Current concepts in glucocorticoid resistance

Steroids 77 (2012) 1041–1049

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Review

Current concepts in glucocorticoid resistance Nan Yang, David W. Ray ⇑, Laura C. Matthews Endocrine Sciences Research Group, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK

a r t i c l e

i n f o

Article history: Received 23 February 2012 Received in revised form 24 May 2012 Accepted 29 May 2012 Available online 20 June 2012 Keywords: Glucocorticoid Glucocorticoid receptor Glucocorticoid resistance Inflammation

a b s t r a c t Glucocorticoids (GCs) are the most potent anti-inflammatory agents known. A major factor limiting their clinical use is the wide variation in responsiveness to therapy. The high doses of GC required for less responsive patients means a high risk of developing very serious side effects. Variation in sensitivity between individuals can be due to a number of factors. Congenital, generalized GC resistance is very rare, and is due to mutations in the glucocorticoid receptor (GR) gene, the receptor that mediates the cellular effects of GC. A more common problem is acquired GC resistance. This localized, disease-associated GC resistance is a serious therapeutic concern and limits therapeutic response in patients with chronic inflammatory disease. It is now believed that localized resistance can be attributed to changes in the cellular microenvironment, as a consequence of chronic inflammation. Multiple factors have been identified, including alterations in both GR-dependent and -independent signaling downstream of cytokine action, oxidative stress, hypoxia and serum derived factors. The underlying mechanisms are now being elucidated, and are discussed here. Attempts to augment tissue GC sensitivity are predicted to permit safe and effective use of low-dose GC therapy in inflammatory disease. Ó 2012 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5.

6.

7.

Glucocorticoids as anti-inflammatory agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucocorticoid receptor structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucocorticoid receptor function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of access of GC to cells: P-glycoprotein activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic and post-translational variation in GR structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Familial GC resistance syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. GR polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. GR protein subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. The GR heterocomplex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. GR posttranslational modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental factors and extracellular signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Changes in the cellular environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Transcription factor cross-talk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Glucocorticoid resistance in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Recovery of GC sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author. Address: Endocrine Sciences Research Group, AV Hill Building, Oxford Road, Manchester M13 9PT, UK. Tel.: +44 (0) 161 275 5655. E-mail address: [email protected] (D.W. Ray). 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.05.007

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1. Glucocorticoids as anti-inflammatory agents Despite huge advances in our understanding and more than 50,000 published manuscripts in the last 10 years, a comprehensive understanding of glucocorticoid (GC) action in inflammation remains elusive [1]. As the most robust anti-inflammatory agents known, natural and synthetic GCs are widely prescribed to treat a range of inflammatory and immune diseases in the clinic. Although in recent years, several novel therapies have been introduced, GC remain the first-line treatment for long-term control of asthma, Crohn’s disease and ulcerative colitis [2,3]. However, in addition to an increased susceptibility to side-effects [3], patients also present a significant variation in response. 2. Glucocorticoid receptor structure GCs exert their effects through the glucocorticoid receptor (GR, or NR3C1), a member of the nuclear hormone receptor superfamily [4]. The GR comprises three major functional domains, an N-terminal transactivation domain (NTD), a central DNA-binding domain (DBD), and C-terminal ligand-binding domain (LBD). The DBD and the LBD are linked by a hinge region. During evolution the ancestral corticosteroid receptor has diverged into the GR, and mineralocorticoid receptor (MR, or NR3C2). These share 94% amino acid identity in the DBD, and 56% in the LBD [5,6]. Recent evidence suggests an important pro-inflammatory role for MR in macrophage cells, which contrast strongly with the anti-inflammatory role of the GR, despite such close structural homology [8]. Recent studies revealed that alternative translation start sites in the GR NTD give rise to greater diversity of protein species. These now appear to play a role in regulating cellular sensitivity to GCs [5]. In particular, the GR a-D proteins have less transcriptional activity compared with other GR a translated protein isoforms [9]. In the U2-OS osteosarcoma cell line, expression of the relatively inactive GR a-D3 may contribute to glucocorticoid-induced apoptosis resistance [10]. Phosphorylation is a key factor in modulating the activity and the stability of GR, with the main sites of phosphorylation located in the NTD [11]. The NTD of the human GR spans residues 1–417 and contains the transcriptional activation function-1 (AF1) domain [12]. AF-1 recruits diverse proteins to the GR to regulate target gene expression, including TATA-binding protein, and MED14 [13,14]. The DBD is located in the central amino acid sequence of the GR. Residues 418–487 of the human GR form this domain, which bind to its DNA targets, termed GC response elements (GREs). This specific binding capability is achieved by its two highly conserved zinc finger motifs [15]. The LBD adopts a complex globular tertiary structure, including eleven a helices and four short b sheet that folded as a central pocket for ligands [16]. The LBD gates ligand access, and also recruits chaperones and coactivators [17]. There is a transcriptional activation function-2 (AF2) residue towards its C-terminal end. The AF2 consists of residues 526–556 and has significant liganddependent function, acting to recruit co-activator complexes with the motif LXXLL [18–26]. 3. Glucocorticoid receptor function In the absence of ligand, GR a primarily resides in the cytoplasm as part of a multisubunit complex, including Hsp90, Hsp70, Hsp40, immunophilins, CyP40, and P23 [26]. Hsp90 is the fundamental protein in this complex and combines with the LBD of GR a [12] to stabilize the optimal and high affinity structure of the ligand binding pocket within the receptor [26]. In response to GCs, the GR a complex rapidly undergoes a conformational change and sub-

sequently dissociates from the heat shock proteins. After replacing immunophilin FBK51 with FBK52, ligand-bound GR is able to produce rapid non-genomic actions through interactions with signaling pathways via cytosolic kinases [27]. Subsequently, the ligand bound GR a translocates into the nucleus, driven by the dynein motor protein [28]. However, this simple dogma has been recently challenged, based on the observations that intracellular GR localization under ligand-free conditions is frequently seen to be heterogeneous, with both nuclear and cytoplasmic expression. Nuclear localization in the absence of added ligand requires the first GR nuclear localization signal (NLS1), and is progressive during cell cycle progression through G1. During mitosis GR is excluded from condensed chromosomes, and in early G1 following cytokinesis the GR is strictly excluded from the nucleus. Therefore in addition to the very rapid (minutes) kinetics of nuclear translocation seen in response to ligand binding there is an additional slow (hours) partial nuclear translocation driven by cell cycle [29]. This discovery is important as immunohistochemical analysis of tissue has been used to infer GR activation based on detection of nuclear protein, but this is an unreliable surrogate. Activated GR binds to consensus elements in the host cell genome to activate or repress gene transcription. These sites are celltype specific, and are determined, in part, by chromatin structure [30,31]. Multiple mechanisms have been inferred to explain antiinflammatory GR action. These include transcription of antiinflammatory mediators and transcriptional inhibition of proinflammatory cytokines. The latter occurs through inhibition of the activity of proinflammatory transcription factors via a tethering mechanism [12,32]. Important factors include activator protein 1 (AP-1) and NF-jB [33,34].

4. Regulation of access of GC to cells: P-glycoprotein activation As one of the ATP-binding cassette (ABC) transporters, the drug efflux pump P-glycoprotein 170 is responsible for transporting structurally and functionally unrelated drugs out of cells [3]. This protein is encoded by the multidrug resistance gene MDR1 (ABCB1) [35]. Recent studies on blood lymphocytes reported the high expression level of MDR1 in GC resistant inflammatory diseases [36,37]. Meanwhile, it has been shown that certain single nucleotide polymorphisms within MDR1 are associated with GC resistance [38]. However, to date this is only reported in GC resistant inflammatory bowel disease and rheumatoid arthritis. Therefore future research in other diseases, e.g. GC resistant pulmonary inflammation is needed [39].

5. Genetic and post-translational variation in GR structure and function 5.1. Familial GC resistance syndrome Inactivating mutations of the GR gene cause familial GC resistance [40–52] (Table 1). This syndrome is characterized by hypercortisolism without features of Cushing’s syndrome and was firstly explained as a GR mediated disorder in 1976 [53,54]. High adrenocorticotrophin levels stimulate an over-secretion of non-corticosteroid adrenal steroids, such as aldosterone and androgen. Therefore clinical manifestations of this syndrome are hypertension, hypokalemia and/or symptoms of androgen excess which occur as menstrual abnormalities and hirsutism in females [39]. Familial GC resistance is very rare, in all cases due to mutations in the GR a gene, most of which affect the function of either LBD or DBD [55].

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N. Yang et al. / Steroids 77 (2012) 1041–1049 Table 1 Mutations within the GR gene causing GC resistance. Mutation

Genotype

Molecular mechanisms

Phenotype

D641V (40)

Homozygous

Hypertension Hypokalemic alkalosis

4-bp deletion at exon/intron 6 (41) V729I (42)

Heterozygous

Ligand affinity;(3); transactivation;; delayed nuclear translocation GR a number;; inactivation of the affected allele

Homozygous

I559N (43;44)

Heterozygous

R477H (45;46)

Heterozygous

G679S (45;46)

Heterozygous

I747M (47)

Heterozygous

V571A (48)

Homozygous

L773P (49)

Heterozygous

F737L (50)

Heterozygous

2-bp deletion at exon 9 (51) Single base deletion at exon 6 (52)

Homozygous Heterozygous

A

Hirsutism male-pattern hair loss; menstrual irregularities Precocious puberty Hyperandrogenism

Ligand affinity;(2); transactivation;; delayed nuclear translocation GR number;; transactivation;; delayed nuclear translocation; dominant negative activity Transactivation;; complete lack of DNA binding; delayed nuclear translocation Ligand affinity;(2); transactivation;; delayed nuclear translocation Ligand affinity;(2); transactivation;; dominant negative activity Ligand affinity;(6); transactivation;; delayed nuclear translocation Ligand affinity;(2.6); transactivation;; delayed nuclear translocation Ligand affinity;(1.5); transactivation;; delayed nuclear translocation Complete lack of ligand binding; null GR mutation Transactivation;; dominant negative activity

Hypertension; oligospermia; infertility Hirsutism; fatigue; hypertension Hirsutism; fatigue; hypertension Cystic acne; hirsutism; oligo-amenorrhea Ambiguous genitalia; hypertension; hypokalemia; hyperandrogenism Anxiety; acne; hirsutism; fatigue; hypertension Hypertension; hypokalemia No endocrine abnormality Fatigue; hirsutism

GR genetic polymorphisms ER22/23EK

5’

3’ 1

2

3

4

5

6

7

9α

8

9β

N363S Bcl I

B

GR protein isoforms 1

77

GR α NH2

262

420

480 520

NTD

DBD Hinge

NTD

DBD Hinge

NTD

DBD Hinge

777 aa

COOH

LBD

1

742 aa

GR β NH2

LBD

1

COOH 778 aa

GR γ NH2

1

GR-A NH2

COOH

LBD

592 aa

NTD

COOH

DBD exons 5-7

1

GR-P NH2

676 aa

NTD

COOH

DBD Hinge exons 8-9

Fig. 1. GR polymorphisms and variant protein isoforms. (A) GR polymorphisms related to GC sensitivity. GR ER22/23EK mutation located in exon 2 and results in GR resistance. GR N363S mutation located in exon 2. GR BclI polymorphism was observed in intron 2. Both of these two induce GC hypersensitive. (B) Variant GR protein subtypes result from alternative splicing of GR mRNA. NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain; aa, amino acids.

5.2. GR polymorphisms Several single nucleotide polymorphisms (SNPs) within the GR gene locus are associated with altered GC sensitivity (Fig. 1A). The ER22/EK23 mutation within GR exon 2 is associated with relative

resistance to GC therapy [56], accompanied by an elevated ratio of GR a-A to GR a-B. Since GR a-A is less transcriptionally active than GR a-B this may explain the observed effect on GC insensitivity [57]. Further downstream in exon 2 is the polymorphism N363S. This is associated with surrogate measures of increased GC sensitiv-

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tected abnormal expression levels of hsp90 protein in certain GCresistant cell lines [82,83]. Conversely, Tissing et al. showed there is no significant correlation between expression levels of hsp90/ hsp70 protein and GC sensitivity when compared with control subjects [84].

ity, including increased probability of low bone density and metabolic disorders, together with increased risk of cardiovascular disease [58]. The N to S substitution apparently alters interaction between GR and transcriptional coregulators [59]. Within intron 2 there is a frequent polymorphism which gives rise to a BclI restriction fragment length polymorphism (RFLP). This polymorphism is also associated with increased sensitivity to GCs in some individuals. However, neither the BclI RFLP, nor N363S was associated with therapeutic advantage in acute lymphoblastic leukemia [60].

5.5. GR posttranslational modifications GR signaling is determined by a combinatorial mechanism involving ligand accessibility, GR expression, subcellular trafficking and posttranslational modifications (PTM). Posttranslational modifications of GR play a key role in its activation, although the mechanisms remain poorly defined [85]. These modifications are summarized in Fig. 2. In human GR six serine residues can be phosphorylation targets, including S113, S141, S203, S211, S226 and S404 [87], and in addition, serine and threonine residues, T8, S45, S134, S234, and S267, can be phosphorylated in a cell cycle dependent manner [86]. Phosphorylation of GR is regulated by various targeted kinases, for instance mitogen activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs). The cyclin-dependant kinases, Cdk2/cyclin A kinase complexes phosphorylate S203 and S211, whilst Cdk2/cyclin E only targets S203. Generally, the transactivation of GRE-regulated promoters is increased in response to S203 and S211 phosphorylation [87]. S211 phosphorylation is regarded as a hallmark for GR transactivation. The peak of GR transactivation was found when the relative phosphorylation of S211 exceeds that of S226 [88]. In lymphoid cells, the p38 MAPK may be partly responsible for S211 phosphorylation [89]. The GR S404 phosphorylation, which is regulated by glycogen synthase kinase 3b (GSK3b), may be important in regulating GR nuclear export [90]. The GR S226 phosphorylation, which is mediated by c-Jun N-terminal kinases (JNKs), acts to inhibit GR transactivation function [91]. The GR is also a target for acetylation, at a motif identified at aa 492–495 within the human GR hinge region showing homology to the consensus K-X-K-K/R-X-K-K [92]. GR acetylation is liganddependant and regulates GR function, depending on targeted gene and cell type. Ito et al. showed human GR acetylation at K494 and K495 and revealed the key role of histone deacetylase (HDAC) 2 induced deacetylation for the transcriptional repression of NF-jB p65 and its downstream target genes [92]. On the contrary, Nader et al. reported that acetylation of GR by the circadian rhythm transcription factor CLOCK, which has intrinsic acetyltransferase activity, enhanced GR repression of NF-jB [93]. There are three SUMO-target sites within GR, K277 and K293 in the NTD and K703 in the LBD. The NTD SUMOylation inhibits GRmediated activation. It is also reported that GR undergoes SUMOy-

5.3. GR protein subtypes The human GR gene is located on chromosome 5q31–q32 and consists of eight protein-coding exons, and multiple upstream non-coding exons, so giving rise to multiple transcripts capable of generating the same full-length protein [61–63]. In addition, the final coding exon, exon 9, gives rise to a major splice variant, GR a, and a minor variant, GR b [64]. Additional GR protein diversity is generated by use of different translation initiation sites in exon 2 [65] (Fig. 1). Alternative splicing of precursor GR mRNA induces five different GR isoforms, including GR a, GR b, GR c, GR-A and GR-P [5] (Fig. 1B). GR a is a ligand-dependent transcription factor and expressed in most cell types [1]. Increased expression of GR b, GR c, GR-A and GR-P has been shown to contribute to GC resistance in some, but not all experimental studies [66–72]. GR b has a disrupted LBD, and is incapable of binding agonist ligands. However, it is reported to bind the partial agonist RU486 [73]. GR b may form heterodimers with GR a, which may explain the reported dominant negative activity reported [74]. GR c contains an additional arginine in the DBD, and was originally defined as having reduced transactivation function [75], and expression in childhood leukemia results in resistance to administered GC therapy [70,76]. Expression of GR-A and GR-P (also known as GRd) are increased in myeloma and leukemia. GR-A lacks exons 5–7 meanwhile GRP lacks exons 8–9, both of which abolish ligand binding [69,77]. 5.4. The GR heterocomplex Studies on certain New World primates have identified GC resistance due to increased expression levels of the GR chaperone complex protein FKBP51 together with decreased levels of FKBP52 [78,79]. In vitro, high level of FKBP51 has been shown to inhibit transcriptional activity of GR and this could be overcome by coexpression of FKBP52 [80]. The role of other chaperone proteins, hsp90 and hsp70, in GC resistance is controversial. GC resistance related to mutated hsp90 protein and low expression levels of hsp70 protein [81]. Subsequently, other research groups also de-

A T8

S45

P

P

S113

P

NH2

S234 S211 S134 S267 S226 S141 S203

P P

P P P P

P

S404

P

DBD

NTD 1

77

B NH2

262

AF-1

420

77

AF-1

480

COOH

LBD

520

777 aa

K277

K293

K419

K494 K495

K703

S

S

U

A A

S

NTD 1

Hinge

DBD 262

420

Hinge 480

520

LBD

COOH 777 aa

Fig. 2. The posttranslational modification sites within GR. (A) Reported human GR phosphorylation sites are demonstrated in terms of location within the receptor. (B) The posttranslational modification sites located in GR are shown, except for its phosphorylation sites. NTD, N-terminal domain; DBD, DNA-binding domain, LBD, ligand-binding domain; AF-1, activation function-1 domain; aa, amino acids; P, phosphorylation site; S, SUMOylation site; U, ubiquitination site; A, acetylation site; K, lysine.

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diminishes GR translocation and binding affinity in target cells. In monocytes, IL-13 alone was also found to reduce GR activity [100–102]. The impaired GR activity was rescued by inhibiting p38 MAPK activity, suggesting p38 MAPK mediated GR phosphorylation is a mechanism explaining such cytokine induced resistance [100]. Investigations in alveolar macrophages revealed a greater degree of p38 MAPK activation in GC resistant asthma, further supporting a role for p38 MAPK in regulating GR function, and GC sensitivity [103]. In GC resistant ulcerative colitis, there are increased mucosal levels of tumor necrosis factor (TNF)-a, IL-6 and IL-8, which downregulate GR expression. This decrease is proposed to explain the lack of clinical response [104]. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and is genetically associated with several inflammatory diseases [105]. This cytokine is proposed to cause GC resistance by blocking GC induction of the DUSP1 phosphatase which limits action of the MAPK family [106]. Increased MIF expression has been reported in numerous GC resistant inflammatory diseases, including asthma, ulcerative colitis, rheumatoid arthritis and systemic lupus erythematosus [39]. Therefore, MIF expression is proposed as a mechanism for acquired GC resistance in inflammation, although the findings are not always replicated [107]. Finally, animal experiments have identified inhibition of GR activity resulting from the actions of IL-1 [3].

lation in a ligand-independent manner; however, the intact DBD of GR is still essential to the SUMO-dependent transcriptional inhibition [85]. SUMOylation of GR results in various functional outcomes, with mutation of the potential SUMOylation sites enhancing GR transactivation in a gene-selective manner [94]. GR SUMOylation is stimulated by S226 phosphorylation, suggesting phosphorylation perhaps directs GR SUMOylation [95], and further suggest that JNK induced GC resistance is mediated by a cascade of GR modifications. The ligand-bound GR is a target for ubiquitination, at lysine 419, which located in a PEST motif (Proline [P], Glutamine [E], Serine [S] and Threonine [T]) [96]. It appears that GR phosphorylation is coupled to ubiquitination as phospho mutant GR molecules are not targeted [97]. Ubiquitination drives GR degradation, but also appears to be important in regulating GR transcriptional function [98]. 6. Environmental factors and extracellular signals 6.1. Changes in the cellular environment In chronic inflammatory diseases, many patients develop resistance to GC treatment. Clinically, this condition is termed as GC resistant inflammatory disease [2]. There are several mechanisms proposed for such acquired GC resistance. These are thought to be the result of significant changes in the cellular microenvironment, which occur over time as the disease progresses. These include alterations in GR translocation, and P-glycoprotein regulation of cellular ligand accumulation [3] (Fig. 3).

6.3. Oxidative stress Oxidative stress is proposed to limit GC response, possibly by modification of HDAC2 [108]. Oxidative stress significantly attenuates HDAC2 activity and expression, thereby potentially limiting recruitment by GR to sites of action in the genome. In GC refractory asthma, there is a markedly reduced expression of HDAC2 both in

6.2. Cytokines In GC resistant asthma, there is augmented production of IL-2 and IL-4 in the airways [99]. The combination of IL-2 and IL-4

Cytokines

Oxidative Stress

(e.g. IL-2, IL-4, IL-13, MIF)

Hsp Hop

FKBP5

GR

SH

NO

KEAP1

Nrf2

SNO

HDAC2

Nrf2

P GR P

Kinases, e.g. p38

HDAC2

Cytoplasm

MPK-1

Nucleus

P GR P

P P

CBP HAT

P P

HDAC2

NF-кB

GRE

DNA binding

Deacetylation

No mRNA

Core histones

Tethering

Fig. 3. Mechanisms of GC resistance induced by cytokines, oxidative stress and P-glycoprotein activation. CBP, CREB-binding protein; FKBP5, FK506-binding protein 5; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid responsive elements; HAT, histone acetyltransferase; HDAC2, histone deacetylase 2; Hsp, heat-shock protein; Hop, Hsp70/Hsp90 organization protein; KEAP1, kelch-like ECH-associated protein 1; NF-jB, nuclear factor jB; NO, nitric oxide; Nrf2, nuclear factor erythroid 2-related factor 2; P-gp, P-glycoprotein; SNO, S-nitrosylation.

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peripheral blood mononuclear cells (PBMC) or alveolar macrophages [109]. A reduction in HDAC2 expression was also reported in the airways of smoking asthma patients, a group difficult to treat due to lack of GC efficacy [110]. The role of oxidative stress is well studied in patients with chronic obstructive pulmonary disease (COPD). Among patients with COPD, there is a dramatically decreased HDAC2 activity in alveolar macrophages, airways and peripheral lung [111]. Furthermore, it has been shown in vitro that the GC resistance of bronchoalveolar macrophages can be reversed by overexpression of HDAC2 [92]. Recent studies suggest the HDAC2 reduction in COPD is due to oxidative stress activation of peroxynitrite and phosphoinositide3-kinase (PI3K)-d. Since both of these factors are crucial for inactivation and degradation of HDAC2, as a result, HDAC2 activity is reduced under oxidative stress [112,113]. 6.4. Hypoxia Recent studies have reported conflicting results for the crosstalk between GC action and hypoxia. This is relevant in inflammation, as hypoxia is observed at many sites of inflammation due to increasing numbers of activated inflammatory cells and subsequently increased oxygen demand [114,115]. In endothelial cells chronic hypoxia induced cellular dysfunction after treatment with dexamethasone [116], and GR transactivation was found to be impaired under low oxygen tension [117]. Similar impaired GR transactivation was observed in hepatic cells, cultured in a hypoxic environment of 3% O2 [118]. On the contrary, there are several studies reporting increased GR activity in hypoxia [119,120]. 6.5. Transcription factor cross-talk As the most abundant AP-1 complex in activated cells, the cFos:c-Jun heterodimer has a key role in the transcriptional regulation of asthma-relevant cytokines [121–123]. The transcriptional

activation of c-Fos is a marker of AP-1 activity [124]. Compared with GC sensitive asthma, increased c-Fos production in PBMC and bronchial biopsies was measured in GC resistant asthma, with loss of GC suppression of JNK activity [125]. The c-Jun component of AP-1 is capable of binding to GR, thereby inhibiting its actions [125,128]. This interaction between GR and AP-1 was originally reported to result in mutual antagonism [126–129] (Fig. 4). However, surprisingly AP-1 has emerged a key partner protein for GR regulated transcription of endogenous target genes. AP-1 regulates basal chromatin structure, and accessibility, and so enhances GR binding to specific sites in the genome [30]. This suggests that the physiological interactions between GR and AP-1 are both more extensive than previously realised, but also more complex, with evidence for enhancement, and diminution of GR activity [130]. In addition, proteins capable of regulating the cytoskeleton (e.g. cofilin-1) are reported to decrease the transcriptional activation of GR [39]. Indeed, gene array studies revealed that overexpression of cofilin-1 was associated with GC resistance in T cells [131]. 6.6. Glucocorticoid resistance in cancer GCs are widely used in the initial induction phase of antileukemia therapy, due to their profound pro-apoptotic effects on T lymphoblasts. However, chronic treatment frequently provokes development of resistant clones. In-vitro models suggest that the strong selection pressure causes either GR mutations, or deletions. In contrast, in vivo resistance to GC therapy is usually accompanied by down-stream changes in the expression and/or function of apoptosis regulating proteins of the BCl2 family. These proteins are the physiological target of GCs in lymphoblasts, and regulate the pro/anti apoptosis status of host cells. This subject has been extensively reviewed recently in [132]. In non-lymphoid malignancy low expression of GR in human small cell lung carcinoma cell lines suggests a possible role in reg-

Cytokines Hypoxia Oxidative Stress

P

AP-1

GR

NF-кB

P

Cytoplasm

Nucleus

P GR P

P P

GRE

Transactivation

mRNA

P GR P P P

p65

mRNA

p50

NRE

Transrepression

Fig. 4. Cross-talk between GR and transcription factors. Increased AP-1 and NF-jB are capable of directly interacting with GR, subsequently induce GC resistance. AP-1, activator protein 1; GRE, glucocorticoid responsive elements; NF-jB, nuclear factor-kappa B.

N. Yang et al. / Steroids 77 (2012) 1041–1049

ulating malignant phenotype [66]. Accordingly, studies were performed to show that both in vitro, and in vivo restoration of GR expression in these cells powerfully induced apoptosis [133,134]. This suggests a broader role for GR and GC signaling in regulating cell fate decisions, but in a cell type specific manner. 6.7. Recovery of GC sensitivity A number of studies have sought to enhance GC sensitivity, particularly in the context of inflammatory disease. Co-treatment with inhibitors of the MAPKs, especially p38, may prove useful in GC resistant asthma [100], and there is some evidence that theophylline, by inhibition of PI3Kd, is able to reverse GC resistance in COPD patients [135,136]. In addition, a number of approaches to target Pglycoprotein and MIF are now being developed [137,138]. In this context selective GR modulators (SGRMs) are an attractive pharmacological approach to treat inflammatory disease. These agents would ideally preserve useful anti-inflammatory activity but lack the side-effect profile of conventional GCs. Indeed, a number of compounds have been proposed, and tested in vivo, but so far none completely fulfil this criterion [139]. However, if such agents could be identified their therapeutic use would still be improved by other approaches to target GC sensitivity.

7. Concluding remarks GCs are the most potent anti-inflammatory agents known. Despite their impressive therapeutic utility, a major factor limiting their clinical use is the wide variation in responsiveness to treatment between individuals and over time. Clinically, GC resistance presents disease independent manner and more common than expected. Recent investigations on GR provide a novel insight to understand mechanisms of GC resistance. This is crucial for predicting patient steroid responsiveness so that optimize GC therapy. Meanwhile, detection of GC resistance model is essential for screening new GR modulators. Consequently, maximum GC antiinflammatory efficacy could be achieved whilst minimize their potential side effects.

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