Abstract: Glucocorticoids are among the most widely prescribed drugs used for human diseases, and are especially commonly used in autoimmune diseases. Their use reflects their rapid and broad spectrum actions on immune cells, which in turn reflect the multiple mechanisms of cell activation upon which glucocorticoids impact. While inhibition of pro-inflammatory gene expression is a major effect of glucocorticoids, they also induce the expression of numerous molecules that exert regulatory influences on the immune system. Among these is glucocorticoid induced leucine zipper (GILZ), a recently described, highly glucocorticoid-induced, transcriptional regulatory protein which has important inhibitory effects on immune and inflammatory cell functions. In this review, we summarize knowledge of the actions of glucocorticoids relevant to autoimmune disease, and focus on the potential for greater understanding of the function of GILZ to facilitate discovery of new therapeutic options for these diseases.
Introduction: Efficacy and Toxicity of Glucocorticoids
Autoimmune diseases are characterized by unwanted increased activity of the immune system. Much research into these diseases has understandably focused on stimulatory aspects of immune cell activation that contribute to inflammation. However, as is the case for most physiological systems, activity of the immune system is modulated in both stimulatory and inhibitory directions, by competing pathways of activation and regulation. Among the many regulatory mechanisms which act to temper the activity of the immune system, perhaps the most powerful are those entrained by adrenal gland production of glucocorticoid hormones (Chrousos, 1995a). Numerous studies demonstrated the powerful effect of endogenous glucocorticoids on inflammation and immune responses. For example, removal of endogenous glucocorticoids by adrenalectomy results in a dramatic exacerbation of inflammation in the rat adjuvant arthritis model and converts this model of arthritis into a fatal systemic inflammatory response (Leech et al., 2000b). Models of immune-mediated glomerulonephritis are also dramatically worsened by adrenalectomy (Leech et al., 2000a). In humans, opposing effects on lymphocyte function in the setting of Cushing’s and Addison’s disease (Sauer et al., 1994), and cases such as remission of systemic lupus erythematosus (SLE) after the development of Cushing’s disease and relapse after its treatment (Arima et al., 1998), attest to the importance of endogenous glucocorticoids in the modulation of immune function.
The therapeutic anti-inflammatory and immunosuppressive effects of synthetic glucocorticoids were first observed in rheumatoid arthritis (RA), in work which led to the awarding of a Nobel prize (Slocumb et al., 1950), and these effects have thereafter been exploited through the use of glucocorticoid drugs for over 60 years (Hillier, 2007). Indeed, a community practice survey in 2000 indicated that up to 1% of the adult population are taking systemic glucocorticoids at any given time (van Staa et al., 2000), and even in the era of targeted biological therapies for RA, more than 50% of patients with RA take regular oral glucocorticoids (Emery et al., 2008; Huscher et al., 2009; Kremer et al., 2008). In SLE, the reported prevalence of systemic glucocorticoid use is even higher, of the order of 60-75% (Furie et al., 2011; Urowitz et al., 2008). Among the very wide range of other diseases in which they are used, systemic glucocorticoids are commonly used in organ-specific autoimmune disease such as myasthenia gravis (Sathasivam, 2008), systemic vasculitides (Miller et al., 2010), idiopathic inflammatory syndromes such as sarcoidosis (Lazar and Culver, 2010), as well as other acute and chronic inflammatory diseases such as asthma and chronic obstructive pulmonary disease. As a result, synthetic glucocorticoids are among the most frequently prescribed of all medications in humans (Hillier, 2007).
The widespread use of glucocorticoids reflects their unique combination of broad-spectrum anti-inflammatory and immunosuppressive effects, and their rapid onset of action. As subsequent sections will review, this breadth and rapidity of action is explained by the multiple pathways of immune cell activation upon which glucocorticoids impact, many of which are signal transduction pathway events. These actions of glucocorticoids exploit mechanisms that we presume have evolved to permit the dynamic physiological modulation of immune system activation by the hypothalamo-pituitary-adrenal axis.
As was noted very early in the history of their use, glucocorticoids have significant dose-dependent toxicity that also mimics the physiological metabolic effects of glucocorticoids. Taking RA patients as an example, several papers have informed on toxicity of glucocorticoids. Saag et al. (1994) reported that the adverse events most strongly associated with glucocorticoid use were bone fracture, infection, and gastrointestinal events such as peptic ulceration. Huscher et al. (2009) reported a very large survey in which the most common features reported by patients included weight gain, bruising, edema, infections, depression, cataracts and glaucoma, skin fragility, hypertension, and dyspnea. Another well-described toxicity of glucocorticoids is diabetes mellitus, which together with weight gain and hypertension, may contribute to the increased risk of cardiovascular events observed in RA patients who take glucocorticoids (Davis et al., 2007). However, not all studies found an association between glucocorticoid use and the metabolic syndrome or surrogate markers of atherosclerosis (Hafstrom et al., 2007; Toms et al., 2008).
In general it is accepted that glucocorticoid toxicity is increased in proportion to exposure. Interestingly, two patterns of glucocorticoid toxicity were observed by Huscher et al. (2009): a linear pattern in which reported toxicity increased proportionally with dose, and a “threshold” pattern in which toxicity was only observed above a certain dose. As a result, it has become a major theme of basic and clinical research to discover the means to reduce glucocorticoid dosing. Indeed, the very widespread use of topical and inhaled glucocorticoids represents the result of attempts to reduce systemic exposure to glucocorticoids while retaining a therapeutic effect.
To date, so called “steroid sparing” therapies are simply additional non-glucocorticoid immunosuppressive drugs permitting partial withdrawal of glucocorticoids, and no mechanism-based approach to improving glucocorticoid sensitivity, or producing glucocorticoid beneficial effects with reduced adverse effects, has been identified. Clearly, while the mechanisms of action of glucocorticoids remain incompletely understood, developing therapies that match their profound immunoregulatory effects but avoid their significant metabolic toxicity remains an unachieved goal. Enhanced understanding of this significant physiological pathway may unveil targets which can be addressed therapeutically in autoimmune disease. In the following sections, we review the mechanisms of action of glucocorticoids, and discuss the potential for a recently reported molecule, glucocorticoid-induced leucine zipper (GILZ), to represent a means to this end.
Mechanisms of Action of Glucocorticoids
Glucocorticoids are members of a class of molecules known as steroids, which also includes mineralocorticoids and sex steroids that are derived from cholesterol. Steroids, whether produced endogenously as hormones or as synthetic chemical substances, are normally water-insoluble and are transported in the blood to target cells by protein carriers. Steroids then dissociate from their protein carriers and pass through the cell membrane of the target cells. In the cytoplasm, steroids bind to ligand-specific receptors, and the receptor-bound steroid molecule translocates into the nucleus, as shown in Figure 1. The effects of the steroids and their receptor complexes can be classified into genomic and non-genomic effects (Dietrich et al., 2011). Genomic effects include transactivation, transrepression, and post-transcriptional modification, whereas non-genomic effects include interference with the activation of signaling cascades via protein-protein interactions.
Among the steroids, glucocorticoids, secreted by the adrenal cortex, have the most widespread systemic effects, including on the immune system (Barnes, 2006b; Chrousos, 1995b). Much effort has been expended identifying glucocorticoid anti-inflammatory mechanisms of action (Barnes, 2006b; Schäcke et al., 2002). Most cellular responses to glucocorticoids are attributed to their binding to the intracellular glucocorticoid receptor (GR). In the cytoplasm, glucocorticoids bind to GR, found in its inactive form in a complex with molecular chaperones including the heat shock protein 90 (Hsp90), Hsp70, the p59 immunophilin molecule, and the small p23 phosphoprotein (De Bosscher et al., 2003; Lovgren et al., 2007; Schmitt et al., 1993). After binding to a glucocorticoid molecule, the GR detaches from this multi-protein complex and translocates into the nucleus, where the majority of glucocorticoid actions to modulate target gene expression are exerted. The following sections outline the multiple mechanisms of glucocorticoids on inflammatory and immune cell functions.
Transcriptional effects of glucocorticoids
The steroid-activated receptor is able to bind to specific steroid response elements in DNA chromatin, such as, in the case of glucocorticoids, glucocorticoid response elements (GRE). The binding of the glucocorticoid-glucocorticoid receptor (GC/GR) complex to GRE results in gene transcription, the production of messenger RNA (mRNA) molecules, and the synthesis of specific proteins. As recently reviewed (Chinenov and Rogatsky, 2007), the GC/GR complex modulates gene transcription by three different molecular mechanisms involving binding to three specific types of GRE. One mechanism involves the activation of gene transcription by direct binding of so-called simple GREs in the promoter regions of target genes that typically bind homodimeric GR. The classically reported palindromic dimeric GRE is an example of a simple GRE, but this type of GR binding site may represent a minority of the targets of the GC/GR complex. Composite GREs in gene promoters also bind the GC/GR complex, but do so together with other transcription factors. In contrast, tethering GREs are in fact sites on DNA for other transcription factors (such as NF-κB, AP-1, and Stat3) which in turn bind the GC/GR complex via protein-protein interactions (Chinenov and Rogatsky, 2007). This ability of the GC/GR complex to interact with DNA as a transcription factor, and with proteins to affect the function of other transcription factors, provides a great diversity of possible cellular responses to glucocorticoids. In addition, transcriptional coregulators, such as Src homologous members (Src-1, Src-2, and Src-3), methyltransferases, and histone acetyltransferases, are also reported to play a crucial role in chromatin remodeling, assembly of transcriptional factors, and target gene transcription in response to glucocorticoids (Chinenov and Rogatsky, 2007; Xu and Li, 2003). A number of genes whose products function as pivotal anti-inflammatory factors, such as annexin-1, MAPK phosphatase 1 (MKP-1), glucocorticoid-induced leucine zipper (GILZ), and secretory leukocyte peptidase inhibitor (SLPI), are activated via GC/GR interaction with GRE (Barnes, 2006b). For example, MKP-1, a member of the dual-specificity phosphatase family, acts to dephosphorylate, and therefore deactivate, MAP kinases (Liu et al., 2007), resulting in inhibition of inflammatory cell activation. MKP-1 is induced by glucocorticoids (Toh et al., 2004), and has important inhibitory effects on both innate and adaptive immune responses, including in a model of autoimmune arthritis (Chi et al., 2006; Hammer et al., 2006; Salojin et al., 2006; Zhao et al., 2005). Interestingly, the glucocorticoid-antagonizing pro-inflammatory protein, macrophage migration inhibitory factor (MIF), modulates glucocorticoid sensitivity via inhibition of glucocorticoid-induced MKP-1 (Aeberli et al., 2006). One glucocorticoid-induced protein may regulate the expression of others, such as annexin 1, MKP-1, and GILZ (Yang et al., 2009; 2006). Further discussion of the effects of GILZ on immune activation is provided in subsequent sections.
In addition to transcriptional activation, the GC/GR complex can also regulate gene transcription via negative GREs (nGREs) that differ in structure and function from positive GREs (Dostert and Heinzel, 2004). So far, only a relatively small number of genes, such as interleukin-1β (IL-1β), osteocalcin, and corticotropin releasing hormone, are known to contain nGREs. The contribution to the control of inflammation of the direct negative regulation of IL-1β by glucocorticoids is probably of relatively minor importance.
In contrast, when GC/GR binds to other transcription factors, such as AP-1, NF-κB, and interferon regulatory factor 3 (IRF3), rather than to DNA (Heck et al., 1994; Ogawa et al., 2005; Reily et al., 2006), the GC/GR complex is able to powerfully inhibit gene transcription entrained by these proteins. Importantly, all of these factors, which act as so-called tethering GREs (Chinenov and Rogatsky, 2007), modulate the transcription of pro-inflammatory genes, and this mechanism is thought to underpin a substantial portion of the immune regulatory effects of glucocorticoids.
As shown in Figure 1, the GC/GR complex physically interacts with the transcription factor NF-κB in the cytoplasm to block its nuclear translocation (Widén et al., 2003) or in the transcription complex to prevent gene transcription activated by NF-κB (McKay and Cidlowski, 1998; Ray and Prefontaine, 1994). The GC/GR complex also represses transcriptional activation mediated by AP-1, through a direct interaction between GR and c-Jun/c-Fos, the two subunits which comprise AP-1 (Schule et al., 1990; Touray et al., 1991). Protein-protein interactions between the GC/GR complex and NF-κB and AP-1 result in repression of the production of cytokines relevant to autoimmune disease, including IL-1β, IL-2, IL-3, IL-6, IL-8, TNF, and granulocyte macrophage colony stimulating factor (GM-CSF) (Almawi and Melemedjian, 2002) as well as other mediators of inflammation such as cyclooxygenase-2. In summary, negative transcriptional regulation by GR, either through nGRE binding or transcriptional interference, is a significant alternative means through which the anti-inflammatory and immune suppressive actions of glucocorticoids are mediated.
Post-transcriptional effects of glucocorticoids
After transcription, mRNAs encoding many pro-inflammatory genes, such as IL-1β, IL-6, IL-8, TNF, and GM-CSF are unstable, and are rapidly degraded by RNases (Anderson et al., 2004; Shaw and Kamen, 1986; Smoak and Cidlowski, 2006). All these mRNAs contain AU-Rich elements (ARE) in 3′ un-translated gene regions (Barnes, 2010). In active inflammation, inflammatory factors such as IL-1β and TNF activate pathways such as p38 MAP kinase, which results in the stabilization of mRNA via effects on ARE and hence enhance translation of pro-inflammatory cytokines (Chikanza and Kozaci, 2004). Glucocorticoid induction of MKP-1 is associated with reduced p38 MAP kinase activity (Toh et al., 2004) and this results in impaired pro-inflammatory mRNA stability and reduced cytokine expression (Yang et al., 2006). Glucocorticoids can also reduce mRNA stability through increasing the expression of proteins, such as tristetraproline (TTP), HuR, and T cell intracellular antigen-1 (TIA-1), that destabilize mRNAs encoding inflammatory proteins (Anderson et al., 2004; Barnes, 2006a). Smoak and Cidlowski (2006) first reported upregulation of TTP by the glucocorticoid dexamethasone in vitro, a finding which provided a novel glucocorticoid-induced post-transcriptional anti-inflammatory pathway. Silencing TTP expression counteracted the inhibitory effect of dexamethasone on TNF expression, and accordingly TTP-deficient mice developed more severe arthritis than wild-type controls (Anderson et al., 2004). Together these findings indicate that glucocorticoids can impact on post-transcriptional modification.
Non-genomic effects of GCs
Apart from classic genomic effects, GCs are also reported to have rapid non-genomically mediated effects through alternative pathways. So far, four alternative mechanisms have been proposed: (i) a signaling pathway through a membrane-bound GR (mGR), (ii) direct membrane effects, (iii) interaction of the GR with other proteins in the cytoplasm, and (iv) GR translocation on the mitochondria. With regard to the first mechanism, cell membrane glucocorticoid binding was first reported in mouse and human lymphoid cell lines (Gametchu et al., 1999) and termed mGR. mGR+ frequency in monocytes is correlated with clinical status in SLE, RA, and ankylosing spondylitis, suggesting a role of the mGR in immunological diseases (Bartholome et al., 2004; Spies et al., 2006; Tryc et al., 2006). Of note, no specific molecule has been identified which subserves the mGR function and further work is required to define this apparent functionality. Direct membrane effects of glucocorticoids have been described for more than thirty years. Synthetic glucocorticoid analogues were reported to increase membrane lipid mobility in lymphocytes and some cancer cell lines (Keating et al., 1985), suggesting that glucocorticoids can directly modulate plasma membrane physicochemical properties to regulate cell functions such as membrane Na+ and Ca2+ ion channels, cell fluid shear response, and cell tight junction formation. The binding of glucocorticoids to the cytosolic GR has been reported to lead to rapid intracellular signaling events that are independent of interaction with DNA-bound transcription factors (Croxtall et al., 2000). Finally, the GC/GR complex can directly translocate into the mitochondria, impacting on sensitivity to glucocorticoid-induced apoptosis (Talabér et al., 2009).
Better understanding of the genomic and non-genomic effects of glucocorticoids and their relative place in control of immune cell activation is still required. However, it is clear that glucocorticoid-induced anti-inflammatory molecules represent an opportunity for mimicking glucocorticoid effects and may offer promising new therapeutic approaches.
A Potential Steroid Sparing Target: GILZ
Structure and function of GILZ
GILZ expression is extremely sensitive to induction by glucocorticoids. For example, in human RA synovial fibroblasts, dexamethasone induced a more than 10-fold increase in GILZ transcripts at a concentration of only 1 nM, while 100 nM dexamethasone increased GILZ mRNA by over 100-fold (Beaulieu et al., 2010). In vivo, exogenous glucocorticoids induce GILZ expression, while blockade of endogenous glucocorticoids inhibits GILZ expression in mouse spleen; and in humans GILZ expression is reduced in response to reductions in circulating cortisol (Beaulieu et al., 2010; Lekva et al., 2009). The dramatic effect of glucocorticoids on GILZ is mediated via the direct binding of the GC/GR complex to six GC-responsive elements (GREs) located in the promoter region of the GILZ gene. The GILZ promoter also contains two functional forkhead-responsive elements (FHREs), which when bound to the FoxO3 transcription factor facilitate maximal GILZ expression induced by GC/GR binding.
GILZ impacts upon multiple signal transduction pathways relevant to immune responses and inflammation, as shown in Figure 2, and through these mechanisms it is postulated to act as a mediator of glucocorticoid actions. GILZ was first identified as a glucocorticoid-induced immune regulatory protein in 1997 in studies in which GILZ inhibited T cell receptor (TCR)-activation of T cells (D’Adamio et al., 1997). Since then, GILZ has been shown to be expressed in other immune cells, including monocytes/macrophages, mast cells, and dendritic cells (Berrebi et al., 2003; Cohen et al., 2006b; Godot et al., 2006; Hamdi et al., 2007), and to have numerous anti-inflammatory functions in these cells. For example, Berrebi and colleagues found that upregulation of GILZ by dexamethasone and IL-10 led to decreased MIP-1a and CCL5 expression, NF-κB activation, and CD80 and CD86 co-stimulatory molecule expression in THP-1 macrophages (Berrebi et al., 2003). Cohen et al. (2006a) demonstrated that dendritic cell maturation and antigen presentation were impaired in the presence of increased GILZ, resulting in reduced antigen presentation, decreased T helper cell activation and increased activation of regulatory T cells. GILZ is also reported to be constitutively expressed in human mast cells, where it is also induced by glucocorticoids and IL-10 (Godot et al., 2006).
GILZ, which is a protein of 137 amino acids in humans, consists of three major domains: the N-terminal, leucine zipper (LZ), and C terminal domains (Beaulieu and Morand, 2011). GILZ, also known as TSC22 domain family protein 3, also contains a tuberous sclerosis complex (TSC) domain. The LZ motif of GILZ is located in the central part of the protein and mainly mediates the homodimerization of GILZ required for many of its functions (Di Marco et al., 2007), while the other domains are responsible for protein-protein interactions between GILZ and transcriptional and signaling molecules.
The C-terminal of GILZ is a proline-rich region necessary for direct binding of GILZ to the p65 subunit of NF-κB (Di Marco et al., 2007; Riccardi et al., 2001). In 2001, Aryoldi and colleagues showed that the over-expression of GILZ in T cells inhibits the activation of NF-κB by binding the p65 subunit of NF-κB and preventing its nuclear translocation (Ayroldi et al., 2001). GILZ inhibition of cyclooxygenase-2 in bone marrow mesenchymal stem cells is also mediated by effects on NF-κB p65 nuclear transport (Yang et al., 2008). GILZ was co-precipitated with the p65 subunit of NF-κB in macrophages stimulated with glucocorticoids, and co-expression of GILZ with an NF-κB reporter inhibits reporter activity (Berrebi et al., 2003).
Mittelstadt and Ashwell (2001) demonstrated in Jurkat T cells that GILZ similarly binds to the AP-1 transcription factor through its N-terminal region. The binding of GILZ inhibits AP-1’s binding to its target DNA. Whether GILZ binds to AP-1 subunits in the cytoplasm or nucleus has not been established. Furthermore, GILZ’s modulation of AP-1 has been implicated in the regulation of IL-5 production in T cells by glucocorticoids, through the CLE0 transcription site, which is a major target for glucocorticoid inhibition of IL-5 (Arthaningtyas et al., 2005). GILZ is also critical for the regulation of Fas ligand (FasL) expression, via modulation of Egr-2 and Egr-3, which are transcription factors under the regulation of the NFAT/AP-1 complex (Mittelstadt and Ashwell, 2001).
GILZ has also been shown to bind to and interfere with Raf-1 (Ayroldi et al., 2002) and Ras (Ayroldi, 2007) to modulate the extracellular signal-regulated kinase (ERK) signaling pathway. In T-lymphocytes, Ras binds to GILZ within the TSC domain and forms a trimer with Raf binding to the N-terminal of GILZ (Ayroldi et al., 2002) to mediate cell proliferation and survival by reducing the activation of downstream targets ERK, Akt, Cyclin-D1, and Rb (Ayroldi, 2007). Gupta et al. (2007) showed that increased GILZ mRNA expression due to DEX does not lead to any changes in protein expression of Raf-1, ERK, p38, or JNK, but does lead to decreased phosphorylation of Raf-1, ERK, and p38 (but not JNK). Thus GILZ can inhibit AP-1 activation indirectly, via effects on Ras/Raf and MAP kinases, as well as through direct binding to c-Jun and c-Fos.
Finally, GILZ has also been suggested to play a pivotal role in controlling cell survival, via modulation of the expression of apoptotic proteins. Induction of GILZ expression inhibits the expression of Bim, a pro-apoptotic protein, through modulation of FoxO3 signaling, to prevent apoptosis, but had no effect on Bcl-xL, an anti-apoptotic protein (Asselin-Labat et al., 2004). In parallel, knockdown of GILZ increased levels of Bim and accelerated IL-2-deprivation-mediated apoptosis in an IL-2-dependent T-cell line. However, it was shown previously that GILZ over-expression leads to an increase in the spontaneous apoptosis in CD4+CD8+ double positive thymocytes, involving an interaction with Bcl-xL (Delfino et al., 2004), and a recent paper suggests that GILZ enhances Bim expression and inhibits cell survival in an Akt-dependent fashion. Whilst these apparently opposing regulatory effects of GILZ on cell apoptosis have yet to be fully elucidated, these studies suggest the possibility of a duality of action of GILZ in T cell survival and apoptosis, similar to that described for GCs (Zacharchuk et al., 1990). It is likely that differences in cells, context, and conditions impact on the effects of GILZ on apoptosis.
Besides these protein-protein interactions, Shi et al. (2003) reported that GILZ directly binds to CCAAT/enhancer-binding protein (C/EBP) DNA binding sites in the PPARg2 promoter, with consequent inhibition of mesenchymal cell adipogenesis. This observation suggests a potential role of GILZ as a direct transcriptional repressor of gene expression.
Roles of GILZ in inflammatory and autoimmune diseases
Clearly, the reported actions of GILZ suggest it as a key regulator of the immune response. Studies in models of disease, or human pathology, remain limited, but favor a role for GILZ as a key modulator of immune-inflammatory responses. A murine model of colitis was significantly inhibited in mice over-expressing GILZ in T cells (Cannarile et al., 2009). Beaulieu and colleagues investigated the role of endogenous GILZ in RA (Beaulieu et al., 2010). GILZ was potently induced by glucocorticoid in cultured human RA synovial cells in vitro, and in murine arthritis in vivo. GILZ silencing by in vivo siRNA administration resulted in increased severity of the collagen-induced model of RA in mice, and in parallel GILZ over-expression inhibited chemokine and cytokine expression in human synovial cells. These results confirm GILZ as a key endogenous regulatory molecule in chronic inflammation and as a potential therapeutic target in RA. Another study found that GILZ was noticeably absent in granulomas in Crohn’s disease and tuberculosis (Berrebi et al., 2003), which suggests that GILZ is regulated in chronic inflammatory disease, while human asthma patients demonstrated increased GILZ expression in response to glucocorticoid therapy (Kelly et al., 2011).
These studies, together with the evidence of GILZ upregulation by glucocorticoids and its protein binding interactions with key pro-inflammatory signal transduction molecules, strongly suggest that GILZ is a key mediator of the anti-inflammatory effects of glucocorticoids. Demonstration of the necessity for GILZ in the actions of glucocorticoids in vivo awaits the development of a GILZ-/- mouse model.
Future Directions and Therapeutic Opportunities
Just as research in immune disease has focused on activating pathways, so has research on solutions to glucocorticoid toxicity focused on the transcriptional pathways that the GC/GR complex directly inhibits. Selective glucocorticoid receptor agonists (SEGRA) have been postulated on the basis of the potential to achieve selective transrepressive actions without activating the expression of GC-induced genes thought to be responsible for glucocorticoid toxicity (Cole and Mollard, 2007; Schacke et al., 2007). These discoveries were made possible by observations using a mutated GR in which DNA-binding, and hence activation of GR-induced genes, could be separated from binding to other transcription factors (Reichardt et al., 2001; Tuckermann et al., 1999). The first SEGRA molecules are now in clinical trials in asthma (http://clinicaltrials.gov/ct2/show/NCT01225549). If the theoretical promise of these agents is delivered, they have the potential to transform the treatment of autoimmune diseases. However, medium to long term clinical studies determining any reduction in glucocorticoid toxicity would be required, and such therapies would still be expected to be sufficiently immunosuppressive to be associated with increased infections.
SEGRA are based on the concept that transcriptional activation of glucocorticoid-induced genes is chiefly relevant to the toxicity of glucocorticoids. As we have noted, however, many important anti-inflammatory molecules, such as GILZ, annexin 1, and MKP-1, are induced by glucocorticoids, and evidence that synthetic glucocorticoids lose their effectiveness in the absence of these molecules has been adduced (Furst et al., 2007; Ralph and Morand, 2008; Yang et al., 2009; 2004; 2006). Perhaps, attention to the molecules that glucocorticoids amplify will permit discovery of the means to develop a surrogate for glucocorticoids’ beneficial impact on immune activation without their toxicity. A GILZ-based therapeutic approach, for example, could potentially offer profound glucocorticoid-like regulatory effects in autoimmune disease. Importantly, the presence of GILZ exerts immune and inflammation modulatory effects in the absence of glucocorticoids. Investigation of the metabolic effects of a GILZ based therapy is required in order to ensure that these undesirable effects of glucocorticoids are not recapitulated. Early results are encouraging in this regard. In mesenchymal stem cells, differentiation towards osteogenic precursors is enhanced by GILZ, whereas silencing of GILZ reduced MSC osteogenic differentiation (Zhang et al., 2008), suggesting that a GILZ therapy might have protective rather than harmful effects on bone. In another study, GILZ expression was associated with osteoblast development, and GILZ silencing increased osteoblast expression of OPG and RANKL in favor of osteoclastogenesis (Lekva et al., 2010), further suggesting that GILZ-based therapy might have a bone-protective effect. Studies of the role of GILZ in glucocorticoid-induced osteoporosis in vivo are eagerly awaited.
GILZ-based therapies could be based on the administration of recombinant protein or NF-κB binding peptides. Srinivasan and Janardhanam (2011) have described a novel NF-κB p65 binding GILZ peptide which exhibited therapeutic potential as a small molecule NF-κB inhibitor in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Alternatively, a gene therapy approach, which has already been successfully used in vivo to suppress arthritis via delivery of the anti-inflammatory cytokine IL-10 (Apparailly et al., 2002), could be applied to GILZ. Inducing GILZ expression other than through the use of glucocorticoids, for example by modifying FoxO3 transcription factor activity, could represent a further means to increase available GILZ protein, as could inhibition of the as-yet unidentified mechanisms of GILZ protein turnover. Finally, structure-function analysis of the molecules with which GILZ interacts in order to achieve its immune modifying effects could reveal targets for synthetic GILZ mimetics. Although considerable work remains, the first proof of concept studies of an in vivo GILZ-based therapeutic approach are under development in the authors’ laboratory (unpublished observations).
In conclusion, glucocorticoids remain among the most widely used drugs in human diseases, and in particular in autoimmune disease. Their effectiveness is increasingly well understood, based on their effects on inflammatory signal transduction, but their use is constrained by toxicity, which also relates to their specific physiological actions. Research into the mechanisms of action of glucocorticoids has the potential to lead to new therapeutics for autoimmune disease, either through devising alternate ligands for the GR, or through devising non-glucocorticoid means to increase the expression of anti-inflammatory proteins induced by the GC/GR complex. Pre-clinical and clinical studies currently underway have the potential to deliver these breakthroughs.
The authors’ work was supported by the National Health and Medical Research Council of Australia and Arthritis Australia.
The authors report no conflicts of interest.
Eric F. Morand, MBBS, Ph.D., FRACP, Professor, Centre for Inflammatory Disease, Southern Clinical School, Monash University Faculty of Medicine Nursing and Health Sciences, Monash Medical Centre, Block E Level 5, 246 Clayton Road, Clayton, Victoria 3168, Australia.
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