Molecular Mechanisms of the Glucocorticoid Receptor in Steroid Therapy

Review

Molecular mechanisms of the glucocorticoid receptor in steroid therapy – lessons from transgenic mice

Sabine H ü bner and Jan Tuckermann* or diffi cult to combat (1) . This is surprising because due to Leibniz Institute for Age Research , Fritz Lipmann Institute their undisputed severe side effects, GCs would not pass cur- (FLI), Beutenbergstr. 11, D-07745 Jena , Germany rent regulations for drug approval in most countries. The side effects include type 2 diabetes, osteoporosis, muscle weakness, * Corresponding author skin atrophy, and even depression upon long-term use (2) . e-mail: jan@fl i-leibniz.de Their receptors, in particular, the classic GC receptor (GR), act in a versatile manner from altering gene expression as transcription factors to interfering with signaling pathways in Abstract a non-genomic fashion. Therefore, research on the molecular mechanisms of GC action is of importance to shed light on Glucocorticoids (GCs) are potent anti-infl ammatory agents physiological processes under GC control. This should gener- that are used to treat chronic infl ammatory diseases, allergic ate rationales to achieve specifi c drug development with the conditions, and some cancers. However, their therapeutic goal of anti-infl ammatory therapy with fewer side effects. effects are hampered by severe side effects, such as muscle Here, we summarize the pivotal fi ndings and recent prog- weakness, insulin resistance, fat redistribution, and osteopo- ress regarding cell-type-specifi c GR mechanisms of action rosis. GCs act on many cell types that express the GC recep- revealed by genetic alterations of the GR in mice. tor (GR) via several modes of action. One of them includes GR homodimers recognizing binding sequences in the DNA of gene promoters. Another mode involves the modulation of Endogenous GCs other DNA-bound transcription factors via dimer-independent mechanisms. To what extent these mechanisms contribute to GCs are elevated in a diurnal rhythm and during acute stress GC-mediated effects is currently being elucidated from analy- responses (3) . Their secretion is pulsatile and controlled by a ses of mice with conditional and function-selective mutations feedback mechanism involving hormones of the hypothala- of the GR and is summarized in this review. Whether GR mus and pituitary (hypothalamus-pituitary-adrenal axis) (4) . homodimerization or its monomer activity is decisive in the The hypothalamus releases corticotropin-releasing hormone therapeutic effectiveness and associated side effects of GCs for (CRH) triggered by the circadian rhythm, systemic infl am- the treatment of infl ammatory conditions depends on the type mation reactions, or mental stress. Subsequently, CRH causes of the pathological condition. Thus, the classic criterion for secretion of proopiomelanocortin (POMC) from the anterior selective GR modulators, discrimination between GR dimer- pituitary, which in turn is proteolytically cleaved into adreno- and GR monomer-dependent protein-protein interaction, will corticotropic hormone, the latter acting on the adrenal gland not help in any condition to avoid side effects and maintain cortex to enhance production of corticosteroids (4) . High con- anti-infl ammatory activity. Rather, novel criteria for selective centrations of GCs lead to a suppression of POMC and CRH GR modulators have to be defi ned that take into consideration in the pituitary and hypothalamus, respectively, resulting in the tissue-specifi c mechanisms of the GR to achieve optimized a decreased GC release. In rodents, the main GC is corticos- anti-infl ammatory therapies with reduced side effects. In the terone, whereas in humans, it is cortisol or hydrocortisone case of avoiding osteoporosis as a side effect, a fi rst example (5) . Within cells, the restricting factor for the production of of such optimized compounds can be provided. active cortisol is 11- β-hydroxysteroid dehydrogenase type 1 (11 β-HSD1), which converts inactive cortisone to the active Keywords: conditional knockout mice; glucocorticoid metabolite. Conversely, active cortisol can be modifi ed by receptor; infl ammation; osteoporosis. 11- β -hydroxysteroid dehydrogenase type 2 (11β -HSD2) via conversion of the hydroxyl group to a keto group at position 11, which in turn leads to inactive cortisone (6) . Introduction One important function of GCs is to allow the mobilization of glucose for rapid energy supply to the brain and by support- Since the middle of the last century, glucocorticoids (GCs) ing gluconeogenesis (7) . In addition, decisive roles in behavior have been widely used as anti-infl ammatory agents for the (8) and infl ammation (9) have been uncovered. Two disease treatment of allergic conditions, chronic infl ammatory dis- states demonstrate the involvement of GCs in the regulation eases, and some cancers in which the initial cause is unknown of glucose and energy metabolism (10) . Addison ’ s disease 242 S. Hü bner and J. Tuckermann

patients with chronic low GC levels experience stress sensiti- sequences are unmasked, resulting in an import of the ligand- vity, lymphoid tissue hypertrophy, weight loss, and hypogly- bound GR into the nucleus (25) . Localization of the GR appears cemia (11) . Cushing’ s disease patients with chronic GC excess to be dynamic, because cyclic administration, withdrawal of display hyperglycemia, liver steatosis, hypertension, elevated ligands in tissue culture as well as pulsed GC treatment of cholesterol, immunodefi ciency, and insulin resistance (10) . adrenalectomized rats lead to a cyclic localization between Further studies have been performed with rodent models of the nucleus and the cytoplasm. This GR shuttling is associated impaired GC secretion by adrenalectomy, recapitulating the with cyclic GR target gene expression over time (26) . Addison phenotype (10) and demonstrating an early role of Within the nucleus, the ligand-activated GR binds to palin- endogenous GCs in systemic infl ammation (12, 13) . dromic response elements of the DNA [GC response elements In addition, multiple aspects of the role of endogenous GCs (GREs)] as homodimers in a head-to-tail fashion via hydro- during development and in adult physiology have been revealed phobic motifs present in the LBD (27) and interactions by the after introducing loss-of-function mutations for the GR in mice DBD (28) . The DBD consists of two zinc fi ngers. The amino and inactivating GC activity in mice overexpressing 11β -HSD2 terminal zinc fi nger of the GR molecule makes contact with or being devoid of 11β -HSD1. With these genetically modifi ed the DNA. The D-loop adjacent to the second zinc fi nger motif mice, it could be shown that endogenous GCs are required for interacts with the D-loop of the dimerization partner (28, 29) . the differentiation of chromaffi n cells, lung maturation during So far, crystal structures of the entire GR bound to DNA are development (14, 15) , and bone integrity (16 – 18) . lacking, leaving the possibility open that additional dimer Thus, it is not surprising that because of the multiple roles interface exists. Recently, it was elegantly demonstrated by of endogenous GCs in various physiological processes, the structural, biochemical and cell-based analyses that varia- presence of excessive GCs during anti-infl ammatory therapy tions in the GREs result in differential GR conformational leads to multiple side effects. In the following section, we changes that affect recruited coactivators, leading to different briefl y summarize how GCs act at the molecular level. We transactivation functions on different GC target genes (30) . In then describe the contributions of different molecular mecha- addition, experiments by the Hager group demonstrated that nisms to GC actions that have been elucidated in animal a preoccupied GR binding site by the GR helps to assist bind- models. ing of additional GR molecules that exchange in a dynamic fashion (31) . Transactivation of transcription is dependent on the AF domains of the GR. The AF-1 domain is respon- The GC receptor sible for transcriptional activation and association with basal transcription coactivators in a ligand-independent manner by Non-genomic effects interacting with ATPase BRG1 and recruiting histone acety- lases like p/CAF and CBP/p300 (32) . In contrast, the AF-2 GCs bind to the GR, a member of the nuclear receptor fam- domain with its helix 12 acts in a ligand-dependent manner ily. Nuclear receptors are ligand-induced transcription fac- by recruiting coactivators from the p160 family (e.g., SRC-1, tors and share a common homology and domain structure: TIF2/GRIP1) (Figure 1A). These coactivators possess histone an N-terminal transactivation domain (AF-1), followed by acetylase activity and thereby help to open chromatin to allow a DNA-binding domain (DBD), a subsequent hinge region transcriptional activation (33, 34) . and a C-terminally located ligand-binding domain (LBD) Binding to DNA, however, can also lead to repression of associated with a second transactivation domain (AF-2) (19) the transcription of genes. Negative GREs (nGREs) were (Figure 1 A). initially described as palindromic sequences separated by a Despite being considered a transcription factor, rapid non- three-base pair spacer and being present only in a few genes, genomic effects have been described. In particular, inter- such as POMC, prolactin, α-fetoprotein, CRH, osteocalcin, ference of the GR with components of signal transduction interleukin 1β , and proliferin (35) . However, it was recently pathways, such as PI3K, JNK, and 14-3-3 proteins, has been reported that additional nGREs exist as inverted repeats with reported (20 – 22) . Recently, Lowenberg et al. (23) suggested none, one, or two spacing base pairs, leading to recruitment a mechanism underlying non-genomic GC-induced immuno- of corepressors, such as silencing mediator for retinoid and suppression in T cells by membrane-associated liganded GR thyroid receptors and nuclear receptor corepressor (36) . disrupting the protein kinase Fyn complex and impairment of Recent genome-wide binding studies of the GR in mamma- T-cell receptor (TCR) activity. GC effects may also be medi- lian cells have revealed that binding motifs for the GR might be ated by non-specifi c interactions with cellular membranes more diverse than previously anticipated, which explains in part or by specifi c interactions with membrane-bound GR (24) . the diverse response of genes dependent on the cellular con- However, a clear identifi cation of these GR types is lacking text (37) . In particular, cell-type-specifi c predetermined open and remains a challenge. chromatin sites in the absence of ligand revealed by genome- wide DNAse I hypersensitivity profi ling seem to determine the Regulation of gene expression by DNA binding majority of GR occupancy in the presence of hormone (38) . To of the GR a minor degree, the occupancy of GR binding motifs is determined by binding sites for certain other transcription factors – In the presence of hormones, the GR chaperone complexes again, dependent on the cell type as revealed by the comparison in the cytoplasm are disrupted and the nuclear localization of a mammary cell line with a pituitary cell line (38) . Glucocorticoid receptor in steroid therapy 243

CBP/p300 complex p/CAF SWI/SNF complex p160 CBP/p300 Trip6 BRG-1 complex LXXLL helix 12

NH2 AF1 DBD HD LBD AF2 COOH

Ligand 2 Zinc finger domains NLS 1 Ligand dependent Independent - P-box: DNA binding NLS 2 - D-loop: GR dimerization

p160 GRIP1 CBP Trip6/ CBP STAMP AP-1 NP-κB IRF3 GRE Pol Pol

Figure 1 Primary structure of the glucocorticoid receptor (GR). (A) The transactivation domain AF-1 is followed by the DNA-binding domain (DBD) with two zinc fi ngers involved in DNA binding (P-box) and dimerization of the receptor (D-loop), a hinge domain (HD) with two nuclear localization sequences (NLS), a ligand-binding domain (LBD), and a C-terminal transactivation domain AF-2. Coactivators with histone acetyltransferase activity such as CBP/p300, ATP-dependent chromatin remodeling complexes like switch/sucrose non-fermenting (SWI/SNF) proteins enhance the transcriptional activator function of the receptor in a ligand-independent manner. The AF-2 domain has a ligand-dependent coactivator function that recruits coactivators with the LXXLL motif in helix 12. Those coactivators belong to the p160 family, which further recruit multiple coactivators among the CBP/p300. Integrators such as TRIP6 interact with the DBD to mediate transrepression of AP-1. (B) The GR dimer binds to the palindromic glucocorticoid responsive elements (GRE) in regulatory sequences of GR target genes and facilitates transcription by recruitment of coactivators (p160, CBP). The GR as a monomer infl uences the activity of pro-infl ammatory transcription factors like AP-1, NF-κ B, and IRF3 by tethering mechanism with co-integrators (Trip6, STAMP). The liganded GR also could sequester GRIP1, usually, a coactivator for IRF3, from these transcription factors. By this mechanism, the pro-infl ammatory gene activity is repressed or modulated.

Tethering of the monomer GR to other transcription (Figure 1B). Loss-of-function studies also point to a role of factors tumor suppressor protein p53 being involved in transrepression by an unknown mechanism (54) . Another major mode of gene regulation is the association of Recent genome-wide studies by Rao et al. (55) measuring GR as a monomer with pro-inﬂ ammatory-acting transcription p65/NF-kB and GR occupancy on the DNA of TNF and GC factors [AP-1 (39 – 41) , NF-κ B (42, 43) , IRF3 (44 – 46) , STAT triggered HeLa cells revealed a more detailed picture of the proteins (47) , CREB, NFAT, T-bet, and GATA3 (48 – 50) ] and crosstalk of those two transcription signaling pathways. Both thereby modulating their activity and target gene expression. Therefore, this tethering mechanism is also considered to be DNA-binding-dependent mechanisms utilizing partially pre- a transrepression mechanism. occupied DNA sites for GR and p65 as well as presumably The cointegrator proteins SRC1, TIF2-associated-binding DNA-binding-independent mechanisms of the GR in TNF protein (STAMP) (51) , thyroid hormone receptor interactor and GC triggered cells seem to be involved. More genome- 6 (TRIP6), GR-interacting protein 1 (GRIP-1), and positive wide studies of inﬂ ammatory acting cells with GR mutations transcriptional elongation factor b (P-TEFb) had been shown unable to dimerize (see below), for example, are required to to be decisive to ensure transrepression (45, 46, 52, 53) resolve the mechanisms more comprehensively. 244 S. Hü bner and J. Tuckermann

Contribution of the molecular functions lacking the GR in myeloid cells (GRLysMCre ) are resistant to GC of the GR therapeutic and side effects therapy (62) . in steroid therapy Thus, in different types of skin infl ammation, different requirements of GR functions are involved in the anti-infl ammatory Because pro-infl ammatory genes under the control of AP-1 activity of GCs. and NF-κ B can be repressed by the GR monomer, there is a long-standing hypothesis that selective-acting GR-modulating Systemic infl ammation: septic shock compounds that address the monomer function of the GR represent selective anti-infl ammatory drugs with fewer side Usually, infl ammatory responses against infections are effects (56) . Mouse models had been developed to prove the counterbalanced by a resolution of this infl ammation, where relevance of this concept. endogenous GCs are considered to participate. Hence, Two different types of modifi cation of the GR allele in patients with adrenal insuffi ciencies (64) have a decreased the mouse were designed to achieve these goals. First, con- survival rate of septic shock as well as rodents subjected ditional GR knockout mice were generated, harboring a GR to adrenalectomy (12) . Moreover, mice lacking the GR in allele with loxP sites fl anking exon 3 (8) or exon 2 (57) , so- macrophages are highly sensitive to septic shock induced called GRfl ox mice. GRfl ox mice were crossed with trans- by a bolus injection of lipopolysaccharides (LPS) (9) . Their genic mice expressing Cre recombinase in a cell-type-specifi c enhanced lethality is explained by an impaired expression manner in adult mice to reveal the critical cell types involved of dual specifi c phosphatase 1 (DUSP1/MKP1) in mac- in an organismic response to GCs (58) . Second, GR dim mice, rophages by endogenous GCs, leading to a suppression of a knock-in strain carrying a point mutation in the dimeriza- p38 activity, thereby blunting TNF- α expression. However, tion interface of the DBD encoded in exon 4 (59) , were char- suppression of macrophage activity by GCs during this dis- acterized to abrogate dimerization-dependent regulation of ease appears to occur on different levels. First, the degree GRE-containing genes but allow DNA-binding-independent of suppression of pro-infl ammatory-acting MAP-kinases tethering mechanisms to AP-1 and NF-κ B (29, 43, 59, 60) . depends on the type of involved toll-like receptors with Interestingly, this germ line mutation has been shown to res- respective adaptors (44, 65). Second, GR dimerization-de- cue a number of defects observed in complete knockout mice pendent functions in these cell types appear to be decisive, (14) , such as lung maturation, development of the adrenal because macrophages from GRdim mice have a less stringent medulla, and thus perinatal lethality (59) . Gene expression effi ciency to suppress cytokines (63) . Furthermore, they are profi ling revealed that the induction of metabolic genes is largely resistant to changes in cell shape, NO synthesis, and strongly reduced, but a subset of genes is regulated in a simi- surface expression of classic activation markers (66) and fail lar manner in the liver of wild-type and GRdim mice following to up-regulate GC and LPS synergistically regulated genes. systemic prednisolone treatment (61) . The differential regula- Moreover, GRdim mice display an enhanced lethality in vari- tion of genes by GCs in GR dim mice makes these mice very ous septic shock models (66) . Interestingly, in these mice, suitable for studies determining the molecular mechanism in TNF-α is not misregulated in septic shock, but IL-1β and disease models where GCs are applied. This is of particular IL-6 levels are increased. Indeed, inhibition of IL-1 activ- importance for functional target genes of the GR in therapeu- ity by recombinant IL-1 receptor antagonist administration tic and side effects of GC application. rescues GRLysMCre mice completely and GRdim mice partially from death after LPS administration (66) . This demonstrates GC effects on skin infl ammation that IL-1β is an important target of endogenous GCs protect- ing against septic shock. A classic animal model of acute irritant infl ammation in the GCs have been reported not only to diminish pro- skin is phorbol ester-induced ear infl ammation. It is char- infl ammatory features of macrophages but also to induce acterized by edema formation, swelling and increased vas- ‘ alternatively ’ activated macrophages (67) . These have been cular permeability, infl ux of neutrophilic granulocytes, and implicated in the resolution of infl ammation by express- mononuclear cells into the skin and can be determined by ing IL-10 with anti-infl ammatory activity and enhancing an increase of ear thickness. Topical treatment with GCs the expression of scavenger receptors, such as CD163, FcR severely suppresses this infl ammation in wild-type and GRdim (CD16 and mannose receptor) (68) . Therefore, the clearance mice (62) . Thus, the dimerization-defi cient GR is suffi cient to of apoptotic cells (69) , a crucial process for the resolution mediate an anti-infl ammatory response. of infl ammation, is enhanced. Studies of genome-wide gene In contrast, contact allergy, which can be modeled by con- expression and phagocytosis in GC-exposed human mono- tact hypersensitivity (CHS) in mice, requires GR dimeriza- cytes – the precursors of macrophages – have corroborated tion for anti-infl ammatory effects by GCs (63) . GRdim mice the GC-induced ‘ anti-infl ammatory ’ , presumably alternative, display a complete refractory response toward GCs, although phenotype (70) . Furthermore, GCs enhance the survival of some cytokines, such as TNF-α , are suffi ciently suppressed. anti-infl ammatory-acting monocytes by up-regulation of the IL-1 β , MCP1, and IP10 fail to be repressed in GRdim mice. A3 adenosine receptor to prevent apoptosis (71) . Using tissue-specifi c GR knockout mice in the CHS model, Thus, myeloid cells, in particular, monocytes/macrophages, it has been revealed that the GR in keratinocytes and the play a key role in systemic infl ammation, which needs to be GR in T cells are dispensable for GC treatment. Only mice addressed by GCs in order to prevent it. Glucocorticoid receptor in steroid therapy 245

GC action in suppression of infl ammation Taken together, the GR in T cells, most likely in the IL-17- in models of autoimmunity producing subset, is critical for the suppression of infl ammation in the hitherto analyzed models of autoimmune diseases. Multiple sclerosis is the most prevalent autoimmune dis- However, myeloid cells may also play a role depending on the ease of the central nervous system. High-dose GC therapy is availability of GCs. applied during acute phases of the disease; however, accompanied complications and sometimes incomplete recov- Side effects ery can occur (72) . Using an animal model, experimental autoimmune encephalomyelitis (EAE), Wust et al. (73) and The most prominent side effects of GC excess, either applied Schweingruber et al. (74) could demonstrate that GC action pharmacologically or being present in disease conditions, are in T cells and myeloid cells is decisive in a therapeutic set- muscle weakness, hyperglycemia, hepatic steatosis, insulin ting. GRLysMCre mice were found to be curable by free acces- resistance, and bone loss (10) . sible GCs, whereas GRLckCre mice, lacking the GR in T cells, displayed an earlier onset of the disease and were completely Muscle weakness GC-induced muscle loss occurs resistant to GC application, remaining heavily infl amed (73) . via the GR in muscle (88) , in part by up-regulation of the In contrast, when GCs encapsulated in liposomes that are muscle degradation initiating E3-ubiquitin ligase Murf1/ taken up by phagocytotic cells, macrophages were identifi ed Atrogenin (89) . Despite the requirement of GR dimerization to be the main targets (74) . This impressively demonstrates to up-regulate Murf1/Atrogenin, muscle loss is not affected that dependent on the formulation of corticosteroids, different in GRdim mice. This could be due to non-genomic effects, cell types could become critical for anti-infl ammatory action. because overexpression of a nuclear localization defective It remains a challenge to underpin the exact mechanisms GR in muscle fi bers of muscle specifi c knockout mice could behind these differences. confer decrease of muscle fi ber size (88) . Paradoxically, also, Concerning the involvement of GR dimerization, no stud- Murf1/Atrogenin was in these mice still induced, in contrast ies have been performed yet, but it is noteworthy that ‘ com- to GR dim mice (89) . Thus, more unrecognized GC-regulated pound A ’ (CpdA), a GR-selective ligand favoring monomer genes contribute to muscle weakness. Furthermore, the GR in GR function, has been reported to be successful in suppress- muscle contributes to diabetes-induced muscle wasting (88) . ing EAE (75, 76) . GC effects on T cells and their precursors are long estab- Insulin resistance Insulin resistance as one of the major + + lished. Double positive (CD4 , CD8 ) T cells are very vul- complications is rapidly evoked by GC administration, which nerable to GC-induced apoptosis. GRdim mice fail to show is in part due to the suppression of insulin secretion from β apoptosis of double positive cells (59) . Potential target genes cells by GCs (90, 91) . Moreover, mice overexpressing the of the GR are the proapoptotic proteins Puma and Bim (77, GR in β -islets display an increased insulin resistance (90) . 78). Mice lacking the GR in T cells do not display altered Furthermore, GCs have direct effects on insulin receptor thymocyte subsets or TCRβ expression (57, 79), excluding signaling by affecting the expression of insulin receptor a role of the GR during thymocyte selection. Interestingly, substrate 1 and 2, reduction of the activity of PI3K, and thus GCs produced by the thymus stroma and/or epithelial cells Akt phosphorylation, which may be mediated by ceramides. appear to mediate post-pubertal thymus involution in male Rodents with impaired ceramide synthesis are rendered mice dependent on androgen action (80, 81) . resistant to GC-induced insulin resistance and do not show GCs can suppress activation-induced cell death of reduced Akt phosphorylation (92) . peripheral T cells (82, 83) and inhibit T cell activation, Nonetheless, loss of GR function in the liver of mice illus- suppress a plethora of cytokines, induce apoptosis, and trates features of the role of endogenous GCs in metabolism. shift from Th1 to Th2 cells, reviewed elsewhere (84) . The In the liver, GCs, together with glucagon, regulate glucose actions of GCs on Th17 cells and regulatory T cells (Tregs ), output, which is antagonized by the actions of insulin (93) . both important modifi ers of autoimmune diseases, have Mice lacking the GR in the liver show a strong hypoglyce- been less studied to date. T regs are potent suppressors of mia after fasting (94) , which is accompanied by a failure autoinfl ammatory T cell responses and have been reported of the up-regulation of mRNA encoding key gluconeogenic to be elevated upon GC treatment (85, 86). Despite the enzymes tyrosine aminotransferase and phosphoenol pyru- observation that Tregs primed in vitro by GCs impede EAE vate carboxykinase. Both are characterized by GREs in the in mice after transfer, endogenous T reg numbers are reduced promoters and require dimerization-competent receptors for during GC therapy of EAE (73) . Nonetheless, a reduction up-regulation of mRNA by GCs (59) . Indeed, unpublished of IL-17 cells accompanies GC treatment in EAE, indi- work in our laboratory favors GR dimerization-dependent cating a potential mechanism for GR action to suppress processes in GC-induced glucose intolerance. infl ammation in this model. Hyperglycemia is further augmented by the breakdown of Inhibition of IL-17-producing cells by the GR dimer protein and fat stores to enhance the substrates for gluconeo- appears to be in part also important for the treatment of another genesis (93) . Long-term GC treatment up-regulates PPARα autoimmune disease, rheumatoid arthritis, as revealed by the (95) , which is involved in insulin resistance. PPARα -defi cient resistance of GRdim , GR LckCre, and IL-17 knockout mice to GC mice display a reduced insulin resistance after 5 months of treatment in antigen-induced arthritis (87) . dexamethasone treatment. Interestingly, interruption of vagal 246 S. Hü bner and J. Tuckermann

nerve innervation in the liver reduces insulin resistance of incidences in clinics are associated with high GC intake or GC-treated wild-type mice, but not of PPARα -defi cient mice GC excess (104, 105) . The bone loss observed in GIO is due (96) . How PPAR α activity links vagal nerve innervation to to an adverse imbalance of bone formation by osteoblasts and GC-induced insulin resistance and whether this mechanism bone resorption by osteoclasts. Systemic effects of GCs such applies to short-term GC exposures require investigation. as lowering of calcium levels and those of anabolic-acting Very recent liver X-receptor β was recognized as a mediator hormones, for example, sex steroids and growth hormone, for GC-induced insulin resistance by participating in gluco- can contribute to bone loss to a certain extent (106) . However, neogenetic gene regulation (97) . Thus, mechanisms involved direct effects on bone cells have been shown to be crucial in in GC-induced insulin resistance are complex and require fur- causing bone loss. ther comprehensive investigations. GCs are potent inducers of osteoclastogenesis-promoting receptor activator of NF-κ B ligand (RANKL) and suppress the Lipid metabolism Within the liver, GCs exert profound osteoclast inhibitor osteoprotegerin (OPG) (107) . Confl icting effects on lipid metabolism and contribute to liver steatosis results exist over GC effects on osteoclast activity in mouse via the GR at least in obese mice (96) . In particular, they are models. Whereas increased resorption has been reported in involved in the elevation of plasma non-esterifi ed fatty acids, GC-treated Balb/c mice (108) , other studies have reported induction of lipolysis in fat tissue, and therefore enhanced decreased resorption (approx. 20 % – 30 % ) in mice of differ- triglyceride synthesis, which is in combination with a decrease ent backgrounds (17, 109). Jia et al. (109) suggest that GCs of fatty acid oxidation; lipid accumulation in the liver triggers enhance the lifespan of mature osteoclasts, whereas osteo- for hepatic insulin resistance (93) . GCs exert profound effects clastogenesis from osteoclast progenitors is inhibited. The on fat tissue. Redistribution of peripheral fat, which is poorly inhibition of osteoclastogenesis by pharmacological GC con- centrations observed in co-cultures of osteoblast and osteoclast understood, and gain of visceral fat are augmented by GCs. precursors is dependent on GR expression in both cell types Lipoprotein lipase is up-regulated upon GC exposure; the (17) . This observation is in line with the observed decrease of uptake of non-esterifi ed fatty acids and triglycerides into resorption in vivo reported by Rauch et al. (17) and Kim et al. adipocytes is augmented (93) . (110) . The cell-autonomous inhibition of osteoclastogenesis by In addition, the generation of adipocytes from mesenchy- GCs is explained by impairment of the reorganization of the mal stem cells is facilitated by GR activity. Further adipogenic cytoskeleton (actin rings for resorptive osteoclast activities) differentiation involves the actions of several transcription (110) . Importantly, total inhibition of resorption by bisphos- factors, most importantly, those of the CCAAT/enhancer- phonates or by RANKL inhibitory antibodies in humanized binding family of proteins (C/EBPs) and PPARγ . C/EBPβ RANKL knock-in mice ameliorates GC-mediated bone loss and C/EBPδ are early induced genes that precede the expres- (108) . sion of the key adipogenic transcription factors C/EBPα and Recently, it was shown that the GR in osteoblasts is crucial PPARγ for the terminal differentiation of adipocytes (98) . for GC-induced bone loss. Mice lacking the GR in osteoblasts Derepression of C/EBPβ by the GR ligand domain at the (GRRunx2Cre mice) are resistant against GC-mediated inhibition promoter of C/EBPα in the preadipocytic model cell line of bone formation and loss of bone mass (17) . Inhibition of 3T3-L1 (99, 100) as a non-genomic mechanism had been bone formation is accompanied with reduced osteoblast and proposed. However, genomic effects of the GR appear to osteocyte numbers (111) . The reduction of osteoblast num- play a major role. Very recently, Siersb æ k et al. (101) studied bers could be due to inhibition of proliferation, induction of the transcription factor recruitment to open chromatin sites apoptosis or inhibition of differentiation. during adipocyte differentiation in 3T3-L1 cells. Here, they The proliferation of osteoblasts can be inhibited by GCs identifi ed that the GR in concert with retinoid X receptor, by antagonizing the Wnt pathway (112) , GC-induced MKP1/ α β δ STAT5 , C/EBP , and works as pioneering factors to pre- DUSP1, leading to a reduction of mitogenic signaling (113) pare transcription factor hotspots a few hours after differen- that requires GR dimerization (17) . Nevertheless, GRdim tiation has been initiated. These factors in later differentiation mice have impaired bone formation upon GC exposure, indi- γ events facilitate in part PPAR binding (101) . Additionally, cating that effects of GCs on proliferation are involved in δ dexamethasone stimulates C/EBP expression and subse- GC-induced bone loss to only a minor extent (17) . Apoptosis γ α quent PPAR and C/EBP expression in 3T3-L1 cells (102) . of osteoblasts and osteocytes is observed after prednisolone Recently, we found that GR dimerization is required for this treatment (114) , is independent of GR dimerization (17) , is dim process (103) . Mouse embryonic fi broblasts of GR and GR accompanied with caspase 3 activation (115) , has induction knockout mice, cultured under adipogenic conditions, dis- of the pro-apoptotic protein BAX (116) and might depend, play a strong reduction of adipogenic differentiation capac- in addition, on the activation of the FAK-related kinase Pyk2 dim ity. GR and GR knockout cells fail to up regulate KLF15, (117) . Furthermore, an overall bone loss is still displayed in which is required for adipogenesis in vitro (103) . However, mice with impaired GC activity and consequently reduced whether the generation of de novo fat involves the GR in vivo apoptotic rates in late differentiated osteoblasts and osteo- requires further investigation. cytes in vivo (118) , indicating that induced apoptosis is not the only mechanism of GC-induced bone loss. Osteoporosis GC-induced osteoporosis (GIO) is a major High dosage of GCs impairs the differentiation capacity side effect of steroid treatment: 25 % of all clinical osteoporotic of primary osteoblasts by about 70% to 90% (17) and is Glucocorticoid receptor in steroid therapy 247

accompanied by reduced differentiation marker gene expres- a discrimination between interference of NF-kB vs. AP-1 sion (119) . Following reduced differentiation, osteoblast might be important to protect osteoblast differentiation while function (collagen production) declines (120) in vitro and in still promoting some anti-infl ammatory activity. vivo and, consequently, bone formation is reduced. This GC Our analysis of the activity of the GR ligand CpdA on bone effect is absent in mice specifi cally lacking the GR in osteo- cells demonstrates that these criteria can be met in osteoblasts (GRRunx2Cre ), but not GRdim mice (17) and prevents bone blasts (123) . CpdA displays potent anti-infl ammatory actions loss only in GR Runx2Cre mice. Taking these fi ndings together, in collagen-induced arthritis (131) . In addition, CpdA is it can be concluded that the inhibition of differentiation is a capable of suppressing pro-infl ammatory cytokines, such as major mechanism of GIO (17) . CXCL10 and IL-6, and does not infl uence the RANKL/OPG GCs can affect differentiation by engagement of multiple ratio in osteoblastic cell lines and primary cells (123, 132), mechanisms and factors, including suppression of BMP/ whereas expression of Il11 and osteoblast differentiation are TGF β signaling (121, 122) , growth hormone/IGF-1 acti- unaffected by CpdA. Finally, mice with collagen-induced vity and affecting AP-1-dependent LIF and particular IL-11 arthritis receiving an immunosuppressive dose of CpdA have expression by the GR monomer in osteoblasts. IL-11 is a strikingly higher serum osteocalcin levels compared with potent inducer of osteoblastogenesis in vitro and in vivo via dexamethasone-treated animals (123) . Therefore, although the Jak2/STAT3 pathway, and when applied in excess, it can CpdA has a narrow therapeutic window, such compounds counteract the GC-induced suppression of osteoblast differ- with optimized pharmacology could be of major clinical use entiation (123 – 125) . to suppress infl ammatory bone diseases and maintain bone integrity. This example demonstrates that for the future screening Novel criteria for selective GR modulators of optimized GR ligands, a more specifi c screening approach for therapeutic effi cacy and prevention should be undertaken. In particular, cellular readouts need to of osteoporosis be developed, which resemble therapeutic and side effects in vivo . The most promising candidates will be required to avoid Based upon the observation that the monomer GR can repress particular side effects but maintain anti-infl ammatory effi - the activity of pro-infl ammatory transcription factors, in par- cacy. Moreover, selective GR ligands may only be success- ticular, AP-1 and NF-kB, selective ligands for the GR have ful in certain types of infl ammation, as revealed from studies been developed on the principles that they should retain with conditional mutant GR mice. monomeric function of the GR but disrupt dimerization functions. The so-called selective GR modulators or activators (SEGRM, SEGRA) (2, 126) should maintain immune sup- Expert opinion and outlook pression but avoid side effects. In the early years, potential compounds were screened for Advanced understanding about the molecular basis of the their ability to suppress a cytokine promoter and for their physiological effects of GCs was obtained by the analysis inability to transactivate GRE-containing promoters. Selected of conditional knockout and knock-in mice for the GR. The compounds could dissociate between transactivation and requirement of the GR in T cells for immune suppression in transrepression of the GR in vitro. However, some of them a model of multiple sclerosis, but in myeloid cells for models exerted side effects (127, 128), such as weight loss, shrink- of contact allergy and septic shock (66) , could be unequivo- age of the thymus, and bone turnover (128) or the induc- cally demonstrated. In part, the immune suppressive effects tion of gluconeogenetic enzyme genes in liver cells (129) . are dependent on GR dimerization. Also, side effects of GC Nonetheless, for local topical application to treat infl ammatory medication seem to employ different modes of GR activ- skin diseases, the non-steroidal compound ZK245186 looks ity. For GIO, interaction of the GR monomer with AP-1 is to be a promising candidate to maintain anti-infl ammatory required, whereas for induction of adipogenesis, a dimeriza- effi cacy and to avoid brittleness of the skin (130) and is cur- tion competent GR is necessary. rently in clinical trials. Studies using cell-type-specifi c and Albeit these analyses are far from being complete, it function-selective knockout mice have revealed different GR becomes evident that for different infl ammatory disease mechanism requirements for the therapeutic and side effects models and for different side effects, specifi c modes of of classic GR ligands (Figure 2 ). For some anti-infl ammatory GR action in particular cell types are involved. Further activities, such as irritant skin infl ammation, a GR defec- insights of the tissue-specifi c molecular mechanisms of the tive in dimerization is able to perform full anti-infl ammatory GR will be obtained in the near future by combining the activity. In other infl ammatory types, contact allergy, sep- physiological analysis of the transgenic mouse models with tic shock, and arthritis, dimer-dependent mechanisms are the genome-wide determination of gene regulatory activity required. Furthermore, the activity of the GR in distinct cell and functional interference of GR target genes in a tissue- types appears to be critical. Side effects also utilize GR dimer- specifi c manner. The comprehensive understanding of GR dependent and GR dimer-independent mechanisms. The biology will give novel rationales for the development induction of hyperglycemia appears to be GR dimer depen- of specifi c GR modulators that may reduce side effects dent (Rauch and Tuckermann, unpublished data) as expected, and keep anti-infl ammatory effi cacy in particular disease whereas GIO occurs in the absence of GR dimerization and conditions. 248 S. Hü bner and J. Tuckermann

Rheumatoid arthritis Fat redistribution Endotoxin ecific cell type Sp Sepsis in vitro T-cells adipocytes ular mech Contact lec an ? o is Type 2 diabetes hypersensitivity M m

t Myeloid

D c cells e e f GR dimer Search for GR dimer t f

e i

m l selective

a osteo- e

i AP1,N F-κB, AP1 n

c glucocorticoid blast

i t

f others? GR a

e GR receptor

Irritant monomer Osteoporosis l

monomer e e

inflammation agonists f

B B

c c

t t

s s

? T-cells ? ? ? Hypertension

EAE ? ?

Depression

Asthma Skin atrophy

Figure 2 Different physiological processes in steroid therapy require cell-type-specifi c modes of GR action. Shown is a graphical summary of the results from analyses of GR dimer-defi cient and cell-type-specifi c GR knockout mice in benefi cial (left) and detrimental effects (right) of GC therapy. For benefi cial anti-infl ammatory effects of GCs (left), GR dimerization is required in animal models for arthritis, sepsis, and contact allergy. For irritant infl ammation, the monomer GR activity is suffi cient; however, the involved interacting partners remain to be identifi ed. The GR in T cells is required for the immunosuppressive effects in an animal for model experimental autoimmune encephalomyelitis (EAE) and arthritis, whereas cell-type requirements for asthma have not been demonstrated. Detrimental side effects (right) of the GR dimer-dependent processes in adipocytes are important for differentiation and possibly for fat redistribution. Type 2 diabetes seems to be GR dimer dependent, which has to be further investigated. Monomer GR-AP-1 interaction is critical for GC-induced osteoporosis. Models for hypertension, brittleness of the skin and depression remain to be analyzed with conditional GR knockout mice. Selective GR modulators should fulfi ll the molecular requirements, specifi cally for individual infl ammatory diseases, and then should exert distinct capacities to reduce side effects.

Highlights • Selective GR modulators addressing cell-type-spe- cific mechanisms of the GR are required for reduced side effects in the therapy of particular inflammatory • GR acts by different mechanisms to suppress infl ammation diseases. dependent on the disease type. • Side effects may depend on GR dimerization but also employ GR monomer dependent activities as in the case of GIO. Acknowledgments • Molecular mechanisms of GR activity in a genome-wide manner are just becoming uncovered and reveal novel The authors thank Dr. Ulrike Baschant for critical reading of the DNA recognition sequences for direct or indirect binding manuscript. The authors are grateful for the support from the funds of of the GR. How cell-type specifi city governs gene regula- the Boehringer Stiftung, the Deutsche Forschungsgemeinschaft (TU tion by the GR remains a challenge in the fi eld and needs 220/3 and TU220/6), and the EU (BrainAge). The authors declare no to be addressed. confl ict of fi nancial interests. Glucocorticoid receptor in steroid therapy 249

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Sabine H ü bner studied Jan Tuckermann studied Biology at the University of Biology in Karlsruhe, Jena in Germany. She is pur- Germany, and obtained his suing her PhD in the group PhD at the German Cancer of Jan Tuckermann study- Research Centre in Heidelberg, ing tissue-speciﬁ c hormone Germany. He was appointed action at the Fritz Lipmann as a Junior Group leader at Institute for Age Research in the Leibniz Institute for Age Jena, Germany. Research in Jena, Germany. On April 1, 2012 he becomes the Department Chair of the Institute for General Zoology and Endocrinology at the University of Ulm, Germany.