR is stable as a result of its association with its chap-erone complex, this data brings into sharp focus the need for futurestudies to investigate potential alterations in the GR levels inducedby SGRM binding, as an extended or shortened half-life would beexpected to have varying effects on both activation and repression.Indeed, in terms of GR dimerisation, very little is known as towhether the various homodimer or heterodimer structures differin their stability. Given the suggestion that GR conformation islikely to determine its degradation [107], it would seem logicalthat alternate homodimer or heterodimer structures would exhibitchanges in their half-life. The functional consequence of GR dimerstability also needs to be addressed, with the speculation beingthat an increased half-life would lead to increased transcriptionalactivity. Indeed, studies investigating proteasomal degradation ofGR found proteasome inhibition increased GR-mediated activationof MMTV templates in a number of cell lines [108]. Furthermore,the trafficking of the GR homo- and heterodimers into the nucleusis also an important consideration when assessing transcriptionalactivity. One must also take into account the role of phosphoryla-tion when considering functional consequences of GR homo- orheterodimers. In particular whether phosphorylation of key resi-dues are required for different forms of dimerisation and if so,whether they are induced in a ligand-specific manner.

7. Conclusions

GR dimerisation is crucial for many if not most GC-mediated ac-tions, yet it is still unclear as to whether there is a universal GRhomodimer conformation that mediates these effects, or multiple

conformations with specific downstream actions. Current knowl-edge of the role of GR dimers has come from work with the ‘dimer-isation-deficient’ GRdim model, yet the demonstration that thesemutant GR molecules are in fact capable of forming GR homodi-mers has allowed new interpretation of the data. Indeed, overthe past number of years evidence has grown in support of analternative homodimer conformation, distinct from the conven-tional GR homodimer on palindromic GREs. Of course it remainsto be determined if such alternate conformations truly exist andwhat, if any, their functional relevance is. Future studies must alsotake into account the role of heterodimer formation between GRand MR as well as the possibility of heterodimerisation with othernuclear receptors.

A current major goal is the development of SGRMs that are po-tent anti-inflammatory agents, but lack the adverse side effectsassociated with traditional GC therapy. The original concept thatdivergence between GC-mediated gene activation and repressioncould be mechanistically explained and thus separated by GRdimerisation has proved over-simplistic. However, it has providedthe basis for research that has revealed a multi-factorial regulationof GR action, with both the hormone and the DNA ligands playingan active role in determining the transcriptional outcome of GRbinding. Ligand binding induces conformational changes in theGR that alter its ability to form dimers and thus probably impactcellular localisation, stability and ultimately, transcriptional activ-ity. In order to investigate such outcomes, future studies with po-tential SGRMs should make use of combined chromatinimmunoprecipitation (ChIP) and RNA-Seq analysis to determinethe effects of potential GR ligands on GR-DNA-binding and tran-scription of GC-regulated genes. Furthermore, the effects of SGRMson GR stability should not be neglected. In conclusion, the path-ways underlying formation of GR dimers and their various struc-tural conformations are likely to hold the key to understandinghow GR both activates and represses gene transcription.

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