The FASEB Journal article fj.14-254755. Published online September 9, 2014.
The FASEB Journal • Review
Targeting glucocorticoid side effects: selective glucocorticoid receptor modulator or glucocorticoidinduced leucine zipper? A perspective Emira Ayroldi,*,1 Antonio Macchiarulo,† and Carlo Riccardi* *Department of Medicine, Section of Pharmacology, and †Department of Chemistry and Drug Technology, University of Perugia, Perugia, Italy Glucocorticoids (GCs) are steroid hormones that are necessary for life and important in health and disease. They regulate crucial homeostatic functions, including metabolism, cell growth, and development. Although GCs are regulated by circadian rhythm, increased production is associated with stress. Synthetic GCs are a valuable resource for anti-inflammatory and immunosuppressive therapy. Natural and synthetic GCs transduce signals mainly through GC receptor (GR) activation. Extensive research has explored the downstream targets of the GR, and optimization of GC therapy has required collaborative efforts. One highly promising approach involves new dissociative GR modulators. Because transrepression and transactivation of GR genes induce beneficial and adverse effects, respectively, this approach favors transrepression. Another approach involves the use of GC-dependent genes to generate proteins to mediate therapeutic GC effects. In a third approach, drug discovery is used to identify agents that selectively target GR isoforms to obtain differential gene transcription and effects. In this review, we focus on mechanisms of GR function compatible with the use of dissociative drugs. We highlight GC-induced leucine zipper (GILZ), a gene cloned in our laboratory, as a mediator of GC antiinflammatory and immunosuppressive effects, to out-
ABSTRACT
Abbreviations: AP-1, activator protein 1; CIA, collageninduced arthritis; CpdA, compound A; DEX, dexamethasone; DNBS, dinitrobenzene sulfonic acid; DUSP1, dual specificity phosphatase 1; ERK, extracellular signal-regulated kinase; GC, glucocorticoid; GR, glucocorticoid receptor; GILZ, glucocorticoid-induced leucine zipper; GRE, GC-response element; HDAC, histone deacetylase; HPA, hypothalamic-pituitary-adrenal; Hsp, heat-shock protein; IKK, IB kinase; IB, inhibitor of NF-B; KO, knockout; L-GILZ, long GILZ; LZ, leucine zipper; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; miRNA, microRNA; MKP-1, mitogen-activated protein kinase phosphatase-1; MSC, mesenchymal stem cell; NF-B, nuclear factor-B; PEPCK, phosphoenolpyruvate carboxykinase; PER, proline/glutamate rich; PI3K, phosphatidylinositol-3-kinase; PMA, phorbol 12myristate 13-acetate; RA, rheumatoid arthritis; RBD, Rasbinding domain; RUNX2, runt-related transcription factor 2; SGRM, selective GR modulator; TAT, transactivator of transcription; TG, transgenic; TNF-␣, tumor necrosis factor ␣; TSC22, TGF-–stimulated clone 22 0892-6638/14/0028-0001 © FASEB
line our perspective on the future of GC therapy.— Ayroldi, E., Macchiarulo, A., Riccardi, C. Targeting glucocorticoid side effects: selective glucocorticoid receptor modulator or glucocorticoid-induced leucine zipper? A perspective. FASEB J. 28, 000 – 000 (2014). www.fasebj.org Key Words: GR activation 䡠 transrepression hypothesis 䡠 inflammatory gene regulation Glucocorticoids (GCs) are produced and secreted by the adrenal cortex. GC secretion is controlled by release of adrenocorticotropic hormone (ACTH) from the pituitary, which, in turn, is stimulated by hypothalamic corticotropin releasing hormone (CRH). The hypothalamic-pituitary-adrenal (HPA) axis is controlled by a negative-feedback loop that guarantees normal levels of GCs in the circulation. Multiple physical and psychological stresses stimulate the hypothalamus and induce augmented secretion of GCs (1). Plasma concentrations of cortisol, the main natural glucocorticoid, follow a circadian rhythm, ranging between 16 g/100 ml (8 AM) and 4 g/100 ml (4 PM). The GCs have numerous physiological effects and influence the function of most cells in the body (2). Cortisol exerts a wide range of biological effects, including regulation of the intermediate metabolism, cardiovascular function, and growth, and it controls the physiological development of the immune system (3). Endogenous GCs play a role in the physiological control of inflammatory and immune responses (4 – 6). For example, abrogation of endogenous glucocorticoids by adrenalectomy leads to a worsening of symptoms in rat adjuvant arthritis and immune-mediated glomerulonephritis (7, 8). At pharmacological doses, GCs have anti-inflammatory and immunosuppressive activities that involve nearly all arms of the inflammatory response (9). The therapeutic effects of synthetic GCs were first discov1 Correspondence: Department of Medicine, Section of Pharmacology, University of Perugia, Piazza Gambuli, S. Andrea delle Fratte, 06132 Perugia, Italy. E-mail:
[email protected] doi: 10.1096/fj.14-254755
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ered in 1950 in rheumatoid arthritis (RA; ref. 10) and have been studied through the present day, through both basic and clinical research. Today, 50% of patients with RA are treated with GCs, and GC therapy is used to treat many other diseases, such as systemic or organspecific autoimmune disorders (lupus, sarcoidosis, and myasthenia gravis); chronic inflammation of bone, liver, lung, thyroid, eyes, and skin; organ transplants; most allergic reactions; asthma and chronic obstructive pulmonary disease; neurological and hematological disorders; and inflammatory bowel diseases (11, 12). Most physiological and pharmacological effects of natural and synthetic GCs involve activation of the glucocorticoid receptor (GR). The GR is a transcription factor belonging to the nuclear receptor superfamily that, in its inactive form, is found in the cytoplasm as a complex with molecular chaperones, such as heatshock protein (Hsp)90, Hsp70, FKBP4, and the small phosphoprotein p23 (13). The GR, once activated by hormone binding, homodimerizes, migrates to the nucleus, and binds to specific palindromic DNA sequences, the GC-response elements (GREs), in the promoters of target genes, thus modulating protein synthesis, either positively or negatively (14, 15). The binding of GR homodimers to simple GREs (sequences containing 2 hexameric half-sites separated by 3 base pairs) induces histone acetylation, allows chromatin remodeling and RNA polymerase II association, by recruitment of coactivator molecules (i.e., cyclic AMPresponsive element– binding protein) with intrinsic acetyltransferase activity, and results in transactivation of GC target genes. However, on binding of homodimeric GR to the less well-characterized negative GRE, either by displacement of coactivator complexes or recruitment of corepressors and histone deacetylases (HDACs), the local chromatin structure closes, and the transcription of GR-responsive genes is inhibited (Fig. 1). Furthermore, homodimeric (and monomeric) GR induces or inhibits target gene transcription by tethering—that is, by binding to transcription factors that are in turn linked to their own consensus sequences in the promoters of target genes (Fig. 1). For instance, activated GR in the nucleus may induce recruitment of HDAC2 to an activated inflammatory gene complex (transcription factor/DNA). HDAC2 reverses histone acetylation induced by the transcription factor, resulting in inhibition of chromatin remodeling and suppression of gene transcription. Finally, homodimeric GR modulates gene expression by binding both a GRE and a transcription factor located on an adjacent consensus site (the so-called composite GRE; refs. 16 –18 and Fig. 1). In contrast, monomeric GRs mediate GC effects indirectly, mainly through protein–protein interactions with other transcription factors, cofactors, regulators, and signaling proteins. The GR has, in fact, been copurified with signal transducers and activators of transcription (STATs), nuclear factor-B (NF-B), activator protein 1 (AP-1), 14-3-3 proteins, and Raf-1 proteins; all of these interactions led to inhibition of target 2
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gene expression (4, 19 –21). Many of these transcription factors are involved in the transcription of proinflammatory molecules (Fig. 1). Furthermore, a decrease in anti-inflammatory gene expression occurs when the GR binds a GRE half-site on tristetraprolin, an mRNA-destabilizing gene (22). A new and interesting viewpoint is that described by Revollo et al. (23), who suggested that hairy and enhancer of split-1, a key regulator of development and organogenesis, acts as a master repressor of GR genomic signaling, in that its repression is essential for proper glucocorticoid signaling.
IS THERE ONLY 1 GR? Although the GR is encoded by a single gene (composed of 9 exons in humans), recent studies have demonstrated the presence of multiple GR proteins, derived by alternative splicing, an alternative translation mechanism, and posttranslational modifications. The GR gene produces two receptor isoforms, GR␣ and GR, by alternative splicing of exon 9. Furthermore, GR␥ alternative splicing has been recently described (14, 18, 24). GR␣ is the most highly expressed and extensively studied of the isoforms and mediates most effects of GCs. GR␣ exhibits several functional domains, through which the GR homodimerizes, binds to DNA, and interacts with hormones (25). Furthermore, multiple start codons in GR␣, GR, and GR␥ mRNA may allow the generation of multiple GR translation isoforms; several, particularly for GR␣ (e.g., GR␣-A, GR␣-B, and GR␣-C1), have been identified and investigated (26). In addition, each of these isoforms may undergo different degrees of phosphorylation, acetylation, ubiquitination, and sumoylation, which further modify the functional properties of the GR (18). As expected, these receptor proteins are differentially expressed in different tissues and cells, thus conditioning the GC-induced biological responses. GR, for example, is constitutively expressed in the nucleus and does not bind GCs or activate GC-induced transcription, but antagonizes the activity of GR␣. This last activity may contribute to GC resistance. In fact, increased GR expression has been found in many inflammatory and autoimmune diseases that are insensitive to GCs, although not all of the molecular mechanisms involved in GR function are fully understood. It is known that GR functions in a dominant-negative manner when coexpressed with GR␣, but this is not the only mechanism that can explain its action. GR, in fact, activates mifepristone-induced transcription, and results of microarray analyses suggest the ability of GR to modulate gene expression, independent of GR␣ antagonism (18, 27). Furthermore, recruitment of HDAC1 by GR has been associated with the repression of IL-5 and IL-13 gene expression (28). Hence, transcriptional, translational, and posttranslational mechanisms produce GR isoforms that have the potential to homo- and heterodimerize and that are
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Figure 1. Transrepression theory. Classic GCs activate the GR and induce transrepression of inflammatory transcription factors, such as NF-B and transactivation of anti-inflammatory proteins, such as GILZ or MKP-1, and thus, antiinflammatory effects, but also induce transactivation of genes encoding enzymes involved in metabolism, such as PEPCK, and thus, physiological metabolic effects or side effects at pharmacological doses of GCs. In contrast, SGRMs induce GR conformational changes, resulting in transrepression of inflammatory transcription factors only. Transactivation is mainly achieved by binding of GR dimers to GREs. Transrepression is mostly mediated by direct binding of GR dimer to negative GRE (nGRE), monomeric GR sequestration of transcription factor (TF) in the cytoplasm/nucleus, or monomeric GR interference with activity of a TF (tethering or composite GRE). SGRM-induced expression of GILZ and MKP-1 may be due to GR dimeric activation of a GRE or monomeric activation of a TF (tethering or composite GRE) or other indirect, unknown mechanism.
differentially distributed in diverse tissues and cells, explaining, at least in part, the multiple physiological actions of GCs. In addition, different affinities of natural or synthetic GCs for the various GR isoforms may account for the differential pharmacological responses to GCs, including hypo- or hyperresponsivity. PREVENTION OF GLUCOCORTICOID SIDE EFFECTS
Long-term therapy with pharmacological doses of GCs is almost always complicated by physiological side effects that, if inappropriately increased, could lead to several adverse effects, including iatrogenic Cushing syndrome. There is a close correlation between the development of side effects and dose and timing of GC 3
administration. The most significant and insidious side effect is osteoporosis, but variously associated fat redistribution, hypertension, atrophy of skin and muscle, cataract, glaucoma, mood disorders, increased susceptibility to infections, diabetes mellitus, and suppression of the HPA axis may also occur.
GC SIGNALING Almost all effects of GC, therapeutic and undesirable, are mediated by GR activity. According to the most widely accepted theory, the anti-inflammatory activity of GCs is due to the interaction of monomeric GR with transcription factors that control the expression of inflammatory factors, resulting in the inhibition of gene expression (GR transrepression activity). In contrast, the activation of gene transcription by GR dimers accounts for both physiological metabolic effects (gluconeogenesis and amino acid catabolism) and side effects of pharmacological doses of GC (GR transactivation activity) (Fig. 1 and refs. 29 –32). For example, GC-dependent up-regulation of the tyrosine aminotransferase, glucose-6-phosphatase, and phosphoenolpyruvate carboxykinase (PEPCK) genes in liver has been associated with anomalies in carbohydrate metabolism (33). Moreover, GC-inducible gene (e.g., ubiquitin ligase muscle-specific RING finger protein 1 and myostatin)-knockout (KO) mice treated with GCs do not develop muscular atrophy (31). Nevertheless, transrepression may also be mediated by GR homodimers. In some experimental diseases induced in GRdim/dim mice, which have reduced GR dimerization as the result of a mutation in the dimerization interface (D loop), GR homodimer-dependent transcription appears to be necessary for effective GC therapy, whereas it is dispensable for other experimental diseases (31, 34). Furthermore, based on this mouse model, dimerization seems to be needed for certain side effects, such as hyperglycemia, but dispensable for skeletal muscle atrophy and osteoporosis (35). This model, although questionable, since GRdim/dim mice express normal mRNA levels of the strictly GR-dependent phenylethanolamine-N-methyltransferase (36), further suggests that the schematic separation of GR dimer-dependent transactivation activity as the cause of side effects and GR monomer as the mediator of transrepression of anti-inflammatory effects, neither explains the conflicting results reached with mice nor accounts for the anti-inflammatory effects induced by proteins transactivated by GR, such as annexin-1 (37), mitogen-activated protein (MAP) kinase phosphatase-1/dual-specificity phosphatase-1 (MKP-1/DUSP1; ref. 38), and GC-induced leucine zipper (GILZ; refs. 39, 40 and Fig. 1). In addition, although MKP-1 has been associated with the anti-inflammatory and immunosuppressive activities of GCs, a recent observation suggests that this phosphatase may contribute to bone damage in response to prolonged GC therapy (41). On the other hand, posttranscriptional or pleiotropic nongenomic mechanisms contrib4
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ute to the anti-inflammatory effects of GCs (42), reinforcing the conclusion that any schematic division of GC effects based on underlying molecular mechanisms may appear forced. On the contrary, these observations suggest that multiple complex mechanisms—not all known and often involving differentiated functions of different cell types— contribute to both the anti-inflammatory/immunosuppressive and the side effects of GCs. Despite this, many research efforts have focused on understanding the molecular mechanisms underlying GC function, with the goal of designing a drug capable of preserving the anti-inflammatory effects without the burden of side effects. Much progress has been made in this direction over the years. Using a schematic approach once again, it can be said that two major scientific approaches have been applied to the pursuit of this goal. On one side, researchers have sought pharmacologically active molecules able to selectively activate the GR, causing only anti-inflammatory activity without collateral responses. On the other side, research has focused on the possibility that a protein, induced by GCs and responsible for their anti-inflammatory effects, could be used as a drug or drug target. Whereas the second approach is still largely theoretical, the first has produced several compounds, some of which are in phase I clinical trials. In this review, we follow the same approach, focusing on the novel pharmacological strategies to dissociate anti-inflammatory from metabolic side effects and on the anti-inflammatory and immunosuppressive effects of GILZ, a protein identified in our laboratory, and speculate on its possible use as a drug.
SELECTIVE GLUCOCORTICOID RECEPTOR MODULATORS (SGRMs) The GR, once activated, may target multiple proinflammatory transcription factors, inhibiting their activity via protein-to-protein interaction (impeding DNA binding and activity of transcription factors, such as NF-B and AP-1) or by an indirect mechanism involving recruitment of a corepressor. As noted above, transactivation of several genes accounts for side effects, whereas transrepression explains most anti-inflammatory and immunosuppressive effects of GCs. Thus, a drug that can dissociate transrepression from transactivation and preferentially drive transrepression should uphold or even enhance the anti-inflammatory effects and induce a reduction in side effects in comparison to the classic GCs. Starting from this simple idea (oversimplified, perhaps), numerous researchers have designed molecules capable of binding and activating the GR in a manner that may result in transrepression only. Synthetic GCs, with prednisone/prednisolone, dexamethasone (DEX), and budesonide being the most commonly prescribed, have steroid chemical structures resembling the natural glucocorticoids, from which they differ in pharmacody-
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namic and pharmacokinetic properties. Like natural steroids, they work by activating all GR transactivation and transrepression activities. The “story” of SGRMs, far from being over, is based on the fascinating transrepression hypothesis, which provides the grounds for the sought-after uncoupling of therapeutic and adverse GC effects (refs. 30, 43, 44 and Fig. 1). One of the first dissociated GR ligands, in the late 1990s, RU24858 has been shown to induce AP-1 inhibition, but little or no transactivation (45). However, dissociation was based on a weak experimental model (AP-1 and GRE reporter constructs), and later, this compound was shown to induce both transactivation of anti-inflammatory protein and side effects, such as a pattern of osteoporosis similar to that observed with the classic budesonide treatment (46). Subsequently, researchers at Abbott Laboratories (North Chicago, IL, USA) first realized that a steroidal structure, a GR agonist or antagonist, was less likely to dissociate GC side effects, and reported the prototypic nonsteroidal Abbott-Ligand (AL)-438 (47). Studies since then, which were also performed within the pharmaceutical industry, have generated many dissociative compounds effective in in vitro and in vivo models of disease, and some, such as ZK-245186, are currently in clinical trials for treatment of eye and dermatological diseases (48). Analysis of the SGRM literature highlights some essential points: the separation of transrepression (anti-inflammatory effects) and transactivation (side effects) is not always possible and may not be the output of a single pharmacologically active molecule; and the molecular mechanisms of transrepression are not fully understood, so that it is hard to identify clear criteria to define a unique theory of transrepression. As noted above, transrepression (Fig. 1) can mean GR-mediated inhibition of proinflammatory transcription factors that prevents their binding to the promoters of target genes. It also means that tethering of the GR to DNA-bound transcription factors induces conformational change in the receptor that results in recruitment of corepressors or displacement of the activator (25). Recruitment of HDACs by corepressors and a consequent decrease in histone acetylation have been suggested to underlie GR-induced inhibition of transcription (18, 30, 35). However, putative corepressors, such as nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRTs), have not been plausibly implicated in transrepression (e.g., of AP-1 or NF-B), and there is no convincing evidence that HDACs are recruited to the promoters of target genes, mechanisms that generally account for transrepression. In addition, transrepression may involve many other mechanisms that have not yet been discovered. These challenges to fully defining the mechanisms underlying transrepression, together with the current paucity of experimental models (which are primarily in vitro models), compared to the enormous complexity of the molecular mechanisms by which GR functions, have led to a delay in achieving a sufficient knowledge PREVENTION OF GLUCOCORTICOID SIDE EFFECTS
of SGRMs to permit their clinical use. Nevertheless, as more sophisticated approaches to drug design have allowed manipulation of the GR agonist–antagonist equilibrium (49, 50), many new compounds that are more theoretically “dissociated” have been produced that indeed show improved transrepression–transactivation balance in vitro (ref. 51 and Fig. 1), but few have shown a dissociating profile in in vivo animal models. The experimentation with SGRMs in vitro has been based on the analysis of repression or activation of markers characteristic of GC action (ref. 46; i.e., inhibition of IL-6 and IL-8 expression, as a measure of the anti-inflammatory activity of the SGRM, and increased expression of PEPCK, as a marker of induction of side effects; refs. 33, 52). Later, the same or new drugs have been tested in in vivo models of inflammation, to confirm anti-inflammatory efficacy and side effects. However, even though many of these dissociative compounds showed promise in preclinical studies, most failed to reach clinical trials (31). For example, in in vitro experiments, AL-438 showed transrepressed production of tumor necrosis factor ␣ (TNF-␣), IL-1␣, IL-6, and E-selectin and less potent activity in triggering GR-mediated aromatase transcription (47, 53). Compared to DEX or prednisolone, AL-438 showed no reduction in cell proliferation or proteoglycan synthesis in the murine chondrogenic cell line ATDC5 and did not decrease levels of osteocalcin mRNA (a marker of GC-mediated osteoporosis) in the MG63 cell line, suggesting a reduced side-effect profile in chondrocytes (54). In in vivo rat models of acute or chronic inflammation, AL-438 showed therapeutic efficacy similar to prednisolone, but, according to some reports, it also had minor unwanted effects on glycemia and bone (53). LGD-5552 (Ligand Pharmaceuticals, La Jolla, CA, USA) binds the GR with high affinity, but shows antagonistic activity toward the mineralocorticoid receptor (55). In collagen-induced arthritis (CIA) in mice, orally administered LGD-5552 shows full efficacy and prednisolone-like potency, with less effect on certain side effects, such as percentage of body fat and bone formation (55). However, its antagonistic activity toward the mineralocorticoid receptor may induce deregulation of sodium and potassium homeostasis and blood pressure. ZK-216348 and ZK-245186 have been characterized by Bayer Schering Pharma AG (Berlin-Wedding, Germany) and presented as compounds capable of dissociating transrepression from transactivation in vivo. ZK-216348 reduced croton oil-induced ear inflammation in mice and had an attenuated side effects profile, compared to prednisolone (i.e., absence of hyperglycemia and reduced skin atrophy when administered systemically; ref. 43). Topically applied ZK-245186 displays anti-inflammatory activities in animal models of dermatitis and lower potential for cutaneous and systemic side effects compared with the classic GCs (48). It is currently in phase II clinical trials for atopic dermatitis. 5
Compound A (CpdA) has been described as the most “dissociated” SGRM, because it does not induce GR dimerization or transcriptional activation, but exerts selective transrepressive effects on the NF-B pathway. CpdA is able to enhance specific GR-dependent expression in certain cases (56, 57). These are not classic GRE-based transactivation events. It is also the only compound of its kind derived from a natural source, a Namibian desert shrub (58, 59). In mouse models of multiple sclerosis and arthritis, CpdA ameliorated clinical signs of disease without suppressing the HPA axis and blood glucose homeostasis (60, 61). Furthermore, unlike DEX, CpdA does not affect osteoblast differentiation in vitro or in vivo. However, some studies suggest that not all CpdA effects are mediated by the GR alone. CpdA inhibits androgen receptor function, inhibiting growth and survival of prostate cancer cells. The most serious problem with CpdA remains its chemical stability, which can lead to toxic metabolites that induce apoptosis (51). Org 214007-0 binds the GR with an affinity similar to that of prednisolone and shows similar potency. Although Org 214007-0 recruits a coactivator peptide, it acts as a partial agonist of the GR, albeit with lower efficacy. Org 214007-0 is as effective as prednisolone in a mouse model of acute and chronic inflammation, reducing expression of disease-related proinflammatory genes, such as the toll-like receptor 4 ligand S100A8/S100A9 and monocyte chemotactic protein-2. However, Org 214007-0 has shown less of an effect on glucose metabolism, having no effect on fasting blood glucose levels after 28 d of therapy. Moreover, it shows an increased ratio of repression to induction of genes expressed in muscle tissue derived from arthritic mice (62). A number of other compounds claiming dissociative effects have been described in the literature. However, analysis of these compounds is beyond the scope of this review, which is intended only to place SGRMs in the broader context of adverse side effects of GCs and to emphasize a point of view shared by many researchers. The transrepression hypothesis was very helpful when it was first introduced, and it led to the synthesis of new molecules, opening the way to future drug discovery, but it is no longer a suitable explanation for the molecular mechanisms of the drugs that it is responsible for generating. The dissociation on which the synthesis of many SGRMs is based has not been clearly demonstrated and, indeed, some of these compounds demonstrate both transrepressive and transactivating activities, similar to classic GCs. For example, it was demonstrated that two ZK compounds (compound 1, closely related to ZK-216348) and mapracorat (ZK245186, Bayer Schering Pharma), up-regulate MKP-1 in numerous cell types, and this response correlated with their ability to suppress cyclooxygenase 2 (COX-2) expression (63, 64). Furthermore, several anti-inflammatory effects of SGRMs were inhibited or reduced in DUSP1⫺/⫺ macrophages, suggesting that transactivation of anti-inflammatory gene expression is a mechanism underlying therapeutic effects of SGRMs (63). In 6
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fact, RU24858 (65) and Org 214007-0 (62) induce the expression of GILZ, although at a much lower level than that of classic GCs, whereas the dissociated character of CpdA is confirmed by the lack of induction of GILZ mRNA (66). In addition, it has recently been demonstrated that one of the main side effects of long-term GC therapy, osteoporosis, may also be due to GR’s inhibition of runt-related transcription factor 2 (RUNX2), a crucial regulator of osteoblast differentiation and bone formation. The molecular mechanism of this inhibition appears to be the interaction of the GR with RUNX2—namely, a protein-to-protein interaction (67), the mechanism that usually accounts for the anti-inflammatory effects of GCs. Based on this finding, a dissociative compound would not prevent the onset of osteoporosis. Furthermore, the contribution of GR dimerization to GC anti-inflammatory effects is still controversial. A large body of evidence suggests that GR dimers are necessary for full activation of the anti-inflammatory cascade, but the repressive effect of the GR on AP-1 and NF-B is still observed in GRdim/dim mice, again demonstrating the difficulty in pharmacological dissociation of transactivation and transrepression to obtain an anti-inflammatory drug with limited or no side effects (31). Notably, little is known about the possible nongenomic effects of SGRMs. Therefore, progress toward the goal of selective GR modulation should be based on scientific doubt rather than conviction (i.e., that the pharmacological effects of SGRMs may not be fully attributable to their ability to dissociate transactivation from transrepression, (68), but to as-yet-unidentified molecular mechanisms). For example, little if any information is available on the possibility of selective pharmacological targeting of different GR isoforms. It is likely that SGRMs have a pattern of gene activation different from that of the classic GCs; however, despite the SGRM-induced expression of some classic GC-dependent anti-inflammatory genes, such as GILZ and MKP-1 (refs. 62, 63, 65 and Fig. 1), their possible contribution to SGRM anti-inflammatory activity, has not been sufficiently investigated. An innovative line of research has investigated the effects of GCs on microRNAs (miRNAs), small noncoding RNA molecules that inhibit gene expression at the posttranscriptional level. Activation of the GR by GCs may induce or repress specific miRNAs, as demonstrated by studies done primarily on leukemia cells (69). Moreover, miRNAs are also involved in the regulation of GR expression, suggesting the possibility that miRNAs modulate the GC response (70). A promising topic for investigation would be to compare changes in miRNA expression induced by SGRMs and classic GCs. The result could increase the understanding of SGRM activity.
GILZ An old, but ever timely, challenge to separating therapeutic from adverse effects of GC therapy is to use a protein induced by GCs as a drug that may mediate their anti-inflammatory, but not their side, effects. Such
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an approach, still in the preclinical stage and largely theoretical, should resolve the issue of GC toxicity. In this review, we focus primarily on one of the candidate proteins in this category, GILZ, which was cloned and partially characterized in our laboratory (39, 40). We present and discuss the most recent findings supporting a role for GILZ as a mediator of the anti-inflammatory activity of GCs. Structure–function relationships of GILZ GILZ was first isolated as a DEX-responsive gene from a thymus subtraction library (39). It is ubiquitously expressed, primarily under the control of steroid hormones, and regulates cell cycle, apoptosis, and differentiation (71). Mouse GILZ encodes a 137-aa leucine zipper (LZ) and shares ⬎90% sequence similarity with human 135-aa GILZ (38). GILZ was initially proposed to function as a transcription factor. It does not contain a canonical DNA-binding domain, but it is involved in the modulation of the same signaling pathways related to immune responses and inflammation that are implicated in mediating the anti-inflammatory and immunosuppressive activities of GCs. Structurally, GILZ is composed of 3 domains, the N-terminal domain (residues 1–75), a central LZ domain (residues 76 –97), and the C-terminal domain, which is endowed with a proline/glutamate-rich (PER) region (residues 98 –137; Fig. 2). The N-terminal domain contains the TGF-–stimulated clone 22 (TSC22) family signature (residues 58 –74). Members of the
TSC22 family are LZ proteins that can homo- or heterodimerize by the formation of parallel coiled-coil ␣-helices, repressing transcriptional activity (72). Based on these observations, subsequent homology modeling studies assigned a folding structure to GILZ using the crystal structure of one member of the TSC22 family, the delta sleep-inducing peptide (DIP), as the template (73). Docking studies of GILZ and Raf-1 suggested that the GILZ N-terminal domain binds the Ras-binding domain (RBD) of Raf. This hypothesis was confirmed by GST pull-down experiments, using a GST-Raf RBD fusion protein as bait and the full-length or deletion mutants of GILZ as prey. In particular, the truncated form of GILZ, lacking the NH2-terminal region, lost the ability to interact with Raf-1 (73). The N-terminal domain of GILZ is also involved in the interaction with Ras. In this case, mutagenesis experiments demonstrated that the binding cleft for Ras spans residues 60 –75 of the TSC22 family signature, with the binding cleft for Raf-1 being located in a downstream, nonoverlapping region of the N-terminal domain between residues 1 and 60 (74). The presence of two distinct binding regions on the N-terminal domain of GILZ with specificities for Ras and Raf-1 was further supported by the experimental observation that these three proteins may form a ternary complex in COS-7 cells as a function of the Ras activation state. In particular, the activated form of Ras is able to stabilize a conformational state of the protein that has high affinity for Raf-1 and GILZ, shifting the protein–protein binding equilibrium toward the formation of strong
Figure 2. Structure of GILZ and structure–function relationships. GILZ domains that are essential for function and binding to the indicated signaling proteins are depicted.
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Ras/Raf-1 and Ras/GILZ complexes over a weak GILZ/ Raf-1 complex. Conversely, the inactivated form of Ras adopts a distinct conformational state with low affinity for Raf-1, thereby favoring the formation of a GILZ/ Raf-1 complex, as well as the assembly of a trimeric GILZ/Raf-1/Ras complex (74). The central LZ domain of GILZ bears a heptad repeat of leucine residues in position d, according to the nomenclature of leucine zipper motifs (75). This domain is important for GILZ homodimerization. In fact, mutagenesis of leucine residues 76, 83, 90, and 97, as well as asparagine 87 and polar-charged residues within this domain, suggests that all of these residues play pivotal roles in mediating the GILZ homodimerization process. GILZ homodimerization plays a role in binding and inhibition of NF-B transcriptional activity (76). The C-terminal domain of GILZ contains 5 PxxP motifs (polyproline helices) with a hypothetical Src homology (SH)-3-binding function, located in the PER region (77). This region is important for GILZ interaction with NF-B and inhibition of its transcriptional activity, as demonstrated by mutagenesis experiments (76) on truncated forms of GILZ. A combination of mutagenesis experiments and molecular dynamic simulations was used to explore the role of the PER region in NF-B binding. Whereas, on the one hand, it was found that the PxxP motif located at residues 121–123 is essential for the inhibitory effect of GILZ on NF-B, on the other hand, it was proposed that conformational rearrangements of the PER region are also critical, with an extended conformation of the PER region being required for GILZ functions. Therefore, the interaction of GILZ with the NF-B p65 subunit requires GILZ homodimerization and the C-terminal PER region (76). Inflammation, NF-B, GCs, and GILZ The transcription factor NF-B is expressed in almost all cell types and regulates several target genes with multiple functions. The regulation of this transcription factor, which is involved in cellular homeostasis in health and disease, is complex. NF-B is composed of 5 subunits (p50, p52, RelA, RelB, and c-Rel) that differentially associate to form homo- or heterodimers. These are activated by the upstream IB kinase (IKK) complex, which consists of two catalytically active kinases (IKK␣/IKK1 and IKK/IKK2) and one regulatory component (IKK␥/NEMO). The dimers are inhibited by a family of inhibitors of NF-B (IBs; ref. 78). In addition, the NF-B pathway is modulated by other, overlapping signaling pathways triggered by common ligands. In most quiescent cells, NF-B is inactive because it is bound to inhibitory IBs that associate with its DNAbinding domains, mask its nuclear localization sequence, and trap it in the cytosol. The p50 and p52 subunits do not contain a transactivation domain and may act as transcriptional repressors when bound as dimers to NF-B recognition elements of gene promoters; however, when they are bound to a subunit that 8
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contains a transactivation domain, such as p65 or RelB, they became transcriptional activators. Different combinations of NF-B subunits in homo- and heterodimers result in differential induction of target gene activation, which adds complexity and functional diversification. Activation of the canonical NF-B pathway implies the activation of IKK in the IKK complex, which can then phosphorylate IB␣, targeting it for subsequent polyubiquitination and proteasomal degradation. NF-B can thus translocate to the nucleus and activate target gene transcription. Noncanonical and atypical NF-B activation have been also described. Whereas a portion of NF-B protein is naturally localized in the nucleus to perform “tonic” functions, different ligands and physiological and pathological environmental stimuli can induce the activation and translocation of cytoplasmic NF-B to the nucleus. In this way, this transcription factor regulates important functions, such as cell cycle, apoptosis, stress response, inflammation, and immunity. NF-B signaling is, in fact, crucial in modulation of inflammatory and immune responses, and even cancer transformation, through the transcriptional regulation of several cytokines and chemokines. Hence, when NF-B is inappropriately activated by proinflammatory or extracellular stress stimuli, it contributes to the regulation of proinflammatory transcription factors, thus, initiating or perpetuating activation of the inflammatory cascade (78). GCs interfere with NF-B signaling pathways, and this is the most important mechanism underlying GC anti-inflammatory effects. The mechanisms and functional interactions regulating GC–NF-B crosstalk, which involves the GR, result most often in inhibition, or potentiation (e.g., when GCs are clinically ineffective), of NF-B activity (79). GC-dependent inhibition of NF-B activity has been ascribed to increased synthesis of I-B␣ (80), which sequesters NF-B in an inactive cytoplasmic form, or to direct interaction of the GR with p65/RelA, with GR–NF-B mutual nuclear exclusion and inhibition of gene transactivation (81, 82). However, many additional mechanisms underlying GC/NF-B mutual antagonism have been suggested; for example, competition for common cofactors, such as cAMP response element– binding protein (CREB)binding protein and steroid receptor coactivator (SRC1); histone modification; chromatin remodeling; and interference with phosphatidylinositol 3-kinase (PI3K) and protein kinase A (PKA) (32). GC/NF-B mutual antagonism is one mechanism through which GCs induce apoptosis in cells of the hematopoietic system, such as the monocytes, macrophages, and T lymphocytes involved in the inflammatory response, and by which they inhibit proinflammatory cytokines (83). GC-induced up-regulation of GILZ, which in turn interferes with the activation of NF-B, is another mechanism by which GCs control this critical transcription factor. GILZ associates with and inhibits NF-B activation. An initial report showed that GILZ interaction with NF-B subunits inhibits NF-B nuclear translocation, DNA binding, and transactivation (84). The
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overexpression model described in that study was further substantiated by experimental evidence that endogenous GILZ physically associates with p65 in murine thymocytes (84). Subsequent experimental evidence has supported these initial observations, and the relationship between anti-inflammatory activity of GILZ and inhibition of NF-B has been demonstrated in several in vitro and in vivo models. In hematopoietic and epithelial cells, an interesting picture has been drawn of the involvement of GILZ, through NF-B modulation, in mediating anti-inflammatory and immunosuppressive effects of GCs. For example, some effects of GCs on inhibition of macrophage activation are mediated by GILZ via inhibition of NF-B. GILZ is constitutively expressed in mouse and human macrophages and human monocytes, and its expression is upregulated in vivo and in vitro by GCs (85). In GILZtransfected THP-1 macrophages, GILZ mimics the effects of GCs and inhibits the costimulatory molecules CD80 and CD86 and production of the chemokines RANTES and MIP-1 (Fig. 3). In addition, with DEX treatment, THP-1 cells up-regulate GILZ, which binds the NF-B p65 subunit (85). This interaction may account for the inhibitory effects of GILZ on macrophage activation. The immunosuppressive activities of GCs mainly affect T lymphocytes, and GILZ behaves, at least in part, like GCs. GILZ overexpression in CD4⫹ T cells of transgenic (TG) mice (86), induces down-regulation of the Th1 and up-regulation of the Th2 response (87)
(Fig. 3). As a consequence, GILZ-TG mice are less susceptible to dinitrobenzene sulfonic acid (DNBS)induced colitis, a Th1-mediated disease. Again, this effect appears to be mediated by GILZ inhibition of NF-B nuclear translocation in CD4⫹ T lymphocytes of the intestinal lamina propria (88). Accordingly, GILZdeficient mice are more susceptible to DNBS-induced colitis as a result of GILZ-induced promotion of regulatory T cells, another mechanism of GC-mediated immunosuppression (89). In addition to the reported functions in cells of the immune system, GILZ has been implicated in regulation of epithelial and endothelial inflammatory responses. Indeed, GILZ upregulation by GCs in the bronchial epithelium (90) contributes to the therapeutic effects of GCs through inhibition of NF-B. DEX treatment of the transformed human airway epithelial cell line BEAS-2B and normal human bronchial epithelial cells results in up-regulation of GILZ, which significantly inhibits NF-B activation induced by IL-1, lipopolysaccharide (LPS), and polyinosinic-polycytidylic acid (91) (Fig. 3). Furthermore, GILZ is downregulated in the bronchial epithelium by inflammatory cytokines (91) and in alveolar macrophages via toll-like receptor activation (92), suggesting the functional necessity of the inflammatory response to down-regulate an anti-inflammatory protein affecting NF-B activation. Silencing of GILZ in A549 human distal lung epithelial cells resulted in secretion of significantly higher amounts of inflammatory cytokines in response
Figure 3. Effect of GILZ on signaling pathways. GILZ, induced by glucocorticoids, directly interacts with NF-B, inhibits NFB-dependent transcription, and mediates anti-inflammatory and immunosuppressive effects in macrophages, T cells, bronchial epithelium, synovial endothelium, and marrow MSCs. GILZ binds Ras and Raf in dimeric or trimeric conformation and diminishes the activation of Ras/ Raf downstream targets, including ERK-1/2 and Akt, leading to inhibition of Ras- and Raf-dependent cell proliferation and Rasinduced transformation. GILZ enhances Akt phosphorylation in ovarian cancer cells, resulting in increased cell proliferation, whereas it decreases Akt phosphorylation in chronic myeloid leukemia, leading to an increase in apoptosis. LGILZ, which binds and inhibits Ras in undifferentiated spermatogonia, contributes to the regulation of spermatogenesis.
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to IL-1 stimulation (93). This is a significant observation in consideration of the pivotal role of NF-B– dependent induction of proinflammatory cytokines in bronchial asthma. GILZ expression has been detected in the synovium of humans with RA and mice with CIA; the severity of CIA is enhanced by GILZ silencing (94). In fact, GILZ expression in synovial endothelial cells of patients with RA modulates inflammatory leukocyte recruitment, again via NF-B. GILZ overexpression inhibits endothelial cell adhesive function through inhibition of TNFstimulated leukocyte rolling, adhesion, and transmigration and through decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6 (Fig. 3 and ref. 95). The mechanism of GILZ inhibition of endothelial cells involves [in addition to MAP kinase (MAPK), which is discussed below] the inhibition of NF-B, which occurs in the absence of an effect on p65 nuclear translocation, suggesting a different mode of GILZ modulation of NF-B activation in endothelial cells (95). Moreover, Yang et al. (96) demonstrated that GILZ inhibits inflammatory cytokine–induced COX-2 expression by blocking the nuclear translocation of NF-B in bone marrow mesenchymal stem cells (MSCs), which have recently been implicated in the pathogenesis and progression of RA (Fig. 3). On the other hand, the role of GILZ as an inhibitor of immune/inflammatory responses in RA was recently confirmed by the demonstration in vivo that the therapeutic potential of MSCs in CIA is related to GILZ-dependent generation of a new population of nonclassic IL-10-producing regulatory Th17 cells (Fig. 3), with significant decreases in the number of Th1 and Th17 cells, subsets that are mainly associated with the development of autoimmune diseases (97). In a coculture model of MSCs isolated from bone marrow of wild-type or GILZ-deficient mice and T cells from wild-type spleen, it has been demonstrated that GILZdependent production of activin A and activation of Smad3/2 participate in MSC-mediated inhibition of Th17 cell development (98). It would be interesting to investigate, in this MSC coculture (and an in vivo injection) model, the effect of GC treatment on the state of activation of NF-B. Overall, these observations support the hypothesis that GILZ is crucial in regulating NF-B activation, thus mediating the anti-inflammatory effects of GC. Inflammation, Ras, extracellular signal-regulated kinase (ERK), MAPK, Akt, GCs, and GILZ MAPKs modulate a variety of physiological cell processes, such as proliferation, apoptosis, and development (99). However, when these proteins are inappropriately activated by proinflammatory or extracellular stress stimuli, they contribute to regulation of proinflammatory transcription factors, thus perpetuating the inflammatory cascade (100, 101). Most anti-inflammatory effects of GCs involve their direct or indirect interference with the MAPK signaling pathway (102, 103). Notably, MAPKs are one of the major families of 10
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kinases involved in GR␣ phosphorylation and thus in modulation of GR transcriptional activity (18). GILZ expression through protein–protein interactions with different components of the MAPK cascade is one means by which GCs exert inhibitory effects on MAPK pathways. The result of the GILZ interaction with the MAPK system is expressed at two levels: GILZ, as a mediator of the anti-inflammatory effects of GCs; and antioncogenic/antiproliferative GILZ activity, which is not always related to GCs, but is closely linked to the formation of GILZ-Ras dimers. GILZ interacts with endogenous Raf-1 in vitro and in vivo. This interaction was initially regarded as the most important mechanism underlying the GILZ-mediated inhibition of both ERK-1/2 phosphorylation and AP-1dependent transcription (73). The finding that GILZ interacts with Ras and that a Ras/GILZ/Raf ternary complex forms that coexists with Ras/GILZ and Raf/ GILZ dimers led to a reinterpretation of the initial observation. GILZ binds Ras primarily when Ras is activated, inducing inhibition of downstream Ras-dependent signaling pathways; phosphorylation of ERKs, Akt/Pkb serine/threonine kinase (Akt), and retinoblastoma protein; and cyclin D1 expression, and this binding is essential for GILZ antiproliferative and antioncogenic activity (74). However, both interactions are functionally active; GILZ interaction with activated Ras results in inhibition of both the Akt and ERK pathways, whereas its interaction with Raf results in inhibition of the ERK pathway (Fig. 3). These interactions have important biological implications for explaining the effects of GCs in the cells of the immune system. For example, Ras is activated in spleen cells treated with phorbol 12-myristate 13-acetate (PMA) and, by activating downstream pathways, induces proliferation and activation of T lymphocytes. Under these conditions, Ras binds endogenous GILZ, which acts as a physiological brake on proliferation (Fig. 3). This finding was suggested by small interfering RNA (siRNA)-mediated GILZ knockdown, which is accompanied by increased PMA-induced T-lymphocyte proliferation. Likewise, GILZ knockdown in activated T lymphocytes completely inhibits DEX-induced antiproliferative effects, suggesting that GILZ underlies the antiproliferative activity of GCs (74). Further, GILZ may contribute to regulation of the GC sensitivity of thymocytes, in which GILZ expression is strongly upregulated by DEX. GILZ has effects similar to those of DEX in thymocytes: it induces thymocyte apoptosis, but then rescues the cells by anti-CD3–induced apoptosis, as established in GILZ TG mice (86). CD4/CD8 double-positive thymocytes are susceptible to GC-induced apoptosis and, to be selected, should be protected from the apoptotic action of GCs by proper stimulation through T-cell receptor (TCR) Ras/MEK/ERK activation. Because GILZ binds to Ras in thymus, it would interesting to investigate whether this interaction represents a regulatory mechanism of thymic selection. It should be noted, however, that GILZ expression may not always be responsible for antiproliferative
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effects. Indeed, it has been reported that, in ovarian cancer, GILZ induces an increase in cell proliferation through augmented expression and activity of Akt (Fig. 3). Although this finding has been demonstrated only in in vitro experiments showing that GILZ, when overexpressed in an ovarian cancer– derived cell line, enhanced Akt phosphorylation and activity and contributed to cell proliferation (104), it may be supported by epidemiological analysis. In fact, GILZ has not been detected in the epithelium of normal ovaries and benign ovarian tumors; however, it has been shown to be expressed in the cytoplasm of malignant ovarian cancer cells. In addition, GILZ and the chemokine Fractalkine/CX (3) CL1, which plays an important role in tumor growth and metastasis, were coexpressed in epithelial ovarian carcinomas, which have high proliferation rates, and expression levels correlated positively with the Ki-67 proliferation index (105). Opposite effects on proliferation may be caused by multiple biological mechanisms. The simplest to consider in the context of this discussion is that, in ovarian cancer, GILZ does not bind Ras, an essential prerequisite as far as we know, for the antiproliferative effects of GILZ, although other mechanisms must be considered and explored. The GILZ/Akt interaction has been described as being crucial in more than one system. For example, in chronic myeloid leukemia, GC-induced GILZ interacts with mammalian target of rapamycin complex 2, inhibits Akt phosphorylation, and activates FoxO3a-mediated transcription of the proapoptotic protein Bim, which has a critical role in cell death mediated by tyrosine kinase inhibitors (Fig. 3). Through this mechanism, GCs may overcome the drug resistance induced by treatment with tyrosine kinase inhibitor (106). Furthermore, in multiple myeloma, for which GCs are commonly used as effective therapeutics, GILZ is involved in GC-induced cell death (107). In this system, inhibition of the PI3K/Akt pathway enhances GILZ expression, whereas MAPK activation has been shown to be necessary for GILZ expression mediated by bacterial toxins (108), suggesting reciprocal regulation of GILZ and these signaling pathways. The importance of GILZ in the negative modulation of Ras signaling is further supported by the recent model of GILZ-KO mice, which display reductions in testis size and weight due to the loss of germ cell lineages, resulting in male sterility (109, 110). Failure of spermatogenesis is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and hyperactivity of the Ras signaling pathway, as indicated by increases in ERK and Akt phosphorylation. In GILZ-KO mice, this effect is associated with the loss of long GILZ (L-GILZ), a new transcriptional variant that shares several domains with GILZ, including the TSC22 family signature, LZ, and C-terminal regions, but differs in the N-terminal domain, which is encoded by an upstream alternative exon. L-GILZ, unlike GILZ, is highly expressed in undifferentiated spermatogonia (109). Therefore, LGILZ binding to and inhibition of Ras contributes to PREVENTION OF GLUCOCORTICOID SIDE EFFECTS
regulation of spermatogenesis (Fig. 3). This last finding confirms the inhibitory role of the GILZ system in Ras signaling and suggests a new non-GC–related basic function for this protein. Despite the broad spectrum of GILZ effects and interactions, which do not always involve effects of GCs, the more interesting question is, based on our current knowledge, could GILZ mediate the anti-inflammatory activity of GCs without the negative side effects? There are conflicting opinions in this field, because it has been suggested that GILZ may, at least in the case of asthmatic syndrome, mediate both beneficial and detrimental effects. GCs are included in multidrug treatment of asthma, where they exert antiinflammatory effects, but hinder repair of the bronchial epithelium. GILZ is upregulated by inhaled budesonide in allergen-challenged patients with asthma and beclomethasone-treated lung macrophages (90). GILZ-mediated inhibition of NF-B reduces the expression of proinflammatory lymphokines (91), but because GILZ also inhibits the MAPK-ERK signaling pathway, it contributes to the inhibition of airway epithelial cell repair (111). In fact, silencing GILZ attenuates the inhibitory effect of DEX on proliferation and migration of 9HTE SV40-immortalized human tracheal epithelial cells. Because proliferation and migration of cells near the site of tissue damage are the primary mechanisms of repair, Liu and colleagues (111) suggested that GILZ inhibition of these processes contributes to maintenance of persistent damage in airway epithelial cells. In contrast to this last observation (supported only by in vitro experiments in a cell line), studies to date suggest that GILZ mediates the beneficial rather than the harmful effects of GCs. For example, long-term treatment with GCs induces osteoporosis, but GILZ promotes proosteogenic effects. In MSCs, differentiation toward osteogenic precursors is enhanced by GILZ expression and reduced by GILZ silencing (112), and GILZ antagonizes the inhibitory effect of TNF-␣ on osteogenic differentiation of MSCs via inhibition of TNF-␣-induced ERK activation (113). TNF-␣ plays a pivotal role in the inflammatory response in autoimmune diseases, where osteoporosis is often present; in fact, TG mice overexpressing TNF-␣ are severely osteoporotic (114). GILZ has been shown to be a crucial, endogenous anti-inflammatory mediator in arthritis. By inhibiting ERK activation, GILZ counteracts both proinflammatory TNF-␣–mediated effects in the synovium of mice with CIA (94) and TNF-␣–induced inhibition of osteogenic transcription factors in bone (113), thus mediating the anti-inflammatory effects of GCs without inducing side effects, such as osteoporosis. Overall, these observations suggest that GILZ is critical in regulation of the Ras/MAPK inflammatory pathways and, again, as a mediator of GC anti-inflammatory effects. GILZ as a drug? The biggest challenge for researchers dealing with the pharmacology of GCs has always been to find a GCinduced molecule capable of inducing anti-inflamma11
tory effects without the heavy burden of detrimental GC effects (i.e., a “pure” anti-inflammatory). Might GILZ be such a candidate? Although a comprehensive analysis that can establish or exclude the effects of GILZ on metabolism is still lacking, some considerations encourage us to be optimistic. The GST–transactivator of transcription (TAT)– GILZ (TAT-GILZ) fusion protein was constructed by inserting GILZ cDNA in an expression vector containing the transactivator of transcription (TAT) peptide, which was then overexpressed in Escherichia coli. The purified TAT-GILZ fusion protein can transduce mammalian cells (86). A TAT-GILZ fusion protein injected intravenously in mice behaves like a high dose of DEX, protecting mice against the development of DNBSinduced colitis, a form of Th1-mediated experimental colitis, suggesting that the GILZ fusion protein has bioavailability and pharmacokinetic characteristics suitable for distribution to and pharmacological activity in target organs. Indeed, GILZ-TG or wild-type mice treated with the TAT-GILZ fusion protein shows attenuated inflammation (diminished intestinal tissue damage) and immune dysfunction (less proinflammatory Th1 cytokines, interferon-␥, TNF-␣, and interleukin-1 in CD4⫹ T lymphocytes of the lamina propria). This effect is due to GILZ-mediated inhibition of NF-B nuclear translocation and activation (88). The efficacy of GCs in inflammatory bowel disease is due, at least in part, to inhibition of NF-B mediated transcription of proinflammatory Th1 cytokines. TAT-GILZ fusion protein introduced into mice by high-pressure hydrodynamic plasmid injection protects against LPS-induced endotoxemia. This finding is in agreement with the demonstration that LPS resistance in the inbred mouse strain SPRET/Ei is due to GILZ expression (115). Again, TAT-GILZ has pharmacokinetic and pharmacodynamic characteristics that enable its action. TAT-GILZ-injected mice are, in fact, protected against the lethal effects of LPS, showing reduced mortality and lower serum IL-6 levels in comparison with empty TAT vector–injected mice, and these results correlate with increased GILZ expression in the liver (115). The immunomodulatory GILZ peptide (GILZ-P) is a proline-rich segment in the carboxyl terminus of GILZ (aa 115–137) that binds and inhibits NF-B p65 nuclear translocation and activation. It was identified by integrating knowledge derived from GILZ molecular mechanisms with molecular modeling analysis, synthesized as peptide amide and then acetylated. When injected intraperitoneally in mice, GILZ-P induces amelioration of experimental autoimmune encephalomyelitis, a model of human multiple sclerosis. The therapeutic efficacy of GILZ-P is due to the inhibition of NF-B nuclear translocation, which results in the suppression of inflammatory cytokine transactivation and T-cell responses (116). Thus, although GILZ therapy is still in the preclinical phase and animal models are limited to a few experimental diseases, the above considerations suggest that 12
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therapy with intact GILZ protein or GILZ peptides is reasonable, not only for the pharmacokinetic and pharmacodynamic features, but also for therapeutic efficacy.
PERSPECTIVE AND EXPECTATIONS Because the incidence of inflammatory and autoimmune diseases is increasing worldwide, there is a need not only to exploit new therapeutic approaches, but also to improve those that are already in use. GCs are widely used for the treatment of many chronic inflammatory and autoimmune diseases and regulate several signals. These drugs are certainly one of the most important therapeutic tools for the treatment of numerous syndromes with high morbidity and mortality, but when the side effects of the treatment become worse than the disease, the therapist’s choice is not easy. Improving clinical therapy with GCs has become imperative for all scientific researchers who study these drugs. Knowledge, albeit incomplete, of the molecular mechanisms of the GR and the effects of GCs has allowed proposal of new research platforms, some of which are now in clinical use. The purpose of this review was to suggest that different ideological scientific approaches to improving GC therapy and decreasing its side effects can be theoretically pursued. Studies on selective modulators of the GR have aimed to find the “perfect” molecule to induce transrepression (anti-inflammatory effects) without gene transactivation (side effects). Nevertheless, through a mechanism of gene transactivation, GCs induce the expression of some proteins, such as GILZ, that exert anti-inflammatory effects and that ameliorate or prevent certain experimental diseases, with no apparent side effects. At this point in our knowledge of the effects of GCs and the underlying molecular mechanisms, does the transactivation vs. transrepression paradigm represent a philosophical and scientific crossroad? What is the right direction and what is the most feasible? Can we use GR agonists to reduce GC transactivation activity while maintaining transrepression activity, or to selectively transactivate molecules with anti-inflammatory activity— or both? Can these compounds be used directly as medicine? Most important, any approach must take into account that the cellular response to GCs depends, not only on the type and quantity of GR isoforms in tissues, but also on the affinity of specific GCs for the different GRs (18). We think that both approaches are valid and that pursuit of balance, rather than dissociation, is the idea that will guide the direction of therapeutic choices. Achieving balance of the GC-induced mechanisms of transactivation and transrepression means, in our opinion, overcoming a schematic vision of GC effects. We must consider that the desired effects are related to both repression (direct) and activation (indirect), whereas adverse effects, which are essentially induced
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by transactivation, might also be the result of transrepression. Our concept of balance also requires dismissing the concept of the GR as a single receptor protein and recognizing that it is a system of heterogeneous isoforms that brings about the diverse biological effects of GCs. Therefore, if it is not possible to induce selective transactivation or transrepression, might it be possible to regulate the two activities in a manner that generates a more favorable balance of beneficial to adverse effects? Again, is it technically possible to tilt the balance through scientific drug design? A hypothetical, balanced GR agonist should induce transactivation of anti-inflammatory molecules and inhibit the expression of inflammatory cytokines. This may be not science fiction; the study of SGRMs began with a hypothetical consideration and then continued with structural analysis of the GR and modeling studies. Finally, the contribution of anti-inflammatory molecules, such as GILZ, to the activity of SGRMs is still unknown. This, for example, could be a first step toward achieving balance. This study was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy (IG 10677).
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Received for publication April 1, 2014. Accepted for publication August 18, 2014.
AYROLDI ET AL.