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Crosstalk in Inflammation: The Interplay ofGlucocorticoid Receptor-Based Mechanismsand Kinases and Phosphatases

Ilse M. E. Beck, Wim Vanden Berghe, Linda Vermeulen, Keith R. Yamamoto,Guy Haegeman,* and Karolien De Bosscher*

Laboratory of Eukaryotic Gene Expression and Signal Transduction (I.M.E.B., W.V.B., L.V., G.H., K.D.B.), Departmentof Physiology, Ghent University, B-9000 Ghent, Belgium; and Department of Cellular and Molecular Pharmacology(K.R.Y.), University of California, San Francisco, California 94143-2280

Glucocorticoids (GCs) are steroidal ligands for the GC receptor (GR), which can function as a ligand-activatedtranscription factor. These steroidal ligands and derivatives thereof are the first line of treatment in a vast arrayof inflammatory diseases. However, due to the general surge of side effects associated with long-term use ofGCs and the potential problem of GC resistance in some patients, the scientific world continues to search for abetter understanding of the GC-mediated antiinflammatory mechanisms.

The reversible phosphomodification of various mediators in the inflammatory process plays a keyrole in modulating and fine-tuning the sensitivity, longevity, and intensity of the inflammatory response.As such, the antiinflammatory GCs can modulate the activity and/or expression of various kinases andphosphatases, thus affecting the signaling efficacy toward the propagation of proinflammatory geneexpression and proinflammatory gene mRNA stability. Conversely, phosphorylation of GR can affect GRligand- and DNA-binding affinity, mobility, and cofactor recruitment, culminating in altered transactiva-tion and transrepression capabilities of GR, and consequently leading to a modified antiinflammatorypotential.

Recently, new roles for kinases and phosphatases have been described in GR-based antiinflammatory mecha-nisms. Moreover, kinase inhibitors have become increasingly important as antiinflammatory tools, not only forresearch but also for therapeutic purposes. In light of these developments, we aim to illuminate the integratedinterplay between GR signaling and its correlating kinases and phosphatases in the context of the clinicallyimportant combat of inflammation, giving attention to implications on GC-mediated side effects and therapyresistance. (Endocrine Reviews 30: 830–882, 2009)

I. IntroductionA. Inflammation at a molecular levelB. Glucocorticoid receptor-mediated signaling

II. Phosphoregulation of the Glucocorticoid ReceptorA. GR phosphorylationB. GR dephosphorylationC. Other posttranslational modifications of GR

III. Kinases Targeted by Glucocorticoid Receptor-Medi-ated SignalingA. Mitogen-activated protein kinases (MAPKs)B. MAPK-activated protein kinases (MKs)C. Cyclin-dependent kinases (Cdks)D. I�B kinase � (IKK�)

E. TANK-binding kinase 1 (TBK1)F. Other kinases

IV. Phosphatases Targeted by Glucocorticoid Receptor-Mediated SignalingA. Dual specificity phosphatases (DUSPs)B. Other protein Y phosphatasesC. Other phosphatases

V. Kinase/Phosphatase Regulation in Glucocorticoid-Mediated Side EffectsA. Skeleton and muscle effectsB. Hyperglycemia and diabetesC. Other side effects

VI. Kinase/Phosphatase Regulation in GlucocorticoidResistance

VII. Future Perspectives in the Combat of InflammationA. New glucocorticoid receptor ligandsB. Combination therapiesC. MicroRNA-specific modulation of GRD. Epigenetic approaches

VIII. Conclusions

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2009 by The Endocrine Societydoi: 10.1210/er.2009-0013 Received March 31, 2009. Accepted August 18, 2009.First Published Online November 4, 2009* G.H. and K.D.B. share senior authorship.

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I. Introduction

According to the World Health Organization (WHO;2007 report), inflammation and inflammation-me-

diated illnesses are the biggest challenge in current medi-cine because 300 million people worldwide are estimatedto suffer from asthma and 210 million people live withmild or severe chronic obstructive pulmonary disease(COPD), the latter leading up to 5% of global deaths.Furthermore, many people live uncomfortably withchronic inflammatory disorders, such as rheumatoid ar-thritis and inflammatory bowel disease. Moreover, theonset of cancer and cardiovascular diseases has also beenlinked to inflammation, claiming 13 and 30% of globaldeaths, respectively (WHO). As the costs of treating thesedisorders mount up and life comfort and expectancy arethreatened, understanding and resolving inflammation iscurrently one of the main targets in science.

Today, glucocorticoid (GC)-based therapy is still themost commonly used treatment to combat chronic andacute inflammation. Since the discovery of the antiinflam-matory properties of human cortisone in rheumatoid ar-thritis (1) and the cloning of the GC receptor (GR) (2),tremendousprogresshasbeenmade inunderstandinghowGCs inhibit inflammation: the molecular antiinflamma-tory mechanism of GCs consists of GR-mediated transac-tivation and transrepression mechanisms, the latter ofwhich prominently features inhibition of nuclear fac-tor-�B (NF-�B) activation and activity.

GCs have multiple physiological actions. As a conse-quence, a chronic exposure to pharmacological hormonedoses becomes a problem in therapeutic settings, causingundesirable, yet on-target and thus GR-mediated, effects.The challenge is therefore not to develop more specificligands for GR, but to change the spectrum of GR-medi-ated events and try to skew it more toward antiinflamma-

tory pathways. This implies that selective (in terms of func-tionality) GR modulators could eliminate these adverseeffects. Besides the undesirable effects, GC resistance, inwhich the patients do not respond to GCs, may also occur.Therefore, the mainstay of antiinflammatory research ef-forts is focused on further characterizing the antiinflam-matory mechanisms of GCs in detail and developing newtherapeutic strategies to fight inflammation with a betterbenefit-to-risk-ratio.

Protein kinases (afterward referred to as kinases) areenzymes that can rapidly and reversibly phosphorylateS, T, or Y residues of cellular proteins and as such affecttheir structure, function, location or metabolism. Inturn, phosphatases function to revert the action of thesekinases by dephosphorylating specific target residues(3). The GR itself is on the one hand subject to intense phos-phoregulation, thus impacting its role in various antiinflam-matoryprocesses,andontheotherhandthisGRdeploysandaffects kinases and phosphatases as tools to implement itscellular antiinflammatory effects. In this review, we willfocus on the above events, providing a contemporary viewon the overall phosphomodulatory effects of and by theGR in the framework of inflammation. Additionally, therole of various phosphorylation events in the describedGC-mediated side effects and the reported phenomenonof GC resistance will be discussed. Ultimately, we willdiscuss future therapeutic implications of phosphoregu-lation in the context of GR-based antiinflammatorystrategies.

A. Inflammation at a molecular levelInflammation is an initially advantageous response to

intracellular damage or an extracellular challenger, pro-voking the activation of various proinflammatory medi-ators with the purpose to remove the damaging agent andto restore tissue structure and function. Physiologically,

Abbreviations: AF, Activation function; AP-1, activator protein-1; ARE, adenylate uridylate-rich element; ATF, activating transcription factor; C, carboxy; CBP, CREB-binding protein;cdc37, cell division cycle 37 protein; Cdk, cyclin-dependent kinase; C/EBP, CCAATenhancer-binding protein; COPD, chronic obstructive pulmonary disease; COX-2, cycloox-ygenase-2; CpdA, compound A; CREB, cAMP-responsive element-binding protein; CRM1,chromosome region maintenance (synonym, exportin1); CTD, C-terminal domain; DBD,DNA-binding domain; Dexras1, dexamethasone-induced Ras1; DUSP, dual specificityphosphatase; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; eIF-4E/F,eukaryotic translation initiation factor 4E/F; ELKS, protein rich in amino acids E, L, K and S;ENaC, epithelial Na� channel; FKBP, FK506-binding protein; GC, glucocorticoid; GILZ,GC-induced leucine zipper; GM-CSF, granulocyte monocyte-colony stimulating factor;G6Pase, glucose-6-phosphatase; GR, GC receptor; GRE, GC response element; GRIP1,GR-interacting protein 1; GSK, glycogen synthase kinase; H3, histone H3; HAT, histoneacetyl transferase; HDAC, histone deacetylase; hGR, human GR; HPA, hypothalamic-pi-tuitary-adrenocortical; Hsp, heat shock protein; ICAM, intercellular adhesion molecule; IE,immediate-early; Ifit1, IFN-induced with tetratricopeptide repeats 1; IFN, interferon; I�B,inhibitor of NF-�B; IKK, I�B kinase; IL1RI, IL-1 receptor I; IRAK, IL-1 receptor-associatedkinase; IRF, interferon regulatory factor; ISRE, IFN-stimulated response element; JAK, Januskinase; JDP, Jun dimerization protein; JNK, c-Jun N-terminal kinase; KO, knockout; LABA,long-acting �2 agonist; LBD, ligand-binding domain; Lck, lymphocyte kinase; LPS, lipo-polysaccharide; Mal, MyD88-adapter like protein; MED14, mediator complex subunit 14(synonym, DRIP150); MEKK, MKK kinase (synonyms, MKKK, MAPKKK, MAP3K); mGR,mouse/murine GR; miRNA, microRNA; MK, MAPK-activated kinase; MKK, MAPK kinase

(synonyms, MEK, MAPKK, MAP2K); MMP, matrix metalloproteinase; MMTV, mouse mam-mary tumor virus; MNK, MAPK-interacting kinase; MR, mineralocorticoid receptor; MSK,mitogen- and stress-activated protein kinase; MyD88, myeloid differentiation primary re-sponse gene 88; NCoR, nuclear corepressor; NF-�B, nuclear factor-�B; nGRE, negativeGRE; NHE, Na�/H� exchanger; NIK, NF-�B-inducing kinase; NLS, nuclear localizationsignal; p/CAF, p300/CBP-associated factor; PEPCK, phosphoenylpyruvate carboxykinase;PI3K, phosphatidylinositol-3-kinase; PKA, protein kinase A; PKB, protein kinase B (syn-onym, Akt); PKC, protein kinase C; PP, protein phosphatase; P-TEFb, positive transcriptionelongation factor b; PTP, protein tyrosine phosphatase; RANKL, receptor activator of NF-�Bligand; RANTES, regulated upon activation, normal T-cell expressed and secreted; rGR, ratGR; RIP, receptor-interacting protein; RNA pol II, RNA polymerase II; ROCK, Rho-dependentprotein kinase; RSK, ribosomal S6 kinase; SEGRA, selective GR agonist; SEGRM, selectiveGR modulator; SEK1, SAPK/ERK kinase 1; SGK, serum and GC-inducible kinase; S6K,70-kDa ribosomal protein S6 kinase; SLAP, Src-like adaptor protein; SOCS, suppressor ofcytokine signaling; Sp1, specificity protein 1; SP-A, surfactant protein-A; SRC, steroidreceptor coactivator; STAT, signal transducer and activator of transcription; SUMO, smallubiquitin-related modifier; TAD, transactivation domain; TAK1, TGF-activated kinase 1;TANK, TRAF family member-associated NF-�B activator; TBK1, TANK-binding kinase 1; TLR,Toll-like receptor; TNFR, TNF-� receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate;TRADD, TNFR-associated death domain; TRAF, TNFR-associated factor; Tram, translocatingchain-associating membrane protein; TRE, TPA-response element; Trif, TIR domain-con-taining adapter-inducing IFN�; Trip6, thyroid receptor-interacting protein 6; TTP,tristetraprolin.

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inflammation is locally marked by swelling, redness, pain,heat, and loss of function. Although inflammation can bea beneficial reaction, return to homeostasis is of the utmostimportance to avoid the onset of an unfavorable chronicinflammation (4). The inflammatory reaction can be pro-voked by physical injury, tissue damage, or the invasion ofalien pathogens. Alternatively, inflammation can also beprovoked by an unwanted immune reaction of the body toits own proteins, i.e., an autoimmune reaction.

Biologically, inflammation progresses through differ-ent stages. First, local hyperemia is brought about by va-sodilators. Ensuing inflammation is characterized by anexudation or leakage of plasma from the blood vessels intothe inflamed tissue. This process is facilitated by an in-creased permeability of the endothelium and the aug-mented hydrostatic pressure in the blood capillaries. Next,cytokines and chemokines cause leukocytes (macro-phages, neutrophils, etc.) to infiltrate the inflamed tissueto accommodate the phagocytosis of cellular debris andpathogens. Finally, fibroblasts proliferate to reinstate tis-sue structure. All cells involved in the inflammatory pro-cess can sense the environment, responding to variousproinflammatory stimuli, and as a result initiate cytokineand chemokine cascades (5–7).

As such, the increase of inflammatory mediators, suchas cytokines [TNF� (afterward referred to as TNF), IL-1�,IL-6, granulocyte monocyte-colony stimulating factor(GM-CSF), etc.], chemokines [IL-8; regulated upon acti-vation, normal T-cell expressed and secreted (RANTES);melanoma growth-stimulating activity (Gro)-�; etc.],growth factors [fibroblast growth factor (FGF), epidermalgrowth factor (EGF), etc.], lipid-derived mediators (pro-stanoids, leukotrienes,), receptors [TNF receptor (TNFR),Toll-like receptor (TLR)], enzymes [inducible nitric oxidesynthase (iNOS), cyclooxygenase-2 (COX-2), matrix met-alloproteinases (MMPs), phospholipase A2, etc.), adhesionmolecules [intercellular adhesion molecule-1 (ICAM1), vas-cular cell adhesion molecule-1 (VCAM1), E-selectin, P-se-lectin], and peptides (bradykinin, tachykinin, endothelin), isdeemed pivotal for the propagation and progression of in-flammation. Conversely, to control the course of the inflam-matory process, modulatory and antiinflammatory cyto-kines such as IL-10, IL-4, and TGF� are released (8).

On a molecular level, inflammatory diseases are medi-ated by stimulation via proinflammatory signals, like thebacterial lipopolysaccharide (LPS), viral factors, or theself-produced IL-1� or TNF. Binding of these agents totheir respective receptors culminates predominantly inthe activation of activator protein-1 (AP-1) and NF-�B,but also in the activation of other transcription factors.In turn, both NF-�B and AP-1 stimulate the expression

of proinflammatory cytokines, chemokines, and adhe-sion molecules, thus propagating cellular inflammation(Fig. 1) (9 –12).

These activation pathways rely on a signaling cascadeof intermediary factors and especially kinases. For in-stance, soluble TNF binds to a membrane-imbeddedTNFR. Upon binding of TNF to TNFR1, the receptorhomotrimerizes and recruits the TNFR-associated deathdomain (TRADD) protein to the cytoplasmic death do-main of TNFR1 (13–16). Subsequently, the adhered pro-tein complex is supplemented by receptor-interacting pro-tein 1 (RIP1) and TNFR-associated factor 2 (TRAF2) (14,17–19). Ultimately, these adaptor proteins, specificallyTGF-activated kinase 1 (TAK1), NF-�B-inducing kinase(NIK), or MAPK kinase kinase (MEKK3), phosphorylatethe inhibitor of NF-�B (I�B) kinase (IKK) complex andtrigger its dissociation (12, 19–22). This process leads tothe activation of the IKK-NF-�B pathway, critical for in-ducing tissue inflammation. Additionally, TNFR ligandbinding results in the activation of ERK, p38, and c-JunN-terminal kinase (JNK) MAPK via a multilayered kinasecascade (23–25). The MAPK family of protein kinasescomprises ERK, JNK, and p38 MAPKs, of which the ac-tivation and function are regulated by upstream kinasesand stress-related inducers (25). The multilayered activa-tion cascade, enhancing the intracellular signal intensityand integrating various stimuli, is constructed bottom upby MAPKs, MAPK kinases (MAP2Ks or MKKs) andMAPK kinase kinases (MAP3Ks or MEKKs) (see Fig. 5).Preferentially, MAPKs target S/T protein residues, fol-lowed by a P (3, 25). These MAPKs diverge the web ofTNF-affected factors via multiple downstream kinase, co-factor, and transcription factor targets. Interestingly, theMAPK cascade is also involved in the posttranslationalcontrol of NF-�B and activation of AP-1 (26, 27).

Alternatively, IL-1� signals via activation of the type IIL-1 receptor (IL1RI) and its associate intermediates my-eloid differentiation primary response gene 88 (MyD88)and IL-1R-associated kinases (IRAK) 1, IRAK2, andIRAK4 and TAK1 toward activation of the MAPK path-way, AP-1, and NF-�B (11).

Lastly, various viral and bacterial factors can activatethe related Toll-like receptors (TLR1-10). For example,TLR3 is activated by double-stranded RNA and TLR4 bythe bacterial cell wall component LPS (28, 29). TLR3 andTLR4 signal via TIR domain-containing adapter-inducinginterferon (IFN) � (Trif) and MyD88, and via MyD88-adapter like protein (Mal) and translocating chain-asso-ciating membrane protein (Tram), respectively, to onceagain result in the activation and modulation of the MAPKpathway, AP-1 and NF-�B transcription factors (11, 12,

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28, 29). Alternatively, TLR3 and TLR4 can signal viaTANK-binding kinase 1 (TBK1) and IKK� to activate IFNregulatory factor (IRF) 3 and to the subsequent transcrip-tion of type I IFN-inducible genes (30).

1. NF-�BBecause NF-�B is activated by a broad range of inflam-

matory and environmental stimuli, this transcription fac-tor, which plays critical roles in both innate and adaptiveimmunity, serves as a biological sensor. Moreover, NF-�Bis a pivotal regulator of inflammation because its activitylevel is raised in—and defective NF-�B signaling is asso-

ciated with—an ever increasing list of inflammatory andpathological conditions (31–36).

a. NF-�B structure. The transcription factor NF-�Bcomprises a family of five members: p65 (RelA), RelB,c-Rel, NF-�B1 (p50/p105), and NF-�B2 (p52/p100). Allmembers are characterized by an N-terminal Rel-homol-ogy domain, which is required for homo- or heterodimer-ization, sequence-specific DNA binding, and nucleartranslocation of NF-�B via its nuclear localization signal(NLS). Furthermore, RelA/p65, RelB, and c-Rel have C-terminal transactivation domains (TADs), whereas NF-

FIG. 1. Inflammation at a molecular level: a simplified scheme. TNFR activation by TNF, IL1RI by IL1�, TLR3 by double-stranded RNA (dsRNA), TLR4by bacterial LPS, and activation of other TLRs can signal via specific intermediary factors such as TRADD, TRAF2, RIP1, MEKK3, TAK1, TAB2/3, andNIK (for the TNFR), MyD88, IRAKs, TRAF6 and TAK1 (for IL1RI), Trif, RIP1, TRAF6 and TAK1 (for TLR3) and MyD88, Mal, Trif, Tram, RIP1, IRAKs, TRAF6and TAK1 (for TLR4) to the MAPK pathway and to the activation and regulation of NF-�B and AP-1. Additionally, triggering TLR3 or TLR4 signalingcascades can instigate IKK� and TBK1 activation and subsequent IRF3-regulated gene transcription (10–12, 29). TAB, TAK1-binding protein.


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�B1 and NF-�B2 are, respectively, p105 and p100 C-ter-minal ankyrin repeat-containing precursors (10), whichare proteasomally processed to yield N-terminal productsNF-�B p50 and p52, respectively. These mature species thenheterodimerize with a TAD-containing NF-�B family mem-ber or with the TAD-bearing I�B protein B cell lymphoma-3(Bcl-3) to form transcriptionally active complexes (37).

All NF-�B family members can form homo- or het-erodimers directing the dimer to a specific set of targetgenes. Recent evidence suggests that alternative splicing ofNF-�B components provides an additional way of con-trolling NF-�B signaling (38). NF-�B dimer function isspecific because the ablation of a certain NF-�B familymember cannot be compensated for (39). Because specificNF-�B dimer-promoter interaction is both context- andstimulus-dependent, the sequence of the NF-�B recogni-tion site is not the sole determinant of NF-�B dimer-pro-moter interfacing (40–43). The main research target ininflammation is the prevalent heterodimer NF-�B p65-p50, of which p50 can increase DNA binding (44) and p65confers transcriptional regulation (10).

b. NF-�B activation. With regard to the NF-�B activationmechanism, multiple pathways have been described. Themechanism by which NF-�B is activated by proinflamma-tory TNF is commonly referred to as the canonical acti-vation pathway (Fig. 1). In this canonical NF-�B activa-tion pathway, the transcription factor NF-�B dimerp65-p50 is, in its resting state, held in the cytoplasm by aninhibitory I�B molecule, most commonly I�B� (10, 45).I�B associates via its ankyrin repeats domain with NF-�B,which thus masks the NF-�B and I�B NLS motifs, conse-quently restricting inactive NF-�B to the cytoplasm. Whencells are challenged with a proinflammatory signal, such asTNF, the IKK complex becomes activated. The activatedIKK complex can phosphorylate I�B� on S32 and S36 orI�B� on S19 and S23, leading to polyubiquitination ofI�B� or I�B� and ultimately culminating in I�B degrada-tion by the 26S proteasome (10, 46, 47). Because NF-�Bis released from its cytoplasmic constraint, NF-�B trans-locates into the nucleus, guided by its NLS, where it canbind specific genomic target sequences.

The IKK complex comprises the catalytically activesubunits IKK� and IKK� together with the scaffold reg-ulatory subunit IKK� [NF-�B essential modulator(NEMO)] (48). Additional, but most likely transient, com-ponents of the IKK complex are the chaperoning heatshock protein 90 (Hsp90), cell division cycle 37 protein(cdc37), and protein rich in amino acids E, L, K, and S(ELKS) (49–51). Hsp90 is necessary to allow the relocal-ization and thus activation of the IKK complex to themembrane-associated and activated TNFR1 complex,

whereas cdc37 mediates the transient recruitment ofHsp90 to the IKK complex (49, 50, 52). Furthermore,ELKS is a necessary regulatory component of the IKKcomplex, serving as an auxiliary docking protein for I�B�

(51). The IKK complex-mediated phosphorylation of I�Brequires IKK� and IKK�, but does not necessitate IKK�,although IKK� can also phosphorylate I�B (47, 53).

Secondly, the noncanonical pathway, initiated via Bcell-activating factor (BAFF), lymphotoxin �, and otherinducers, entails cytokine, among which TNF, and virus-initiated NIK activation (54, 55). Activated NIK triggersIKK� homodimer activation, and subsequently S865,S869, and S871 of the p100 subunit in the p100-RelBcomplex becomes phosphorylated (56). This phosphory-lation induces proteasomal processing to the p52 NF-�Bsubunit (57–59). The p52-RelB complex, consequently,translocates to the nucleus, where it targets specific genepromoters, such as IL-2 (60, 61), for activation.

c. Posttranslational modifications of NF-�B. Subsequent tothe NF-�B activation process, NF-�B activity is substan-tially modulated by various posttranslational modifica-tions: acetylation (62–65), SUMOylation (66), and phos-phorylation (63, 67), of which the last is best characterizedso far. The intracellular control of NF-�B transactivation(duration and intensity) (64, 68, 69), subcellular localiza-tion (70), DNA-binding affinity (64, 65, 69, 71), andNF-�B interaction with cofactors and I�B� (72–74) viavarious posttranslational modifications forms an intricateweb of NF-�B management. Phosphorylation sites ofNF-�B p65 are spread out over the Rel-homology domainand TAD1 and TAD2 (Fig. 4C). Although some of thesephosphomodifications contribute to the transcriptionalactivity of NF-�B (73, 75–78), others do not (79).

In particular, the phosphorylation of NF-�B p65 S276by p38 and ERK MAPK-activated mitogen- and stress-activated protein kinase 1 (MSK1) or protein kinase A(PKA) is pivotal for proper initiation of specific inflam-matory gene expression (73, 74, 76, 80–84). Phosphor-ylation of NF-�B p65 S276 facilitates association of p65with the coactivators cAMP-responsive element-bindingprotein (CREB)-binding protein (CBP) and p300 (73, 74,83, 85) and the transcription elongation complex P-TEFb(positive transcription elongation factor b), consisting ofcyclin-dependent kinase, Cdk9 and cyclin T1 (81). Fur-thermore, NF-�B p65 S276 phosphorylation can enhancethe displacement of the inhibitory histone deacetylase 1(HDAC1)-NF-�B p50 complex to derepress proinflam-matory gene promoters (85). Therefore, NF-�B S276phosphorylation is a crucial step in NF-�B driven pro-moter activation of specific gene targets (73, 76, 80, 86).However, not all NF-�B-dependent genes require the


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phosphorylation of NF-�B S276 for their transcription.Whereas the transcription of ICAM, VCAM, Gro-�, IL-8,and IL-6 depend on the NF-�B p65 S276 phosphorylation,the NF-�B-mediated transcription of major histocompat-ibility complex-I (MHC-I), mangano-superoxide dis-mutase (MnSOD), and I�B� do not (73, 81, 86). Of note,NF-�B-mediated gene transcription independent of NF-�BS276 phosphorylation shows constitutive binding of RNApolymerase II (RNA pol II) (81). This differentiation sug-gests that a NF-�B phosphorylation code controls NF-�B-mediated transactivation of specific target genes (87),possibly defined by the architecture and topology of thetarget promoter (88). Consequently, the selective phos-phorylation of S276 of NF-�B p65 may be responsible forsome of the differential sensitivity of certain �B sites torepression by GCs. Because the phosphorylation of NF-�Bp50 S337, which appears to be essential to NF-�B p50DNA binding, is mediated by PKAc and possibly MSK1,these kinases are attributed an important role in theregulation of NF-�B p65-p50-dependent gene tran-scription (89).

Various kinases can phosphorylate NF-�B S536: IKK�,IKK�, TBK1, IKK�, ribosomal S6 kinase 1 (RSK1), andglycogen synthase kinase (GSK) 3� (71, 90–96). Also, thephosphorylation of NF-�B p65 S536 can contribute to theactivity of NF-�B p65, most likely via facilitating the in-teraction between activated NF-�B and p300 (62, 93).Additionally, NF-�B p65 S536 phosphorylation is re-ported to weaken the binding of NF-�B to I�B, thus pro-longing the activity of NF-�B in the nucleus (71, 93). Ofnote, acetylation of NF-�B of multiple lysine residues,probably by the histone acetyl transferase (HAT) activityof CBP/p300, is preceded by and requires NF-�B S276 andS536 phosphorylation (62).

A third NF-�B phosphorylation that can positively af-fect NF-�B activity is the protein kinase C (PKC) �-medi-ated phosphorylation of NF-�B p65 S311. Similar toNF-�B p65 S276 and S536 phosphorylation, the phos-phorylation of NF-�B at S311 can enhance the interactionof NF-�B p65 with CBP. Moreover, NF-�B p65 S311phosphorylation augments NF-�B recruitment to �B sitesclose to the promoter of the proinflammatory cytokineIL-6 (75).

Phosphorylation of NF-�B p65 T254 by an unknownkinase can enhance the NF-�B p65 activity via inducingthe interaction of T254 and P255 of NF-�B p65 with thenuclear peptidyl-prolyl isomerase Pin1 [protein NIMA(never in mitosis gene a)-interacting]. This interaction re-sults in the isomerization of the P residue, entailing a con-formational change of NF-�B p65. Consequently, NF-�Bp65’s binding affinity for I�B� is decreased. Furthermore,

this modification stabilizes the NF-�B p65 protein andpromotes the nuclear translocation of NF-�B p65 (97).Conversely, ubiquitination of NF-�B p65, inducing itsproteolysis, is mediated by the E3-ubiquitin ligase sup-pressor of cytokine signaling (SOCS) 1. Because thebinding sites for Pin1 and SOCS1 lie in close proximity,competition for binding might be possible (98). The pro-teasomal degradation of DNA-bound NF-�B p65 pro-motes transcriptional termination (99).

For the phosphorylation of NF-�B p65 S529 by caseinkinase 2 (CK-2), it is still unclear whether this phosphor-ylation can affect NF-�B-mediated transcription (100,101). In contrast, phosphorylation of T505 via activationof checkpoint kinase 1 (Chk1) and ATM/Rad3-related(ATR) checkpoint kinase could decrease the activity ofNF-�B via enhancing its interaction with HDAC1 (102–104). Even so, the IKK�- or GSK3�-mediated phosphor-ylation of NF-�B p65 S468 negatively affects NF-�B ac-tivity (68, 79), whereas IKK�-mediated phosphorylationof this same residue was associated with NF-�B transac-tivation (105).

Conversely, endogenous protein phosphatase (PP) 2Acan associate with and dephosphorylate NF-�B p65 (106).Moreover, pharmacological blockage of PP2A leads toincreased phosphorylation of NF-�B p65 (68, 106). Ad-ditionally, association of PP4 has been linked to the acti-vation of NF-�B via the dephosphorylation of T505 (102,107, 108). Furthermore, it was suggested that a rapid de-phosphorylation of NF-�B S536 could contribute toswitching off NF-�B-dependent gene transcription (109).

d. NF-�B crosstalk. NF-�B-mediated transcription can fur-thermore be coregulated by crosstalk of NF-�B with othertranscription factors and association of NF-�B withvarious cofactors (110). Positive crosstalk of NF-�B-promoting proinflammatory gene transcription has beendescribed for aryl hydrocarbon receptor, specificity pro-tein 1 (Sp1), IRF, signal transducer and activator of tran-scription (STAT), activating transcription factor (ATF),CREB, and AP-1 (111–118). The binding of multiple dis-tinct transcription factor complexes occurs in a highly dy-namic manner (110, 119). An example of negativecrosstalk with NF-�B is shown by GR (120). Cofactorscan either coactivate or corepress NF-�B-mediated genetranscription. Functionally, these cofactors can stimulateor repress the transcriptional activity of the enhanceosome(i.e., the multiprotein complex mediating promoter acti-vation and gene transcription) (121) or alter the chromatinstructure. Many coactivators, including p300, CBP, p300/CBP- associated factor (p/CAF), and steroid receptor co-activator 1 (SRC1) have a HAT domain, capable of acety-lating histones but also other proteins (122–127).


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Interestingly, IKK� can phosphorylate CBP, increasingCBP activity and CBP binding to p65 (128). Conversely,corepressors often include HDAC activity (HDAC1,HDAC2, HDAC3) and can be directly or indirectly re-cruited to NF-�B-dependent gene promoters (85, 129–133). Other NF-�B-dependent gene-associated core-pressors are the NF-�B p65-p50-binding silencingmediator for retinoid and thyroid-hormone receptors(SMRT) and the NF-�B p50-binding nuclear corepres-sor (NCoR) (134, 135).

e. NF-�B-targeted genes. After NF-�B activation, nuclearNF-�B p65 can enhance gene expression of multiple proin-flammatory genes via the occupation of a NF-�B-specificpromoter recognition site (136). Because NF-�B can beconsidered a central regulator of proinflammatory genetranscription, NF-�B function is a highly dynamic signal-ing event, showing differential temporal expression pro-files for different genes (40, 137). Moreover, associationof NF-�B with its specific recognition sites is transient,with a half-life of seconds, suggesting a dynamic regula-tion of enhanceosome composition and gene transcriptionconstantly sensing the inflammatory status of its environ-ment (119).

The canonical or classical NF-�B activation pathwaycontrols among others leukocyte activation/chemotaxis,cellular metabolism, antigen processing, and negative reg-ulation of the TNF signaling pathway (138, 139). Theactivation of NF-�B results in gene transcription of cyto-kines (e.g., IL-6), chemokines (e.g., IL-8 and RANTES),adhesion molecules (e.g., E-selectin), enzymes (e.g.,iNOS), and other inflammatory mediators (8). Further-more, the cytokine TNF, but also IL-1�, can activate NF-�B, and these are thus mediators of a feedforward mech-anism perpetuating inflammation.

However, it is essential to prevent the uncontrolledpropagation of inflammation, which would cause sys-temic disorders. Therefore, NF-�B activation is also sub-ject to an autoregulatory negative feedback loop. First,gene transcription of I�B� is stimulated by NF-�B activa-tion. The newly synthesized I�B� replenishes the formerproteasomally degraded I�B� levels and can bind to activeNF-�B complexes in the nucleus, weakening NF-�B DNAbinding and subsequently transporting NF-�B back to thecytoplasm (10, 140, 141). Furthermore, upregulation ofthe anti-apoptotic protein A20, the A20-binding inhibitorof NF-�B activation, ABIN-1, the TNF�-converting en-zyme TACE, the tumor suppressor cylindromatosis(CYLD), the antiinflammatory cytokine IL-10, and themicroRNA miR-146 ultimately results in a negative reg-ulation of NF-�B activity (142–153).

2. AP-1AP-1 is a transcription factor of general importance for

many cellular processes in different organs, including in-flammation. Among the target genes of AP-1 are impor-tant regulators of cell proliferation, differentiation, andapoptosis. The DNA binding of the AP-1 complex to 12-O-tetradecanoylphorbol-13-acetate (TPA)-response ele-ment (TRE) sequences is rapidly induced by growth fac-tors, cytokines, and oncoproteins, which are implicated inthe proliferation, survival, differentiation, and transfor-mation of cells (154).

a. AP-1 structure. Similar to NF-�B, AP-1 is a homo- or het-erodimeric transcription factor complex that can be tar-geted to its regulatory sites in a sequence-specific manner(Fig. 1). The subunits of AP-1 are selected from the Jun(c-Jun, v-Jun, Jun B, and Jun D), Fos (c-Fos, Fos B, Fra-1,and Fra-2), activating transcription factor [ATF2, ATF3,B-ATF, Jun dimerization protein (JDP)-1, JDP-2], or MAF(MAFA, MAFB, c-MAF, NRL, MAFF, MAFG, andMAFK) families (154–156). The most predominantly oc-curring forms of AP-1 are Fos/Jun heterodimers, whichshow preferential binding to heptameric TREs when com-pared with ATF2/Jun homo- or heterodimers, which bindpreferentially to octameric TREs and are strongly inducedby the tumor promoter TPA (157). AP-1 proteins areknownasbasic leucine-zipperproteinsbecausetheydimerizethrough a leucine zipper motif and contain a basic domainfor interaction with the DNA backbone. While the Fos pro-teins do not form homodimers but can heterodimerize withmembers of the Jun family, the Jun proteins can both ho-modimerize and heterodimerize with other Jun or Fosmembers to form transcriptionally active complexes (155,158). In addition to Fos proteins, Jun proteins can alsoheterodimerize efficiently with other AP-1 family mem-bers, such as the ATF family (159), and other basiczipper-containing transcription factors (156, 160). Al-though members of the Jun and Fos families share a highdegree of structural homology, the individual AP-1dimers exert significant differences in their DNA-bind-ing affinity and their capability to activate or suppressgene expression (155).

b. AP-1 activation and activity. Regulation of net AP-1 ac-tivity can be achieved through changes in transcriptionof genes encoding AP-1 subunits, control of theirmRNA stability, posttranslational processing, turnover ofpreexisting or newly synthesized AP-1 subunits, and spe-cific interactions between AP-1 proteins and other tran-scription factors and cofactors.

Various stimuli, including physiological agents such asgrowth factors and cytokines, pharmacological com-


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pounds, such as anisomycin, phorbol esters and okadaicacid, and stressors such as UV radiation, hyperosmoticand heavy metal stress, rapidly elicit transcription of“immediate early” (IE) genes, such as those of the Fos andJun families, by activation of MAPK cascades (161). Theseso-called “IE genes” are activated directly and require nonew transcription or translation for their induction. TheFos and Jun proteins then activate and repress other genes,thereby producing secondary transcriptional reprogram-ming appropriate to specific stimuli. Because these pro-teins are differentially expressed and regulated in a cell-and stimulus-specific manner, every cell type produces acomplex mixture of AP-1 dimers with subtly differentfunctions (154, 161). In most cases, enhanced expressionof c-jun gene, protein, and function is not a solitary eventbut can be accompanied by an induction of transcriptionfactors that are related to c-Jun (e.g., Jun B, Jun D), Fosfamily members (Fos, FosB, Fra-1/2), or ATF familymembers allowing the formation of functionally differ-ent heterodimers in a cell- and time-specific manner.Whereas some AP-1-regulated genes are preferentially in-duced by cJun-cFos dimers, others are mainly induced byJun D-Fra-1 dimers. Fra-1 and Fra-2 promoters are acti-vated by Jun-Fos dimers. The fos and jun genes are con-trolled by multiple upstream elements; for human c-fos,these include a cis-inducible element, a serum responseelement (SRE), a ternary complex factor site (TCF), anAP-1 site, an AP-1/CRE, a direct repeat, and a cAMP re-sponse element. The human c-jun promoter is controlledby the Jun2 AP-1 site, a footprint (FP), an NF-Jun site, twooverlapping Sp1 sites, a CCAAT box, the Jun1 AP-1 site,a related to serum response factor site (RSRF), and in the5�-untranslated region, two AP-2 sites and a weak AP-1site (158, 161, 162). These regulatory elements are highlyconserved between mouse and human.

AP-1 is induced by several external stimuli that increaseMAPK activity. Expression of c-Fos is induced by TCFs,which are activated through phosphorylation by the ERKMAPKs.

IE gene expression of c-Jun may be achieved throughATF or c-Jun. Alternatively, c fos and myocyte enhancerfactor 2 (MEF2), transcription factors can also induce c-jun expression in other contexts. The targeting of MAPKsto transcription factors controlling c-fos and c-jun geneexpression has been very clearly established, and phos-phorylation of these factors is further implicated in re-cruitment of coactivators such as p300/CBP and p/CAF, tothese promoters (163, 164).

c. Posttranslational regulation of AP-1. Posttranslational ac-tivation of AP-1 is produced by translocation of a stimu-lus-dependent kinase, such as MAPKs or their effector

kinases, which bind to and phosphorylate these factors,effecting transactivation and transcription of the associ-ated gene (154, 158). The mechanism of posttranslationalcontrol is most extensively documented in the case of mi-togen- and cellular stress-induced hyperphosphorylationand, in particular, activation of Jun through the JNK cas-cade (165, 166). Activated by a MAPK cascade, the JNKstranslocate to the nucleus, where they phosphorylate Junwithin its N-terminal TAD at S63 and S73 and therebyenhance its transactivation potential. The JNKs also phos-phorylate and potentiate the activity of JunD and ATF2.Alternatively, p38 MAPK-dependent phosphorylation ofc-Jun S63 and S73 has been demonstrated in response toa UV stimulus (167). Moreover, DNA-binding activity ofc-Fos, FosB, and JunB were also dependent on the p38protein kinase activity, whereas JunD, Fra-1, and Fra-2were not affected. A complex network of signaling path-ways that involves external signals for growth factors-Ras-Raf-MEK-ERK families also regulates AP-1 activity(168). Activated Ras or MEK1 primarily induces Fra-1and c-Jun after N-terminal phosphorylation by JNKs. Incontrast to c-Jun, JunB is not an efficient substrate forJNK. Furthermore, although JunD can be phosphorylatedby JNK, its phosphorylation requires interaction withpartners that provide a docking sequence (169). JunB andJunD are less potent collaborators of Ras in cell transfor-mation than c-Jun, which correlates with their lower tran-scriptional activity. ERKs are persistently activated bygrowth factors and oncogenic Ras in tumors and are pos-itive regulators of tumorigenesis (170). As such, they con-tribute substantially to the increased expression and acti-vation of AP-1 members in many tumor types. Potentialcandidates for kinases that regulate Fos activity are the Fos-regulating kinase (FRK), RSK2, and p38 and ERK MAPK(170). When the AP-1 complexes are present in larger quan-tities, glycogen synthase kinase 3� (GSK3�), RSK2, caseinkinase 2, cdc2, PKA, and PKC phosphorylate Fos and Junproteins, thereby regulating their protein stability, DNA-bindingactivity,andthetransactivatingpotentialof theAP-1family members (154, 158).

d. AP-1 dimers and crosstalk. The activities of AP-1 are par-tially modulated through the differential expression of itsindividual components, which determines their dimercomposition (154, 171), and partially through their spe-cific context—cell type, response element sequences andorganization, modification state, interaction with otherregulatory factors, promoter sequence and organization,etc. (172). Whereas Jun, Fos, and FosB are often associ-ated with a strong transactivation potential, JunB, JunD,Fra-1, and Fra-2 are usually found in a context in whichonly a weak transactivation potential is supported. Under


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specific circumstances, the latter might even act as repres-sors of AP-1 activity by competing for binding to AP-1sites or by forming “inactive” heterodimers with Jun, Fos,or FosB. The crucial and determining aspects of “context”are not yet fully understood. To illustrate the importance,it was described that the composition of AP-1 regulatorycomplexes and the biological activities of the bound fac-tors are dynamic and dependent on cell and response el-ement contexts. Col3A is the response element in the col-lagenase-3 gene that confers activation by phorbol estersand repression by GCs in human U2OS osteosarcomacells. The subunit composition and activity of AP-1, whichbinds ColA3, parallels the intracellular level of c-Fos,which is modulated by phorbol esters and GCs. A similarAP-1 site at the collagenase-1 gene, however, not induciblein U2OS cells, was not bound by AP-1, underscoring theimportance of a context-dependent gene regulation (172).

The decision as to whether AP-1 is oncogenic or anti-oncogenic might depend on the antagonistic activity ofdifferent Jun proteins, but it is probably also influenced bytumor type, tumor stage, and the genetic background oftumors (158, 168). For example, c-Jun-Fra-2, but not c-Jun-Fra-1 or c-Jun-c-Fos, inhibits the growth arrest of im-mortalized fibroblasts at confluence and under low-serumconditions. Using dimer-specific mutants of AP-1 pro-teins, in which manipulation of the leucine-zipper domainallows only specific dimers to form, it was demonstratedthat the c-Jun-induced transformation program can beseparated into two distinct pathways: c-Jun-ATF2 activitytriggers growth factor independence, and c-Jun-c-Fos ac-tivity causes anchorage-independent growth (158). Tofully elicit their oncogenic potential, most AP-1 compo-nents need the activity of “cooperating” oncoproteins,which often induce the expression of Jun and Fos proteinsbut also support AP-1-mediated cell transformation byposttranscriptional mechanisms (173, 174). The main co-operating partner of AP-1 is the Ras pathway because celltransformation by activated Ras or MEK1 (MAPK kinase)induces AP-1 protein expression (168). The oncogenic co-operation of c-Jun with Ras and other oncoproteins, func-tioning upstream of Ras, requires N-terminal phosphor-ylation of c-Jun by JNKs.

B. Glucocorticoid receptor-mediated signalingInflammation can be controlled by the stress-induced

release of GCs, mainly cortisol in humans. Besides beingefficient in combating inflammation, GCs display pleio-tropic effects in the regulation of protein, lipid, andcarbohydrate metabolism; innate and adaptive immunesystems; stress homeostatic regulation; reproductive pro-cesses; and growth and brain functions such as memoryand behavior (175).

1. GR domainsThe GR, which binds and mediates the signals of the

GCs, belongs to the superfamily of nuclear receptors. Thissuperfamily can be categorized according to ontogeny andfunction (176). In that respect, the GR is classified asNR3C1, most proximate to the mineralocorticoid recep-tor (MR). However, GRs and MRs nevertheless affect dis-tinct target genes (177). The GR consists of an N-terminaldomain, encompassing a first TAD [activation function 1(AF-1)] responsible for transcriptional activation and as-sociation with certain basal transcription factors (2, 178),a DNA-binding domain (DBD), in which the dimeriza-tion or D-loop within the two zinc fingers plays a rolein GR dimerization and DNA-binding functions (178 –183), and a C-terminal ligand-binding domain (LBD),containing a second TAD (AF-2) and also protein-bind-ing sites (178, 184 –189). Interestingly, in addition toGR ligands, different GR/DNA-binding sequences candifferentially affect GR conformation and regulatoryactivity; as such, DNA can be considered as a sequence-specific allosteric ligand of GR (190). Furthermore, theDBD can also account for GR-transcription factor as-sociation (191–193). In close proximity of the DBD andat the end of the LBD, two nuclear localization sites, theligand-independent NL1 and the ligand-dependentNL2, have been described that direct the activated GRtoward the nucleus (Fig. 2) (194, 195).

The GR can exist as multiple isoforms due to alternativesplicing (GR�, GR�, GR�, GR-A, GR-P) and differenttranslational start sites (GR�-A, GR�-B, GR�-C1, GR�-C2, GR�-C3, GR�-D1, GR�-D2, or GR�-D3). Both theN-terminal and C-terminal domain of GR can vary de-pending on the isotype, but the DBD most often remainsconstant. GR�, stretching to 777 amino acids in humans,is the most predominant, functional GR and currently themain research target. In contrast, GR� cannot bind GCsand is not ubiquitously expressed. However, GR� can actin a dominant-negative manner to suppress actions ofGR� and is implicated in GC resistance (196, 197). Al-though GR� is expressed throughout the body, the ex-pression pattern of the isoforms can be restricted tocertain cell types, possibly fine-tuning the GC-GR re-

FIG. 2. Structure and domain functions of the GR. NTD, N-Terminaldomain; HR, hinge region; TF, transcription factor.


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sponse in various tissues (196, 198 –200). Additionally,various function-altering polymorphisms have been de-fined for the human GR (hGR) (199 –201). Membrane-associated and mitochondrial GR proteins have alsobeen described (202–206). Alternatively, GCs can actvia G protein-coupled receptors and its downstreamcascades (207). The precise role of GC signaling viathese receptors, however, still awaits further research. Itis expected that GR�, being the most predominant GRspecies, will remain a prime focus of therapeutic atten-tion for some time still.

In an uninduced state, GRs reside predominantly in thecell cytoplasm in association with a multimeric molecularchaperone complex, keeping the ligand-binding pocketreceptive to high-affinity hormone binding and inactivat-ing the NLS. This chaperone complex consists of severalHsps, such as Hsp90, Hsp70, and the Hsp90-bindingprotein p23, the hsp-organizing protein Hop, and tet-ratricopeptide repeat proteins that also bind Hsp90 suchas FK506-binding protein (FKBP) 51, FKBP52, cyclophilin40 (Cyp40), the C-terminus of Hsp70-interacting protein(CHIP), or the phosphatase PP5 (see Section II.B). However,in a single lysate, not all chaperone complexes are equallycomposed (186, 208).

2. Triggering GR-mediated signaling

Once their cellular target is reached, GCs can cross themembrane because they are small hydrophobic molecules.Alternatively, natural GCs can enter the cell via the steroidhormone recognition and effector complex (209). GCbinding to cytosolic GR instigates a conformationalchange in this receptor (185, 210). The active conforma-tional state of GR and its subsequent modifications allowGR to shed most of its chaperone complex, unmasking theNLS. These steroid-dependent changes allow GR to freelyand rapidly move along cytoskeletal tracts to ultimatelytranslocate into the nucleus (186). Subsequently, GR cangive rise to positive or negative transcriptional effects (Fig.3) (120, 211, 212) and rapid nontranscriptional effects(213). Together, these genomic and nongenomic path-ways controlled by GRs culminate in a multilayered andfine-tuned control mechanism for gene regulation. In ad-dition to the classic slow mode of GC action occurringbetween hours to days, increasing evidence is culminatingfor more rapid GC effects on cellular responses, takingplace within minutes. Because these GC effects are too fastto be regulated at the transcriptional level, they are termednongenomic, to distinguish them from the traditionalgenomic mode of GC action (214). Rapid GC effects may

FIG. 3. Activation and nuclear actions of the GR. The unactivated, cytoplasmic GR is complexed with chaperone proteins. Binding of GCs to theGR instigates the nuclear translocation of GR. The binding of dimeric, activated GR onto GREs, DNA binding of GR in a concerted manner withanother transcription factor (TF), or binding of GR onto a TF via a tethering mechanism can all result in GC-directed promoter activation. Thistransactivation results in the expression of metabolic gene products, associated with the occurrence of side effects, and to the expression of anumber of antiinflammatory proteins. The antiinflammatory effects of GCs are predominantly mediated via interference of monomeric GR with thetransactivation capacity of TFs, such as NF-�B, via a tethering mechanism. Otherwise, GC-activated GR can also negatively regulate genetranscription via competition for an overlapping binding site (competitive GRE) or via DNA-binding crosstalk with another TF (composite GRE), orelse via the sequestration of a DNA-bound TF. Although the nature of these sequences is not well-defined, gene repression via direct binding ofGR onto a so-called nGRE has also been reported.


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be transmitted by the GR or, more controversial, non-genomic GC activities might be mediated through non-specific physicochemical interactions with the plasmamembrane at high GC concentrations (215).

Although uninduced GR is mainly found in the cyto-plasm and GC-induced GR is largely nuclear, constitutiveshuttling between nucleus and cytoplasm has been re-ported for both nonactivated and activated forms of GR(216). It is, however, the import or export rate that deter-mines the location of the bulk of GR at any given time.Whereas importin�- or importin�-based nuclear import ofGR is mediated by the GR NLSs NL1 and NL2 (195, 217,218), calreticulin-based and chromosome region mainte-nance 1 (CRM1)/exportin1-based mechanisms have beendescribed to account for the nuclear export of liganded orunliganded GR (195, 219–221). Moreover, the locationof GR is codetermined by the recently discovered nuclearretention signal (NRS), which actively configures GR tothe nucleus (222). Additionally, the DNA-binding abilityof GR and ligand-specific conformational changes in GRhave been previously linked with GR’s nuclear mobilityand cellular location (223–225).

Once activated, several mechanisms are set in motion todiscontinue the GR response. Although GR is assumed tobe constitutively expressed under a vast array of physio-logical conditions, GR mRNA expression is subject to neg-ative regulation by GCs (226–228). This might be ex-plained by the presence of negative GC response element(nGRE), AP-1, NF-�B, and CREB regulatory motifs in thepromoter of GR, all of which are negatively regulated byGCs (196). Alternatively, GCs can destabilize GR mRNA(229). Moreover, ligand-activated GR protein is degradedupon prolonged exposure to GCs by the proteasome-ubiq-uitin degradation pathway (230, 231). However, muta-tion of the N-terminal GR PEST motif abrogates ligand-dependent downmodulation and consequently boostsGR-mediated transactivation (232). Lastly, it was shownthat protein degradation of GR is linked to its export.Although hormone dissociation results in a rapid releaseof GRs from chromatin, unliganded GR is delayed in itsexport. Accelerated nuclear export of a nuclear exportsequence-tagged GR chimera is associated with an in-creased rate of hormone-dependent down-regulation. Theprotracted rate of receptor nuclear export may be a way ofincreasing the efficiency of biological responses to second-ary hormone challenges, via a limitation on receptordown-regulation and hormone desensitization (233).

3. Nuclear activity of the GRa.GRtransactivationandtransrepressionmechanisms.Ligandbinding of GCs to GR results in the nuclear translocationof GR where this receptor can act to modulate transacti-

vation of typical GC response element (GRE)-containingor other promoters.

Ligand-activated, nuclear GR can stimulate the expres-sion of certain genes via DNA binding of a dimerized GR.These GR homodimers bind in the major groove of DNAvia their zinc finger DBD and target the imperfect palin-drome of the consensus GRE (5� GGT ACA nnn TGT TCT3�) (234–236). This GRE can differ among promoters insequence, copy number, and relative location in the pro-moter (in relation to the TATA box or other transcriptionfactor-binding sites), regulating the specificity and mag-nitude of its response. GR may thus be modified in anallosteric manner by its response elements to generate apattern of regulation that is appropriate to an individualgene (237, 238). In DNA-binding GR-mediated transac-tivation research, either a simple GRE or concatamer GREmouse mammary tumor virus (MMTV) reporter gene con-struct is often used, which GCs can transiently activate(239, 240). However, various studies revealed that manyknown GC-inducible genes do not contain consensus GREsites and do not require binding of dimerized GR. Some ofthese could be classified as promoters containing compos-ite elements, in which GR collaborates with another tran-scription factor to enhance transcription in a cooperativemanner (211, 241–245). Essentially all genes have GRbinding motifs reasonably close (at an average of 15 kb) tothe transcription start site, however few of those are func-tional. Many elements lie very far (�50kb) from thestart sites, and at present there are no simple ways toprove that a given element is controlling a given gene.Assignments are done essentially by conservation, dem-onstration of GR occupancy in vivo, and proximity(246). Lastly, tethering, i.e., direct binding of GR toDNA-bound transcription factors, has also been de-scribed to positively regulate DNA transcription uponGC administration (Fig. 3) (247–249).

Various transcription factors [e.g., Sp1, STAT1,STAT3, STAT5, CCAAT enhancer-binding protein (C/EBP), Ets, Egr-1, AP-2, AP-1, and NF-�B] can function inconcerted array with GR to regulate and fine-tune tran-scription in a positive or negative manner (250–252). Forexample, ligand-activated GR can inhibit most, but notall, NF-�B-driven gene expression (211). Indeed, GR actsselectively to inhibit NF-�B at some, but not all, NF-�Bsites. Notably, it inhibits NF-�B action at the IL-8 but notat the I�B� gene. NF�B at IL-8 is phosphorylated atSer276 and recruits P-TEFb to promote elongation. GRrepresses IL-8, where transcription elongation depends onP-TEFb-mediated phosphorylation of pol II C-terminaldomain (CTD) S2, by competing P-TEFb from p65 asso-ciation. In contrast, GR fails to repress I�B gene expres-sion, where CTD S2 phosphorylation proceeds without


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p65 recruitment of P-TEFb, and GR binding to p65 there-fore poses no interference (253, 254). The findings presentedabove represent a good example of a situation in which thecontext dependency is mechanistically understood.

Likewise, activation of the transcription factor NF-�Bp65 can repress most, but not all, GR-transactivated genepromoters (255, 256). The NF-�B-mediated inhibition ofGR transactivation mechanisms is called “reciprocal re-pression” and is partially based on the mutual interactionof GR with NF-�B and partially on the cellular context.

Additionally, SRCs of the p160 family, such as SRC-1and SRC-2, interacting with GR via its LBD LxxLL motif,and various cofactors for chromatin remodeling and his-tone modification [e.g., SRC-1 and TIF-2 associated mod-ulatory protein (STAMP), CBP/p300, p/CAF, switching ofyeast mating type/sucrose nonfermenting (SWI/SNF), andcoactivator-associated arginine methyltransferase (CARM)1] can regulate GC-mediated promoter activation (240,257–261). The nature of the GR ligand impacts GR cofactorbinding. Although administration of the synthetic GC dexa-methasone leads toGRSRC-1binding, ligandbindingofGRto the GR antagonist RU486 stimulates binding of GR to thecorepressor NCoR (262). Furthermore, the requirement forcertain cofactors and different GR interfaces can depend onthe targeted gene promoter (244, 263). Concerning histonemodifications, aGC-inducedphosphorylationofhistoneH3(H3) S10 and acetylation of H3 K14 in MMTV promoterchromatin is associated with a transcriptionally active pro-moter (257).

The DNA binding of ligand-activated GR is, however,not a static phenomenon. Upon activation, GR rapidlyassembles onto known GREs. The chaperones Hsp90 andp23 can localize to GR-bound GREs in a GC-induciblemanner and promote disassembly of a functional GR-GRE transactivation complex (264). Furthermore, the GRtransactivation regulatory complex has proven to turnover in an extremely dynamic manner, via a ligand-dependent “hit and run” mechanism (223, 265, 266).Combined with the release of GCs from GRs (264), thesemechanisms continuously sense cellular stress hormonesand thus allow an appropriate cellular response to varyingGC levels.

b. GR transactivation and inflammation. GC-mediated up-regulation of I�B�, GC-induced leucine zipper (GILZ),dual specificity phosphatase (DUSP) 1 (see Section IV.A),lipocortin-1/annexin A1, secretory leukocyte protease in-hibitor SLPI, IL-10, the decoy IL-1 receptor type II, dexa-methasone-induced Ras1 (Dexras1), downstream of ty-rosine kinase 1 (Dok-1), Src-like adaptor protein (SLAP),p11/calpactin binding protein, thymosin �-4sulfoxide,Clara cell secretory 10-kDa protein (CC10), �-adrenergic

receptors, SOCS1, and tristetraprolin (TTP) have all beensuggested to be involved in the GC-mediated combat ofinflammation (211, 243, 267–270). For example, becauseDNaseI footprinting studies in T47D/A1–2 cells demon-strated that regulatory factors bind to the I�B-� promoterafter GC treatment, Deroo and Archer (271) proposedthat GCs may be required for transcription factor bindingand subsequent transactivation of the I�B-� promoter.Higher levels of I�B-� would then block the activation ofNF-�B. GILZ, a classical GRE-driven target gene, alsoseems to mimic some aspects of GC action and inhibitsinflammatory cytokine-induced COX-2 expression inbone marrow mesenchymal stem cells, via blockage of thenuclear translocation of NF-�B (272). Annexin A1, an-other GC-regulated target gene, is believed to exert itseffects through the FPR receptor family of G protein-cou-pled receptors, of which the implication in the regulationof many inflammatory processes is increasingly being rec-ognized (273). GCs transcriptionally stimulate the syn-thesis of TTP, a zinc finger protein capable of destabilizingseveral proinflammatory cytokine mRNAs by binding toadenylate uridylate-rich elements (AREs) within their 3�untranslated regions, subsequently targeting them fordegradation (see Section III.B.2) (274). The effect of(de)phosphorylation on the functionality of some of thegene products of the above-mentioned list or the effect ofthese proteins on other kinases/phosphatase signalingpathways is discussed further below.

The overall role and contribution of the different de-scribed GC-induced antiinflammatory proteins in the GC-mediated antiinflammatory mechanism remains some-what controversial (211, 275, 276). In some studies, GCscan repress proinflammatory gene expression of IL-6,ICAM1, and COX-2 without the need for de novo proteinsynthesis (277, 278), whereas in other studies urokinaseplasminogen activator, COX-2, and IL-8 mRNA tran-scriptional repression by GCs have been found to partiallyrely on de novo protein synthesis (279–284). Neverthe-less, although a body of evidence supports that the prin-cipal and initial antiinflammatory potential of GR mayreside in its direct repression of proinflammatory gene ex-pression (236, 277, 285, 286), it is apparent that GR-mediated transactivation also plays a role. Moreover, theprecise contributions of transrepression vs. transactiva-tion mechanisms in the antiinflammatory actions of GRseem highly context-dependent and make matters evenmore complex (120). Hence, the idea that dissociating li-gands would remain powerful antiinflammatories whilemodulating side effects is likely simplistic.

c. GR-mediated promoter inhibition. Besides GR-mediatedtransactivation, ligand-activated GR can also act as a


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DNA-binding factor to repress specific gene transcriptionvia composite response elements or competitive mecha-nisms (241, 251, 287–291) or can tether to another tran-scription factor to modulate transrepression of targetedgenes via crosstalk with kinases, cofactors, or other pro-moter-bound transcription factors (Fig. 3) (211, 212,251). In the latter mechanism, GR can bind and modulateDNA-bound transcription factors such as AP-1, NF-�B,STAT5, octamer-binding transcription factor 1 (Oct-1),CREB, Smad3, Smad6, Ets2, T-box expressed in T cells (T-bet), and GATA3 (211, 212, 251, 292). Typically, the teth-ering GR repression of transcription factor activity is re-flected inareciprocal repressionofGRtransactivationbythevery same transcription factors (192, 251, 255, 277).

The transcription factors NF-�B and AP-1 are key tothe propagation of inflammation and are also targets ofGC-dependent repression of proinflammatory gene tran-scription (31). Moreover, chromatin immunoprecipita-tion analysis has shown that GR binds proximal to theNF-�B or AP-1 binding site in various promoters (254,293–295). The nuclear interaction of GR with the C-ter-minal activation domains of NF-�B p65 is pivotal to theGC-mediated repressive effect on NF-�B-regulated geneexpression (192, 255, 277). Mechanistically, GR does notneed to bind the target genes’ promoter DNA to infer in-hibition of NF-�B and AP-1 function, yet mutation anal-ysis revealed that a functional GR DBD is necessary forrepression of AP-1- and NF-�B-regulated genes (192, 193,256, 296). This is explained by the finding that the GRDBD is involved in AP-1 tethering interactions (297). Evenso, the GR LBD has been implicated in repression of NF-�B-regulated genes (298). GR-interacting protein 1(GRIP1) was originally identified as a corepressor for GRduring tethering to AP-1, explaining the LBD requirementfor repression (297). Nonetheless, GR typically does notinhibit binding of AP-1 or NF-�B to its respective responseelement within the endogenous promoters (253, 262, 293,295, 299). In addition, the LIM-domain protein thyroidreceptor-interacting protein 6 (Trip6) is suggested tofunction as an essential intermediary interaction part-ner in the association of GR, and AP-1 or NF-�B, be-cause knockdown of Trip6 or abolishing the interactionof Trip6 with GR abrogates GR-mediated transrepres-sion (295, 300).

Additionally, GCs can directly affect various histonemodifications that combine into a so-called “histonecode,” thus influencing chromatin accessibility and theassociated gene transcription. Effects of GCs on histonephosphorylation will be discussed below. Furthermore,GCs can inhibit cellular TNF-induced histone H4 K8 andK12 acetylation via reducing the HAT activity of CBP.Moreover, GC administration can increase the expression

of HDAC2, target HDAC2 to NF-�B CBP complexes, andtarget HDAC1 to the SP-A gene promoter (130, 294, 301).These histone-deacetylating events are associated with ahalted transcription of NF-�B-driven genes (130, 294,301). At the single promoter level, recent studies revealeda GC-mediated decline of histone H3 and H4 acetylationon the SP-A and IL-8 gene promoters, respectively, whichwas associated with a decreased transcription of thesegenes (294, 302). Lastly, GCs instigate the dimethylationof H3 K9 at the SP-A promoter (294). This histone mod-ification constitutes a transcription-repressive chromatinmark (303).

Because besides DNA-bound transcription factors andthe basal transcription machinery, the activated NF-�B-driven gene promoters recruit various cofactors in a gene-and cell type-specific manner, specific research has aimedto unravel possible GC effects on the composition andmodulation of this enhanceosome. First, it was proposedthat GR competes with NF-�B or AP-1 for a limitedamount of cofactors, such as the HAT CBP/p300 or SRC-1(304). However, overexpression of these cofactors or mu-tation of the coactivator-interacting domains of GR orNF-�B did not lead to a reversal of the marked inhibition(295, 305–308). Furthermore, GC administration did notaffect NF-�B p65 association with CBP (307). Lastly, thehypothesis of involvement of GR-interacting cofactorswas challenged via a mutation experiment. Although themutation of E755A in the GR C terminus, abolishing theinteraction of GR with LxxLL-containing cofactors, sig-nificantly decreased GR-mediated transactivation of aGRE-regulated reporter gene construct, this mutation didnot alter GR-mediated transrepression of Gal4-p65 activ-ity (309). Nevertheless, gradual overexpression of SRC-1or SRC-2 combined with the comodulator STAMP resultsin a lower EC50 value and a higher fold repression forGR-mediated inhibition of AP-1-mediated reporter geneactivity, in which EC50 is defined as the GC concentrationrequired for a half maximal response (310). Involvementof SRC-2 in GR-mediated repression of AP-1- or NF-�B-dependent gene expression was also confirmed for theendogenous genes collagenase-3 and IL-8 (297). How-ever, because most data on cofactor function are derivedfrom overexpression experiments, physiological rele-vance of nuclear cofactor modulation of GR-induced tran-srepression is yet to be determined. Lastly, not only couldcofactor complex assembly be modulated, but like NF-�Band GR, cofactors such as SRC-1, SRC-2, SRC-3, PGC-1,CBP, NCoR, and SMRT are themselves also subject toextensive posttranslational modulation such as phosphor-ylation, methylation, SUMOylation, ubiquitination, andacetylation. These modifications can affect cofactor-nu-clear receptor binding, activity, localization, and half-life


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(311–313). However, the impact of GCs on these modu-lations or vice versa, how these modulations impact GC-mediated mechanisms, is currently not very well known.

II. Phosphoregulation of the GlucocorticoidReceptor

Activational control of GR can be imposed via a combi-natorial mechanism involving ligand accessibility, GRconcentration, subcellular localization, and also post-translational modifications of GR. The activity of GR isaffected by various modifications, among which are phos-phorylation, acetylation, nitrosylation, redox regulation,ubiquitination, and SUMOylation (Fig. 4) (314, 315). Inthis section, we will discuss the impact of phosphomodu-lation of GR on various aspects of GR functionality andsignaling.

A. GR phosphorylationPhosphorylation is the reversible co-

valent association of a phospho groupon a protein. Phosphorylation is regu-lated by the balance between phos-phorylating kinases and dephosphory-lating phosphatases. This modificationmay affect GR hormone and DNAbinding and subcellular localization,alter GR interactions and protein half-life, ultimately affecting transactivatingand transrepressing capabilities of GRs.These phosphorylations of the GR aremediated by specifically targeted kinases.

In murine GR (mGR), researchershave identified eight phosphorylationsites: S122, S150, S212, S220, S234,S315, S412, and T159, most of whichreside in the N-terminal domain (Fig.4A) (316, 317). The rat GR (rGR) phos-phorylation sites correspond to those ofmGR (Fig. 4A) (318). Conversely, inhGR, five Ser residues were character-ized as phosphorylation targets: S113,S141, S203, S211, and S226, and re-cently S404 (319–321). These residuescould be sequence matched to the mGRphosphorylation targets S122, S150,S212, S220, S234, and S412, respec-tively (Fig. 4A). Additionally, recentmass spectrometry analysis of hGR con-firmed the phosphorylation of hGRS226, but also suggested a cell cycle-dependent potential phosphorylation ofT8, S45, S134, S234, and S267 (322), of

which only the latter four S residues present a conservedcounterpart in the mGR and rGR. However, additionalevidence to confirm in vivo phosphorylation of these sitesis yet to be reported. Of note, all phosphorylation sites arelocated in the AF-1-containing N-terminal domain of GR.

Phosphorylation of hGR S203 and S211 can both bemediated by Cdk2/cyclin A kinase complexes, whereasCdk2/cyclin E targets only hGR S203 (323). In support,murine embryonic fibroblast cells devoid of the Cdk in-hibitor p27Kip1, which affects Cdk2 activity, show en-hanced GR phosphorylation at the corresponding mGRS212 and S220 and an increase in GR transactivation po-tential (324). In general, phosphorylation of hGR S203and S211 or their murine counterparts is associated withan enhanced transactivation of GRE-regulated promoters(323, 325–327). However, the interaction of Cdk5 and itsactivator protein p35 and p25 with the GR LBD could also

FIG. 4. Structure and posttranslational modifications of the GR. A, The reportedphosphomodulated sites for the hGR, mGR, and rGR are depicted in relation to the knownfunctional domains of this receptor. B, The reported posttranslational modifications for thehGR, exempt from the phosphorylated sites, are depicted in relation to the knownfunctional domains of this receptor. C, The reported phosphomodulated sites for humanNF-�B-p65 are depicted in relation to the known functional domains of this transcriptionfactor. NTD, N-Terminal domain; HR, hinge region; P, phosphorylation site; h, human; m,murine; r, rat; aa, amino acids; SUMO, SUMOylation site; Ub, ubiquitination site; Ac,acetylation site; Rel-HD, Rel-homology domain; TA, transactivation.


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mediate phosphorylation of hGR S203 and hGR S211,remarkably resulting in a decreased GR transcriptionalactivation of the MMTV and serum- and GC-induciblekinase (SGK) promoter via attenuated GR-cofactor inter-actions (328).

The p38 MAPK could possibly also contribute to thephosphorylation of hGR S211 from 20 h of GC exposureonwards, in lymphoid cells. However, in these cells pre-treatment with p38 MAPK inhibitor SB203580 onlyslightly diminished hGR S211 phosphorylation. Further-more, in these cells, overexpression of the hGR S211Amutant severely impaired the GR transactivation potential(329). Conversely, p38 MAPK-mediated phosphorylationof hGR at an undefined residue was also associated witha decreased GR ligand-binding affinity and a slightly re-duced GC-dependent repression of GM-CSF production(330). Furthermore, activation of p38 MAPK via IL-1�

administration or overexpression resulted in a diminishedGR transactivation function and GR-GRE binding (331–333). Possibly, the p38 MAPK-mediated inhibition of GRactivity is not mediated via a direct GR phosphorylationbut is instigated via phosphorylation of a GR LBD-inter-acting factor (332).

Additionally, the direct interaction of GR via the JNKinteraction motif with JNK MAPK accommodates JNK-mediated phosphorylation of rGR S246, corresponding tohGR S226. This phosphorylation inhibits GR transcrip-tional activation (334, 335), but currently, the possibleeffect of this phosphorylation on GR transrepressionmechanisms has not been researched. In correspondencewith the above findings, overexpression of the JNK up-stream activator MKK7 inhibits GR transactivation to-ward an MMTV reporter gene construct (332). Mecha-nistically, activated JNK-instigated export of GR to thecytoplasm via a leptomycin B-sensitive, CRM1-dependentmechanism could contribute to the inhibition of GR ac-tivity. In support, UV-induced JNK MAPK or overex-pression of JNK expedited GR nuclear export and wasassociated with an inhibition of GR-mediated transcrip-tion, whereas expression of a GR S226A mutant showsno UV-induced export or associated diminished GR ac-tivity (334).

Furthermore, a GSK3-mediated phosphorylation ofrGR at T171 was shown in vitro. Overexpression of GSK3inhibited GR transcriptional activation but does not affectGR-mediated repression of an AP-1-driven reporter geneconstruct. However, this rGR T171 corresponds to a hGRA150, and GSK3 overexpression in human cells thus doesnot affect GR activity and shows a species-specific differ-ence in GR phosphorylation. Nevertheless, when hGRA150 is mutated to T, the GSK3-mediated inhibition ofGR transactivation can be restored (318). In contrast,

hGR S404 was recently identified as a target of GSK3�.The nuclear phosphorylation of this residue would lead tonuclear export of GR, an enhanced down-regulation ofGR and attenuated transactivation of GRE-containingpromoters, and a hampered transrepression of NF-�B-reg-ulated gene promoter activities (321).

Lastly, a ligand-independent association of GR withPKA has been reported, and overexpression of PKA canenhance basal and GC-induced MMTV reporter gene ac-tivity and GR-GRE binding (336–338). Although phos-phorylation of GR by PKAc was suggested in vitro, thiswas never confirmed in vivo (339).

Although GRs display a low basal phosphorylation,these receptors get hyperphosphorylated upon the addi-tion of agonist (316). In quiescent cells, hGR S211 phos-phorylation count is lower than that of hGR S203. TheGC-induced alterations in GR phosphorylation seem todepend on the preexisting phosphorylation status of GRbecause mutation of hGR S203 to A mildly impedes hGRS211 phosphorylation, while slightly enhancing hGRS226 phosphorylation, suggesting a possibly ordered, se-quential phosphorylation of GR and an intersite depen-dency (320, 340).

Although GCs strongly enhance both S203 and S211phosphorylations, hGR S211 phosphorylation is pro-posedasahallmark for the transactivationpotential ofGRbecause the antagonist RU486 still allows for GR S203 butnot S211 phosphorylation (320). It should be noted, how-ever, that RU486 can also behave as an agonist in a con-text-dependent manner (341). GR transactivation func-tion is found to be at its peak when the relativephosphorylation of hGR S211 surpasses that of S226(327). Although overexpression of hGR S211A only di-minishes GR transcriptional activity (329), mutation anal-ysis of mGR showed that S to A mutations for the S212 andS220 residues strongly decreased GR transactivation of aminimal GRE-regulated reporter gene construct (325).Not all phosphorylations lead to an increase in trans-activation. The recently identified interaction of ERK8with GR� via the LIM domain-containing Hic5 inter-mediate was suggested to function as a dampener of GRtransactivation, the mechanism of which remains un-known (342).

The localization of phosphorylated GR can also differ.Ligand-induced S211 phosphorylated GR appears to bemainly nuclear, whereas S203 phosphorylated GR pref-erentially resides in the cytoplasm (320, 326). In contrast,in the absence of ligand, basally phosphorylated GRs atS203 or S211 were both found in the cytoplasm (320,326). Conversely, a nuclear phosphorylation of hGR S404by GSK3� and rGR S246 by JNK seems to expedite nu-cleocytoplasmic transport of these GRs (321, 334). These


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findings demonstrate the impact of GR phosphorylation sta-tus on its subcellular localization. Interestingly, pharmaco-logical inhibition of tyrosine phosphorylation—usinggenistein or tyrphostin AG126—is reported to stimulate nu-clear export of GR (343). Currently, it is not clear whetherthis relocalization effect should be attributed to the directmodulation of an unknown GR Y residue phosphoryla-tion. However, GR phosphorylation is not critical to thereceptors’ nuclear import function because mGR, with allphosphorylatable sites mutated to A, still undergoes li-gand-dependent nuclear translocation (325). In addition,RU486 can also elicit GR translocation (262).

A recent study has provided a link between cell com-partment-specific phosphorylation of the GR, induced byacute or chronic stress, and GR-dependent transcriptionalactivity in rat central nervous system tissue. Only acuteisolation stress resulted in an increase in serum cortico-sterone levels. Under the condition of chronic stress, de-spite unaltered levels of nuclear GR, a significant tran-scriptional activity was still observed. In fact, it turned outthat GR-dependent gene regulation patterns in the centralnervous system, for GR, corticotropin-releasing factor(CRF), and brain-derived neurotrophic factor (BDNF)were similar compared with the acute stress model. Theseresults may suggest that the transcriptional activity of GRis not solely regulated by the levels of hormone. Rather, thetranscriptional activity of GR under chronic isolation wasproposed to result from an increased Cdk5 activation andphosphorylation of the nuclear GR at S232 and a de-creased JNK activity, which was reflected in a decreasedphosphorylation of nuclear GR at S246 (344).

GR phosphorylation can impact its half-life becausehGR phosphorylated at S203 displays a more rapid decaythan GR phosphorylated at S211, and various mutationsablating the phospho-acceptor sites of mGR loweredligand-dependent down-regulation of mGR (320, 325). Inaddition, association of GR with the tumor suppressorgene TSG101 (tumor susceptibility gene 101), which canbind ubiquitin groups and negatively affect ubiquitin-de-pendent proteasomal degradation, occurs preferentiallywith a nonphosphorylated GR. This interaction ofTSG101 and hypophosphorylated GR then leads to unli-ganded GR protein stabilization (345, 346).

The GRE-regulated tyrosine aminotransferase (TAT)and GILZ gene promoters appear to preferentially bindGR phosphorylated at S211 or S226 (326). Mechanisti-cally, hGR phosphorylation of S211 would alter its con-formation and thus accommodate interactions of GR withvitamin D receptor-interacting protein 150/mediatorcomplex subunit 14 (DRIP150/MED14) (346). Indeed, inGR S211A-expressing cells, the expression of MED14-independent genes was not impaired, whereas the tran-

scription of MED14-dependent genes was attenuated,suggesting a link between GR phosphorylation andMED14 involvement in a gene promoter-specific manner(327). Promoter selectivity was also previously shown bymutation analyses of mGR showing differential effects onvarious GRE-containing reporter gene constructs (325)and mutation analysis of hGR showing differential effectson various GC-activated and GC-repressed genes (321). Inthat respect, it is widely accepted that the different mod-ification statuses of GR could lead to a variable cofactorinteraction profile.

Thus, ligand-dependent phosphomodulation of GRcould affect GR ligand binding, gene promoter-selectiveGR transactivation, GR DNA binding, cofactor recruit-ment, subcellular localization, and half-life.

B. GR dephosphorylationBecause phosphorylation is a reversible mechanism,

GR function is also regulated by various phosphatases. Insupport, administration of a pharmacological inhibitor ofPP1, PP2A, and PP5 function augments GR phosphory-lation and blocks nuclear import of ligand-activated GR.These agents, however, allow export of GR to the cyto-plasm but prevent its subsequent return to the nucleus,thus abrogating recycling of GR (340, 347, 348).

The tetratricopeptide repeat domain-containing PP5,via binding to Hsp90 in the GR chaperone complex, formsan indirect binding partner for GR (349–351). Knock-down of PP5 resulted in increased GR binding to DNA andGR transcriptional activity but did not affect the ability ofthe synthetic GC dexamethasone to bind to GR (352,353). In contrast, a similar experiment was recently re-ported to reduce GC-induced transcription of three en-dogenous genes (IRF8, IGF binding protein 1, Ladinin),while leaving GILZ expression unaffected. Concomi-tantly, this PP5 knockdown raised phosphorylation ofhGR at S203, S211, and even more pronounced at the GRactivity-inhibiting site S226 (340). This gene-specific con-trol of PP5 over GR activity may be regulated by promot-ing the ligand-binding affinity of GR (354). The questionarises whether these findings can be reconciled with oneanother on the basis of the existence of different contextsand/or a mix of primary and secondary effects. Alterna-tively, PP5 could mediate GC-instigated nuclear trans-location of GR via the interaction between PP5 and themotor protein dynein (355). In this respect, PP5 is be-lieved to dephosphorylate recycled GR proteins return-ing from the nucleus, thus resetting GR in a ligand-inducible state (340, 356).

Very recently, estrogen has been described to inhibitGC induction of DUSP1 and GSK genes in breast cancercells, providing a plausible explanation for why GC trialsin breast cancer are not overtly successful (357). The


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mechanism involved a reduced ligand-induced GR phos-phorylation at S211, which is associated with the activeform of GR. Estrogen increased the expression of PP5,which mediates the dephosphorylation of GR at S211.After PP5 knockdown, the estrogen-promoted cell prolif-eration was significantly suppressed by GCs, providingproof for a crosstalk between estrogen-induced PP5 andGR action (357).

In short, GR phosphomodulation is thus a flexiblemechanism, integrating cellular stimuli and modulatingmultiple receptor functionalities. Currently, no GR-tar-geting phosphatases have been linked to specific GR res-idues. Moreover, considering the plethora of kinases in-volved in GR phosphorylation, the small number of GR-targeting phosphatases, characterized until now, seemstoo constrained. Future research in this direction couldthus possibly unveil additional information on theseprocesses.

C. Other posttranslational modifications of GRIn this paragraph, we will discuss how other posttrans-

lational modifications of the GR can affect its phosphor-ylation status. SUMOylation involves an E1-activating en-zyme, an E2 conjugation enzyme, and an E3 ligase. Thecovalent attachment of a small ubiquitin-related modifier(SUMO) motif can affect protein stability, subcellular lo-calization, and transcriptional activity (358). The follow-ing SUMO-target sites have been identified in GR: K703in the C-terminal LBD and K277 and K293 in the N-terminal synergy control motif (which is defined to inhibitsynergistic transcription conferred by GREs containingmultiple GR binding sites). These latter SUMOylationscan thus act as inhibitory elements controlling GR activitytoward multiple, but not single, GREs (Fig. 4B) (359–361). Interestingly, the JNK-mediated phosphorylation ofhGR S226 appears to facilitate subsequent GR SUMOy-lation at the N-terminal SUMOylation sites, and thus GRSUMOylation could be considered a phosphorylation-di-rected posttranslational GR modification (359). Overex-pression of SUMO-1 can destabilize GR protein (362),whereas a mutation of the potential SUMOylation sites ofGR or inhibition of the SUMOylation enhances GR trans-activation in a gene-selective manner, with a preference formultiple GRE-containing gene promoters (359–361).Moreover, overexpression of the GR targeting SUMO-2protein decreased GR-mediated expression of endogenousGRE-regulated genes (359). Although SUMOylation ofGR can occur in a ligand-independent manner and doesnot require an intact GR LBD and DBD dimerization mo-tif, the integrity of the latter domains and especially bind-ing of GR to DNA appears necessary to implementSUMO-dependent transcriptional inhibition (360, 362).Although binding of death domain-associated protein

(DAXX) to SUMOylated GR has been proposed to me-diate the described, inhibitory effects (363), a recent reportfinds no effect of death DAXX overexpression or knock-down on SUMOylated GR activity (360). Currently, it isunclear how GR SUMOylation controls its transcriptionalactivity, but SUMO-interacting proteins are most likely tobe considered.

Additionally, it is interesting to note that the phosphor-ylatable hGR S404, which is phosphorylated by GSK3�,mGR S412, and rGR S424 are comprised in a PEST deg-radation motif (316, 321). Ligand-activated mGR couldbe ubiquitinated at K426, leading to degradation of theGR protein (232, 364). The residue K419 is the counter-part in hGR (Fig. 4B). Blocking mGR downmodulationvia proteasomal inhibitors or via mutation of K426 en-hances GR-mediated transactivation and retards GR mo-bility in the nucleus (232, 364). Moreover, because GRbecomes hyperphosphorylated upon ligand binding, it ap-pears that this phosphoregulation is key to the onset ofthe ubiquitination-mediated proteasomal degradationof GR, since a mutant GR with all possible phospho-sites mutated to A does not undergo ligand-dependentdownmodulation (325).

Because SUMOylation and ubiquitination appear to beaffected by a differential phosphorylation of GR (325,359), it would prove interesting to research the possibilityof other phospho-directed posttranslational modifica-tions of GR. In this perspective, interesting research couldfocus on the acetylation of hGR at K494 and K495, whichis a prerequisite for GR association with NF-�B p65 andassists the GC-instigated repression of GM-CSF gene ex-pression (365).

III. Kinases Targeted by GlucocorticoidReceptor-Mediated Signaling

Because kinases such as MAPKs, MSKs, and Cdks are veryimportant in controlling the expression of inflammatorycytokines, recent research has moved its focus to the mod-ulation of these kinases as alternative antiinflammatorytools beside GCs. However, various aspects in kinase sig-naling and expression are already positively or negativelyregulated by GCs. Therefore, this section will focus on theeffects of activated GR on kinase signaling in the frame-work of inflammation.

A. Mitogen-activated protein kinases (MAPKs)A dysregulated activation of MAPKs was traced in nu-

merous inflammatory diseases such as rheumatoid arthri-tis, psoriasis, systemic lupus erythematosus, asthma, andinflammatory bowel disease (3, 366). Interestingly, acti-vated GR forms a complex regulatory loop with the acti-


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vated MAPK signaling pathway. In short, whereas GR candirectly and indirectly inactivate MAPKs, these MAPKscan affect GR via phosphorylating the receptor (see Sec-tion II.A). Depending on the cell type used, GCs have beenshown to suppress p38, JNK, and/or ERK MAPK phos-phorylation. The GC-induced dephosphorylations of p38,JNK, and ERK MAPK have all been suggested to occur viaactions of the GC-induced DUSP1 phosphatase (see Sec-tion IV.A).

1. JNK MAPKThe GR crosstalk with the JNK signaling pathway,

leading to repression of its downstream targets c-Jun,ATF2, and Elk-1, appears to have multiple mechanisticlayers (367, 368). Besides the GC-induced DUSP1-medi-ated dephosphorylation of JNK MAPK (369–371), GRcan also directly interact with JNK and thus interfere withJNK activity without the necessity of de novo gene ex-pression (367, 372). Furthermore, the JNK-binding se-quence of GR seemed to be required for GC-induced JNKinhibition and nuclear import of JNK (372). Notably, theinteraction of GR with JNK can indirectly decrease AP-1activity via binding of inactive JNK, together with GR, tothe AP-1-bound elements of the c-jun gene promoter(372–374). However, binding of GR with the antagonistRU486 does not support a GR JNK interaction or GR andJNK recruitment to the c-jun gene promoter. Also, JNKsignaling can be blocked by activated GR via interruptingthe association between the activating MKK7 and JNK(372). Additionally, activation of the upstream MEKK1can be hampered by GCs via its negative interference withthe MEKK1 Hsp90 association, culminating in a disrup-tion of the JNK activation (375). Recently, Kim et al. (376)demonstrated that the GC-inducible SGK1 kinase inhibitsthe activation of SAPK (stress-activated protein kinase)/ERK kinase 1 (SEK1) (the upstream JNK kinase, alsocalled MKK4), thereby negatively regulating the JNK sig-naling pathway. SGK1 was further found to physicallyassociate with SEK1. Because SGK1 has been implicatedin the promotion of cell survival and the protectionagainst cellular stress, the described mechanism ex-plains how SGK1 negatively regulates stress-activated sig-naling, namely through the inhibition of SEK1 function-ality (376). Finally, a GC-mediated inhibition of JNKMAPK activity has been shown to block the translation ofTNF in murine monocytes (377).

2. ERK MAPKInhibition of ERK MAPK activation can occur via

alternative GC-dependent mechanisms. The interactionof Raf-1, a MAP3K in the ERK signaling pathway, withits chaperone Hsp90 is inhibited by GCs. As a result,

Raf-1 can no longer associate with Ras, the activationof which remained unaffected by GCs. Consequently,the impaired Raf-1 activation leads to a decrease in ERKactivation (375, 378).

GCs can elevate not only DUSP1 expression but also theexpression of GRE-regulated GILZ in various cell lines(270, 280, 379–389). Interestingly, binding of GILZ toRaf-1 inhibits its phosphorylation and thus represses thephosphorylation of Raf-1’s downstream targets MKK1/2and ultimately ERK1/2 MAPK (390, 391). Of note, Raf-1can also associate with liganded GR, together with 14-3-3(392). Additionally, GILZ can directly bind to NF-�B andAP-1, and as such represses NF-�B- and AP-1-directedexpression of proinflammatory genes (270, 384, 386, 393,394). Other GC-induced genes, namely downstream oftyrosine kinase 1 (Dok-1), SLAP and Dexras1, have alsobeen associated with a GC-mediated inhibition of ERKMAPK activation and inflammatory signaling (395–398).

3. p38 MAPKAlthough in many cell lines GC treatment leads to a

decrease in p38 MAPK phosphorylation and activationvia a DUSP1-dependent regulation (275, 399, 400), pro-longed GC exposure of lymphoid cells is actually knownto increase p38 MAPK phosphorylation levels (329). Cell-specific effects should thus be considered.

B. MAPK-activated protein kinases (MKs)Although p38 and ERK MAPKs have their own specific

transcription factor and other protein targets, theseMAPKs can continue the MAPK cascade by activating yetanother level of kinases, the MKs (Fig. 5). Based on se-quence homology, these MKs comprise MSKs, 90-kDaRSKs, MAPK-interacting kinases (MNKs), MK2, MK3,and as a final group MK5 (25). Only ERK MAPKs canactivate RSKs and MNK2, and only p38 MAPKs can ac-

FIG. 5. The layered MAPK signaling cascade. A schematicrepresentation of the signaling cascades initiated by mitogens andstressors that lead to the activation of the MAP2Ks/MKKs, MAPKs, andultimately the various MKs.


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tivate MK2, MK3, and MK5, whereas both ERK and p38MAPKs can activate MSK1, MSK2, and MNK1 (25).

1. MSK1The two MSK proteins, MSK1 and MSK2, consist of

two kinase domains bound together by a linker region.The N-terminal kinase domain of MSK accommodatessubstrate and autophosphorylation, whereas the MSK(CTD) comprises a bipartite nuclear localization sequence(401), a MAPK-docking domain, and an autoinhibitorysequence (402, 403) and can only mediate autophosphor-ylation of MSK (25). At the cellular level, MSK1 andMSK2 are predominantly nuclear kinases, most likely dueto their bipartite NLS (401, 404). MSKs are activated viap38� and/or ERK1/2 MAPK phosphorylation in responseto various stimuli (84, 401, 405–408). Activation of theseupstream kinases leads to the phosphorylation of MSK1T581 or MSK2 T568, which is considered primordial tothe activation of the MSK1 C-terminal kinase domain andthe subsequent full activation of MSK1 (402, 403, 409,410). MSKs are involved in affecting chromatin structure,transcription factor accessibility, and ultimately genetranscription via the phosphorylation of CREB S133,ATF1 S63, and NF-�B S276, leading to their activation(73, 83, 84, 405, 407), and the phosphorylation of H3S10, contributing to chromatin relaxation and thus facil-itating transcription of select gene promoters (411–414).Interestingly, phosphorylation of H3 S10 and H3 S28 byMSKs creates confirmed binding sites for 14-3-3 regula-tory proteins at c-jun, c-fos, and HDAC1 gene promoters(415–417). Furthermore, it was demonstrated that theconcomitant acetylation of H3 K9 and K14 stabilizes theassociation between phospho-acetylated H3 and 14-3-3(416–418). Lastly, the phosphorylation of H3 S10 andattraction of 14-3-3 correlates with dissociation of thetranscription-repressive heterochromatin protein (HP) 1�

and recruitment of RNA pol II (417, 419).Recent investigation into the effect of GCs on MSK1

signaling revealed that, whereas GCs cannot profoundlyaffect MSK1 phosphorylation or activity, these steroidscan effectively abolish MSK1 recruitment and thus H3 S10phosphorylation at specific inflammatory gene promoters(420), which nuances an earlier report that GCs could notimpact the overall H3 S10 phosphorylation in these cells(421). Moreover, the GC-induced lack of MSK1 occu-pancy at these TNF-activated inflammatory gene promot-ers contributes to a decline in NF-�B transactivation(420). Remarkably, GCs not only impede recruitment ofMSK1 at inflammatory gene promoters, but actuallydrive certain MSK1 proteins to the cytoplasm in a GR-and CRM1-dependent manner and furthermore sup-port a physical interaction between GC-activated GRand activated MSK1 (420). However, in-depth mecha-

nistic studies of how GCs affect MSK1 localization arestill warranted. Because the GR modulator compoundA (CpdA), which can transrepress NF-�B-driven geneexpression without displaying any transactivatingproperties (293, 422), can export a fraction of the nu-clear MSK1 to the cytoplasm similar to classical GCs,the active GR-instigated redistribution of MSK1 to thecytoplasm fits in the framework of a general cellularmechanism of GR-mediated transrepression of NF-�B-mediated transcription (420).

MSK1 has also been implicated in the cytoplasmicphosphorylation of the eukaryotic translation initiationfactor 4E-binding protein 1 (4E-BP1) at S64 and possiblyalso T36 (423). In unstimulated cells, 4E-BP1 can bind tothe eukaryotic translation initiation factor-4E (eIF-4E)complex, thus inhibiting functioning of the eIF-4E holo-complex in translation initiation. Phosphorylation of 4E-BP1 relieves the translational block by dissociating 4E-BP1 from the eIF-4F member eIF-4E at the mRNA cap(423). Interestingly, the administration of GCs blockedphosphorylation of the translational repressor 4E-BP1,thus allowing reassociation of 4E-BP1 and eIF-4E (424–428) (see Section V.A). The latter interaction preventsmRNA cap-dependent initiation of translation. Currently,crosstalk of GR with MSK1 in this mechanism has notbeen fully researched. As such, the possible involvementof GC-translocated cytoplasmic MSK1 in these eventsand the overall implications of these inhibitory effectsfor the antiinflammatory potential of GCs are not fullyunderstood.

2. MK2The GC-mediated MAPK inhibition results in an inhi-

bition of proinflammatory gene transcription via repress-ing the phosphorylation of multiple factors, but it alsoaffects proinflammatory protein production becauseMAPKs and the MK2 are involved in mRNA stability.However, only the select mRNAs that contain AREs atthe 3�-untranslated end, which are often cytokine and che-mokine transcripts, are affected by MAPK regulation(429). Mechanistically, TTP can bind and destabilize theseARE-containing transcripts and trigger their degradationvia binding of exonucleases (430, 431). However, TTPfunction can be inhibited via phosphorylation by the p38MAPK-activated kinase MK2, thus stabilizing the ARE-containing transcripts such as TNF mRNA (432–438).Notably, TTP knockout (KO) mice display phenotypicallyinflammatory arthritis due to an increased stability ofTNF mRNA and an enhanced production of TNF protein(439). Because GCs enhance the expression of TTP in var-ious cell lines (243, 269, 274, 440) and concomitantlyinhibit p38 MAPK and thus most likely MK2 activity,these steroids promote the destabilization and degrada-


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tion of, among others, TNF, COX-2, GM-CSF, vascularendothelial growth factor (VEGF), MMP1, MMP3, IL-6,and IL-8 mRNAs (243, 268, 279, 283, 441–443). In sup-port, GCs repress TNF mRNA in lung epithelial cells anddecrease luciferase expression of a TNF 3� untranslatedregion reporter plasmid in an orientation-dependent man-ner. Small interfering RNAs to TTP significantly preventthis effect, and a cell line stably expressing a short-hairpinRNA to TTP showed that TTP is critical for GC-inducedinhibition of TNF mRNA (269, 274). Comparing knock-down approaches with TTP�/� murine embryonic fibro-blasts, differential effects of the lack of TTP on the mRNAturnover of different target genes were observed. Despitethis, a certain degree of loss of GC-induced repression ofCCL2, CCL7, CXCL5, and IL-6, which demonstratedTTP binding, was found to be reproducible. The observeddifferences may be due to different levels of TTP repressionand to the diverse phenotype of cells with a chronic vs. anacute factor depletion (269, 274). Taken together, thesestudies reflect a novel inductive antiinflammatory signal-ing pathway for GCs that acts via posttranscriptionalmechanisms (269, 274). Not only p38 MAPK, but alsoJNK and ERK MAPK signaling has been linked to theposttranscriptional stabilization of TNF, IL-2, and IL-3mRNA (444–446). Combined, the GC-induced mRNA de-stabilization together with the inhibited gene transcriptioncauses a rapid clearance of existing inflammatory signalingmolecules. Of note, the GC-stimulated destabilization of GRmRNAismediatedthroughthissamemechanism(447),pro-viding an adequate feedback response.

Currently, no link has been demonstrated between GRsignaling and the RSKs, MNKs, MK3, or MK5.

C. Cyclin-dependent kinases (Cdks)The proinflammatory stimulus TNF promotes the re-

cruitment of RNA pol II at the IL-8 and ICAM1 promoterand the subsequent phosphorylation of the CTD of RNApol II at S2 and S5. Conversely, GCs can interfere with theS2 phosphorylation of the RNA pol II CTD, while leavingits recruitment to the promoter unaffected (254). This S2phosphorylation was mediated by Cdk9 of the Cdk9/cyclinT1 P-TEFb complex and is absolutely necessary for(NF-�B-mediated) transcription (448, 449). In support,knockdown or pharmacological blockage of P-TEFb se-verely impaired TNF stimulation of IL-8 gene transcrip-tion. Interestingly, at the IL-8 promoter, GR competeswith P-TEFb for binding to NF-�B p65, thus inhibitingIL-8 gene transcription and the phosphorylation of therecruited RNA pol II S2 (253, 254). Recently, it was alsodiscovered that the association of P-TEFb with NF-�Brequired NF-�B p65 phosphorylation at S276 (81), thusintegrating the GC-instigated blockage to recruit MSK1

(420) and P-TEFb (253, 254) at the inflammatory IL-8gene promoter.

Additionally, GCs can cell-dependently repress thegene expression of Cdk4 and Cdk 6 and their associatingcyclin D3 (450) and induce expression of the Cdk inhibitorp21Cip1 (451, 452). Furthermore, GCs can inhibit Cdk2and Cdk4 activity, an event that is associated with theantiproliferative effects of GCs and decreased GRE-mediated transcription (453–455). Experiments withseliciclib, a Cdk inhibitor targeting Cdk2, Cdk7, andCdk9, in a mouse model for the chronic inflammatorycondition systemic lupus erythematosus revealed that thisCdk inhibitor can lower kidney inflammation and prolongsurvival. Upon combining seliciclib with GCs, however,the therapy elicited a greater beneficial effect than eithertherapy could alone (456). Additional research into therole of Cdks in the GC-mediated antiinflammatory mech-anisms is still required.

D. I�B kinase � (IKK�)The multifactorial kinase complex IKK is essential in

relaying proinflammatory stimuli to NF-�B activation.When activated, IKK� and IKK� can phosphorylate I�B,thus targeting this inhibitory molecule for degradation viathe ubiquitin-proteasome degradation pathway and pro-gramming the subsequent release of NF-�B to translocateto the nucleus (10). Furthermore, IKK� can promoteNF-�B DNA binding on specific gene promoters (457).

GCs can, in particular cell lines, up-regulate the expres-sion of I�B�. The replenishment of the depleted I�B� poolthus redirects the activated NF-�B to the cytoplasm andultimately counteracts the former actions of the NF-�B-activating IKK complex. A critical discussion of the role ofGC-induced I�B� in the GR-mediated antiinflammatorymechanism is presented in Refs. 211 and 243.

In addition to MSK1 (411, 414), IKK� has also beendescribed to mediate H3 S10 phosphorylation (458, 459).Constitutive shuttling of IKK� between cytoplasm andnucleus has been reported (460), but upon TNF induction,IKK� translocates into the nucleus (458, 459). Ablation ofIKK� halts H3 S10 phosphorylation and decreases proin-flammatory gene expression, without affecting I�B� deg-radation or NF-�B DNA binding (458, 459, 461, 462).Chromatin immunoprecipitation analysis of the NF-�B-regulated surfactant protein-A (SP-A) gene promoter inhuman fetal lung type II cells recently revealed that GCscan diminish H3 S10 phosphorylation in the SP-A pro-moter and can block the recruitment of the H3 S10 kinaseIKK� (294). In this respect, it may also be interesting toinvestigate the recruitment dynamics of the H3 S10-phos-phorylating kinase MSK1 at the SP-A gene promoter.


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E. TANK-binding kinase 1 (TBK1)In a recent report, TRAF family member-associated

NF-�B activator (TANK)-binding kinase 1 (TBK1) wasidentified as a new target for GCs (463). This kinase isactivated downstream of both TLR3 and TLR4 signalingand functions together with the inducible I�B kinase IKK�

as an intermediate in the activation of the transcriptionfactor IRF3 (464, 465). Upon activation, IRF3 subse-quently dimerizes, associates with CBP, and can thentranslocate into the nucleus where it binds onto specificIFN-stimulated response elements (ISREs), e.g., in theRANTES gene promoter (466, 467). Currently, no effectsof GCs on IKK� have been described. However, GCs candiminish LPS (TLR4)- and poly I-C (TLR3)-stimulatedS127 phosphorylation of TBK1 and thus attenuate TBK1activity. Possible involvement of DUSP10 and the GC-inducible DUSP4 (275), but not DUSP1, was suggested viaanalysis of the effects of their overexpression on ISRE-containing reporter gene constructs. However, in this re-spect, (double) knockdown studies are still lacking. More-over, possible effects of GCs on the SH2-containingtyrosine phosphatase SHP-2, which is known to target thekinase domain of TBK1 (468), have not yet been re-searched. The described GC-mediated inhibition of TBK1would thus contribute to the steroid-initiated inhibition ofIRF3-regulated gene promoter activity of a reporter geneconstruct in U373 cells (463). Correspondingly, the LPS orpoly(I-C)-induced expression of RANTES, of which thepromoter contains an ISRE, can also be inhibited by ad-dition of GCs. Moreover, the poly(I-C)/TLR3-induced ex-pression of other IRF3-dependent genes such as IFN-�,IFN-inducible protein of 10 kDa (IP-10), ISG15, andISG56 and ISRE-luc reporter gene constructs is also sub-ject to a GC-mediated inhibition, which would be insti-gated via the disruption of IRF3 and GRIP1/nuclear co-activator-2) complexes in murine macrophages (469).

However, these events appear to be in contrast withfindings of Ogawa et al. (299), who showed in macro-phages that TLR4- or TLR9-mediated promoter activa-tion of the chemokines IFN-inducible protein of 10 kDa(IP-10) or IFN-induced with tetratricopeptide repeats 1(Ifit1) or of an IRF3-dependent reporter gene constructcan be inhibited by GCs, whereas the TLR3-mediated ac-tivation of the same gene promoters is refractory to GCs(299). Mechanistically, Ifit1 depends on activation via thetranscription factor IRF3, irrespective of the stimulus.However, the interaction of DNA-bound IRF3 withNF-�B p65 only occurs upon TLR4/TLR9-mediated Ifit1gene promoter activation. Under these circumstanceswhere p65 acts as a cofactor, the specific sensitivity of thispromoter to GC-mediated repression in a TLR4- or TLR9-stimulated environment is being guaranteed. The basis of

this repression was attributed to a binding competitionbetween IRF3 and GR for the same site in NF-�B p65, inwhich GR has a greater affinity for NF-�B p65 than IRF3(299). Interestingly, TBK1 is also a TNF-activated kinasefor NF-�B p65 S536, which plays a role in the full tran-scriptional activation of this transcription factor (470).Further research is needed to resolve this apparent para-dox between the results of McCoy et al. (463), Reily et al.(469), and Ogawa et al. (299).

F. Other kinasesThe STAT transcription factors are phosphoproteins,

activated by receptor-associated Janus kinases (JAKs), af-ter cytokine receptor stimulation. Administration of GCscan inhibit the IL-2-induced phosphorylation, nucleartranslocation, and DNA binding of STAT5 via a decreasedexpression of JAK3 and IL-2 receptor �. As a consequence,IL-2, IL-4, IL-7, and IL-15 signaling and ultimately T cellproliferation was impeded (471). Furthermore, GCs caninhibit the IL-12-induced phosphorylation of STAT4 onposition Y693 in T-lymphocytes, however, without af-fecting the IL-12-instigated JAK phosphorylation (472,473). The inhibition of IL-2-induced STAT5 phosphor-ylation by GCs in T cells was associated with a GC-mediated down-regulation of JAK3, but not JAK1, pro-tein levels (471).

PKC is a family of S/T kinases, which are ubiquitouslyexpressed under the control of diverse stimuli and havebeen implicated in the pathogenesis of asthma and COPD(474). As such, PKCs can function as an intermediate inNF-�B-dependent gene transcription (475, 476). An over-all GC-instigated decline in PKC activity was recentlyreported for mesenteric arteries from rats (477). In par-ticular, PKC�, which has been associated with the NF-�B-mediated activation of IL-8 gene promoter activity (476),is an interacting partner for the GC-induced Dexras1. Thisassociation results in a decline in PKC� phosphorylationand activity (478).

Protein kinase B (PKB), also known as Akt, is a phos-phatidylinositol-3-kinase (PI3K)-stimulated kinase, in-volved in cell cycle regulation. The GC-induced PKB ac-tivation can also result in the phosphorylation of itsdownstream target kinase, GSK3�, causing inhibition ofits activity (479, 480).

Upon GC administration, PKB can be rapidly activatedvia a PI3K-dependent pathway, leading to the release ofvasorelaxing nitric oxide (479, 481–484). In support, GRcan interact with the p85� subunit of PI3K (484). Theserapid nongenomic effects typically need high doses of GCsto occur.

Depending on the cell type, however, GCs may differ-entially target particular kinases. An important mecha-nism underlying long-term GC-induced bone loss is the


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impairment of osteoblast function and bone formation.The Wnt signaling pathway has been hypothesized to playa critical role in the osteoblast differentiation-related cellcycle. A key negative regulator in the Wnt signaling path-way is the serine/threonine kinase GSK3�. Consequently,GC-mediated activation of GSK3� in osteoblasts resultedin an inhibition of the Wnt signaling pathway. In osteo-blasts, GCs do not stimulate but further inhibit phosphor-ylation of PKB/Akt on Ser473 and thus disrupt input fromthe PI3K/Akt pathway into the Wnt signaling pathway atthe level of GSK3� (485). GSK3�, a known phosphory-lator of NF-�B S468 (68), has recently also been shown tomodulate GR (321), adding on complexity (see also Sec-tion II.A). In mammary epithelial tumor cells, GCs inducethe phosphorylation of GSK3�, ultimately leading to itsdegradation via the ubiquitin 26S proteasome (480). Thismechanism is believed to be involved in the control of tightjunction formation in mammary epithelial tumor cells.Alternatively, GSK3� can also be activated via serum- andGC-inducible kinase 1 (SGK1). A high level of expressionof SGK in breast cancer cells suggests that this kinase mayfunction to protect tumor cells from apoptosis and thuscan act as an oncogene. As its name suggests, the expres-sion of SGK1 can be induced by GCs (486–488) and re-markably, this kinase can up-regulate also the transcrip-tional activity of NF-�B (489). In that respect, SGK1 wasrecently identified as the mediating kinase for PI3K-initi-ated phosphorylation of IKK� T23, which in turn facili-tates the phosphorylation of IKK� S180 and the subse-quent activation of NF-�B. Concomitantly, SGK1 wasfound to phosphorylate p300 S1834 and enhance NF-�B-mediated transcription (490). However, it is cur-rently unknown how GCs could affect these processesand what their role could be in an inflammatory re-sponse. It may seem paradoxical that GCs are able totranscriptionally stimulate a kinase that subsequentlyserves to activate NF-�B, but it is important to realizethat the functionality and role of SGK1 may be regu-lated not only transcriptionally but also posttransla-tionally by its upstream activating kinases, includingPI3K and MAPK (490).

Additionally, GCs can affect various members of theSrc family of tyrosine kinases. The c-Src kinase can—uponactivation—contribute to I�B� phosphorylation and sub-sequent NF-�B translocation (491), whereas p56lck [lym-phocyte kinase (Lck)] and p59fyn (Fyn) kinases and src-like spleen tyrosine kinase (Syk) have been reported to playa role in T cell receptor-mediated signal transduction(492). In contrast, GCs can rapidly inhibit the activity ofthese Lck and Fyn kinases via a membrane-associated GR,thus contributing to the GC-mediated inhibition of T-cellreceptor signaling (493–495). Moreover, the GC-induc-

ible increase in SLAP expression would inhibit Ag-inducedphosphorylation of the Syk in mast cells (396). In contrast,GCs can augment the phosphorylation of Lck and thedownstream activation of p59Fyn, Zap70, Rac1, and Vavin resting but not in activated T cells (496).

Furthermore, GCs can stimulate the protein and activ-ity levels of the Rho-dependent protein kinase (ROCK) 2but down-regulate the specific activity of ROCK1 in ratepithelial cells (497). The activation of ROCK2 is espe-cially implicated in lung inflammation and the associatedmyosin light chain phosphorylation (498). In support,GCs can impede myosin light chain phosphorylation inairway hyperresponsive rats (499).

The molecular mechanisms that underlie nongenomicGC-induced immunosuppression remain to be preciselydefined.

IV. Phosphatases Targeted by GlucocorticoidReceptor-Mediated Signaling

Phosphatases can be classified according to their substratespecificity. PPs targeting Ser/Thr and/or Tyr can be re-garded as three groups: the tyrosine-specific phosphatases[protein tyrosine phosphatases (PTPs)], the Ser/Thr spe-cific phosphatases, and the DUSPs, targeting phospho-Tyr/Ser/Thr (500, 501). Currently, the most intensivelyresearched GC-regulated phosphatase in the context ofinflammation is DUSP1 (400, 502). However, this phos-phatase is not the only phosphatase that is subjected toGC-mediated modulation.

A. Dual specificity phosphatases (DUSPs)The DUSP family comprises a subfamily of 10 enzy-

matically active DUSPs that target MAPKs, dephospho-rylating T and Y residues, and therefore these phospha-tases are also referred to as MAPK phosphatases. TheMAPK family of protein kinases includes extracellularsignal-related kinases ERK1 and ERK2; p38 MAPKs �, �,� and �; and the c-Jun N-terminal kinases JNK1, JNK2,and JNK3 (Fig. 5) (25, 503). Structurally, these DUSPs aremarked by a C-terminal catalytic domain, and DUSP1,DUSP2, DUSP4, and DUSP5 are considered inducible nu-clear members of this DUSP subfamily (504).

The promoter of DUSP1 can be driven by ligand-acti-vated GR, and overexpression of the dimerization-defec-tive GR mutant (193) is incapable of mediating activationof the murine DUSP1 promoter by GCs (193, 505). How-ever, GCs enhance DUSP1 mRNA expression in murinemacrophages expressing only this GRdim mutant (369).These findings per se are not necessarily in conflict becausethe group of Pearce (506) showed that GRdim can func-tion even better than wild-type GR at promoters with mul-


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tiple GR binding sites. Also, in the case of steroid recep-tors, the DBD dimer interface plays an important rolebecause specific receptor mutations or noncanonical spac-ing of half-sites that disfavor this dimer interface lead toenhanced synergy (507). However, GRE sequences werenever identified in DUSP1 promoters, and GCs failed toenhance human DUSP1 expression in cells solely express-ing a rGR LS7 (P493R/A494S) mutant, which is incapableof transactivating classical GRE-regulated promoters(192, 508, 509). Alternatively, GC-mediated DUSP1 pro-moter activation was attributed to an ERK- and JNK-dependent mechanism involving AP-1 and CREB bindingonto the DUSP1 promoter (510). Additionally, GR bind-ing onto the DUSP promoter, 1.5 kb upstream of the tran-scription start site, was suggested to occur via a tetheringmechanism onto C/EBP�, and mutation of this C/EBPbinding site attenuated GC-inducible DUSP1 reportergene expression (509). In this context, it is interesting tonote that GCs can enhance the function of C/EBP� byinducing its phosphorylation (511). DUSP expression inresponse to GC signaling seems cell type-dependent. Be-sides DUSP1, also DUSP2, DUSP9, the PEST domain-enriched tyrosine phosphatase, and especially DUSP4 areup-regulated in response to GCs in bone marrow-derivedmast cells (275), and GCs can also enhance the expressionof DUSP4 in adipocytes (512), whereas the expression ofDUSP2, DUSP4, and LC-PTP seems to be inhibited by GCsin various pre-B leukemia cell lines (513). Although, theabove-mentioned DUSPs can also modulate ERK, p38,and JNK MAPK phosphorylation, thus impacting the in-flammatory process (504), their role in the GR-mediatedantiinflammatory mechanism is currently unaddressed.To date, no reports have been published on GC modula-tion of the related family members DUSP5, DUSP7,DUSP8, or DUSP16.

Besides GCs, mitogens and stressors can also elevate theexpression of the inducible DUSPs, such as DUSP1, func-tioning as a negative feedback loop in MAPK regulation(504, 514). Mechanistically, evidence from KO cellsshowed an involvement of the p38 and ERK MAPK-activated MSK1 and ATF1, and of the JNK1 MAPK in theLPS-mediated stimulation of DUSP1 gene transcription(514, 515). However, stimulation of GR could furtherenhance the already LPS-induced DUSP1 promoter in asynergistic manner in macrophages (516). Very recently, aDUSP1/GRIP1-dependent combinatorial mechanism hasbeen identified for repression by GR of LPS-inducedCOX-2, CXCL-5, and IL-6. Activated GR could inhibitTLR4-dependent COX-2 gene induction in macrophagesvia a mechanism that involves both DUSP1-mediatedAP-1 inhibition and GR-GRIP1 recruitment to the p65NF-�B DNA complex (517).

Interestingly, SOCS1, which appears to be up-regu-lated by GCs in hematopoietic cells and immune cell can-cers (267, 388, 513, 518, 519), can hamper the transcrip-tion of DUSP1, probably via the association of GR andSOCS1 (520). In the inflammatory process, this SOCSis also involved in NF-�B p65 proteolysis (98) and cannegatively interfere with TLR2- and TLR4-mediatedsignaling (521).

Cell-specific GC-elevated levels of DUSP1 can then tar-get the activational phosphorylations of the MAPKs ERK,JNK, and p38, thus inactivating them (400). JNK and p38MAPK are especially subject to DUSP1-mediated dephos-phorylation because DUSP1 KO mice-derived immunecells display prolonged p38 and JNK activation, whereasthe GC-induced ERK dephosphorylation was not altered(275, 369). However, because the GC-induced ERK de-phosphorylation depends on de novo protein productionin RBL-2H3 mast cells, cell-specific effects may be at playhere or possibly other GC-inducible DUSP family mem-bers, such as DUSP2, DUSP4, or DUSP9, may intervene(275, 505).

Subsequent to the inactivation of the MAPKs, the on-going proinflammatory signaling pathway is halted, andgene expression and mRNA translation of proinflamma-tory cytokines is attenuated (reviewed in Refs. 243, 400,and 522) (see Sections III.A and III.B.2). Therefore,DUSP1 KO mice show an impaired GC-mediated re-pression of various proinflammatory cytokines. Never-theless, GCs can still inhibit the expression of certaincytokines. The difference in GC-induced repression be-tween DUSP1�/� and DUSP1�/� cells depends on theselected cytokine but also cell type. Furthermore, inhibi-tion of de novo protein synthesis in bone marrow-derivedmast cells of DUSP1�/� mice can still partly reverse theantiinflammatory potential of GCs, suggesting the in-volvement of other GC-induced antiinflammatory pro-teins and of direct GC-dependent antiinflammatory mech-anisms (275, 369). Although DUSP1 KO mice are viable,they appear more susceptible to inflammation than theirwild-type counterparts (275, 369, 523–526). However,the inflamed cells of these DUSP1 KO mice remain sensi-tive to the antiinflammatory mechanisms of GCs (275). Inconclusion, GC-instigated up-regulation of DUSP1 is con-sidered an additional layer in the overall antiinflammatorymechanism of GR.

In addition to GR and the MAPKs, DUSP1 itself is sub-ject to posttranslational modifications. Although an ex-treme C-terminal phosphorylation of DUSP1 (S359,S364) by ERK MAPK instigates DUSP stabilization (527),prolonged ERK MAPK activity results in other C-terminalphosphorylations of DUSP1 (S296, S323). However, thelatter phosphorylations promote DUSP degradation (528,


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529). Interestingly, GCs can inhibit the proteasomal deg-radation of DUSP1 (505). Moreover, LPS-mediatedDUSP1 acetylation (K57) via p300 seems to stimulate thebinding affinity of DUSP1 for p38 MAPK (530, 531). It iscurrently unknown whether and how these processes areaffected by GCs and connected to inflammation.

B. Other protein Y phosphatasesIn the PTP family, PTP receptor type C (PTPRC, also

known as LCA or CD45) is also regulated by GCs. As anessential regulator of T and B cell antigen receptor signal-ing, this membrane-associated tyrosine kinase is a markerfor inflammation and is not surprisingly down-regulatedby GCs in synovial membranes of arthritic rats and leu-kocytes of rheumatoid arthritis patients (532, 533).

Alternatively, GCs have been reported to induce theexpression of the SH2 domain-containing protein Y phos-phatase PTP1C (also known as SHP1), but not PTP1Dand PTP1 in pancreatic rat cells (534). This PTP1C hasbeen implicated in the regulation of T cell antigen re-ceptor signaling (535), and a deficiency in PTP1C func-tion was recently linked to an increase in TLR-initiatedinflammation (536).

C. Other phosphatasesThe protein S/T phosphatases PP1 and PP2A family

(PPP) are most abundantly present in the cell. The target-specific activity of PP1 and PP2A is encoded by five cata-lytical subunits and a vast abundance of interacting reg-ulatory subunits (537). Although effects of PP1, PP2A,and PP5 on GR have been reported (see Section II.B),currently only a few effects of GCs on these PPs have beenreported. PP1 and/or PP2A may be involved in the GC-mediated inhibition of translation via a GR-dependent de-phosphorylation of p70 S6K (70-kDa ribosomal proteinS6 kinase) (427, 428) (see Section V.A). However, thisinvolvement is yet to be confirmed via knockdown studies.

The S/T PP2A, which is composed of a catalytical sub-unit PP2Ac, a regulatory subunit PP2A-A (PR65), and avariable regulatory subunit PP2A-B (500), was suggestedto target ERK, p38, JNK MAPK phosphorylations, andNF-�B phosphorylation in various cells (106, 538–544).Furthermore, PP2A can impose a genome-wide dephos-phorylation of H3 S10 in Drosophila (545). In light of thefact that GCs can also target H3 S10 phosphorylation(294, 420), it could be interesting to investigate possibleadditional effects of GCs on PP2A.

PP2B (also known as calcineurin or PP3) is a Ca2�-dependent phosphatase comprising a catalytic A-subunitand a regulatory calmodulin-binding B-subunit (500),which has been implicated in the negative regulation ofMyD88 and Trif and thus TLR-mediated signaling (546).

Functionally, PP2B inhibitors are often used together withGCs as an immunosuppressant and antiinflammatorytherapeutic (547, 548). However, both pathways do notsignal independently as GCs can also modulate PP2B ac-tivity (549, 550). Because GCs can trigger the release ofCa2�, GCs could also rapidly and transiently enhance theactivity of PP2B in T cells (549). Alternatively, GCs canstimulate PP2B activity in a Ca2�-independent mecha-nism, involving a GC-induced association of Hsp90 andPP2B in pancreatic �-cells (550, 551). However, GCs donot seem to affect the protein levels of PP2B in various cells(551, 552).

V. Kinase/Phosphatase Regulation inGlucocorticoid-Mediated Side Effects

Although GCs remain the mainstay in the treatment ofacute and chronic inflammatory afflictions, long-term GCtherapy of chronic inflammatory disorders could lead to adetrimental side-effect profile and possibly also GC resis-tance (see Section VI). Overall, prolonged GC treatmentand high doses of potent GCs are the main risk factors inthe onset of GC therapy-associated side effects. It is likelythat both “desired effects” and “adverse effects” reflectGR actions, not only on transcription/translation directlybut also on phosphorylation.

The most prevalent GC therapy-associated side effect isweight gain, followed by skin bruising/thinning and insom-nia. From the patients’ view point, these skin problems to-gether with a disturbed fat distribution are psychologicallydistressful. Clinically, however, hyperglycemia leading todiabetes mellitus, the enhanced susceptibility to infec-tions, and osteoporosis are most worthy of attention andpose the greatest challenge and management issues in thesepatients. For instance, osteoporotic fractures occur in upto 50% of patients on long-term GC therapy, and theGC-induced patients’ sensitivity to infections could dou-ble their hospitalizations (553). Note that we call thesesecondary effects “side effects,” but it should be kept inmind that all of these effects are actually exaggerations ofnormal physiological GC actions.

Mechanistically, the onset and maintenance of GC-associated side effects is complex. Some GC therapy-as-sociated side effects are mainly attributed to GR transac-tivation mechanisms (e.g., diabetes, glaucoma, myopathy,hypertension), whereas others originate from GR transre-pression mechanisms [e.g., suppression of hypothalamic-pituitary-adrenocortical (HPA) axis]. However, the mech-anisms of some side effects are not completely known orcould be attributed to both GR transactivation and tran-srepression mechanisms (e.g., osteoporosis) (554). Fur-ther clinical and molecular research in this field is required


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to deepen our knowledge about the prevalence and mech-anistic basis of GC therapy-associated side effects.

The GC-enhanced susceptibility to infections stemsfrom the plethora of antiinflammatory and immunosup-pressive actions of the activated GR, as discussed in thesections above. Below, we will highlight the involvementof phosphatase and kinase signaling in the mechanisms ofGC-instigated side effects, with particular attention to theclinically most important ones.

A. Skeleton and muscle effectsLong-term GC therapy-induced osteoporosis origi-

nates from a dual mechanism: a decrease of osteoblastproliferation and activity and an increase in osteoclastactivity (555, 556).

Key to the activation status of osteoclastogenesis is thereceptor activator of NF-�B ligand (RANKL)/osteoprote-gerin (OPG) ratio. GCs can enhance the expression ofRANKL while decreasing the expression of a RANKL-signaling inhibitor, namely osteoprotegerin (555, 556). Inthe GC-mediated stimulatory mechanism of bone catab-olism, abrogated expression of the IL-1R-associatedpseudokinase IRAK-M is coupled to an enhanced differ-entiation of hematopoietic precursor cells into osteoclastsand an increase in osteoclast activity (557). In that respect,GCs can impede the expression of this IRAK-M and thusstimulate bone resorption (558).

In osteoblasts, ERK MAPK is essential to accommodateosteoblast proliferation. However, the GC-inducedDUSP1 expression in these cells can dephosphorylate ERKMAPKs, an event that was thus linked to a tyrosine phos-phatase-mediated inhibition of osteoblast proliferation.GCs directly affect osteoblasts by decreasing prolifera-tion, and therefore this mechanism may contribute to thephenomenon of steroid-induced osteoporosis (559–562).Additionally, the GC-induced activation of GSK3� in os-teoblasts was recently shown to contribute to their apo-ptosis, whereas GC-activated p38 MAPKs rather inhib-ited the apoptosis of these osteoblasts (563). Furthermore,the cell-detachment-induced apoptosis of osteocytes,which are bone-embedded cells that can regulate osteo-blast activity via gap junctions, is regulated via the rapidactivation of protein tyrosine kinase 2� (PTK2beta) (alsoknown as RAFTK or Pyk2) and thus its downstream targetJNK MAPK (564–566). Lastly, a GC-mediated inhibitionof osteocalcin transcription, via a competitive nGRE (290,567) contributes to the inhibition of bone formation.

Long-term GC therapy in juvenile diseased patientsalso causes serious growth retardation (553). The GC-induced apoptosis of chondrocytes, linked to this pro-cess, has been attributed to GC-mediated caspase acti-vation as well as GC-mediated inhibition of PKBphosphorylation (568).

When a high-dose GC treatment is sustained, pa-tients may experience muscle atrophy, leading to my-opathy. This GC-induced loss of muscle tissue is mainlyattributed to enhanced protein degradation (cataboliceffects) and diminished protein synthesis (antianaboliceffects) (554, 569).

One of the factors in the antianabolic effects of GCs,hampering the cellular protein synthesis mechanism, is theGC-mediated inhibition of 4E-BP1 phosphorylation,which facilitates the translation repressing association of4E-BP1 with eIF-4E (424–428). However, okadaic acidcould perturb the GC-mediated inhibition of p70 S6K and4E-BP1 phosphorylation, thus pointing to a possible rolefor PP1 and/or PP2A as the PPs used (427, 428). Alterna-tively, the GC-mediated decrease in phosphorylation of4E-BP1 in myoblasts was suggested to occur via negativeinterference with upstream mammalian target of rapamy-cin (mTOR) signaling (570).

Additionally, the PI3K/PKB/GSK� signaling path-way can play an important role in the regulation ofmuscle atrophy. Not surprisingly, GCs can inhibit PKBactivity in myoblasts, thus allowing GSK3� activation,which is in turn associated with suppressed protein syn-thesis (569, 571).

B. Hyperglycemia and diabetesPatients who are subject to long-term GC therapy have

a tendency to develop hyperglycemia, with the risk of di-abetes because GCs not only decrease the stimulated in-sulin production but also lower their response to circulat-ing insulin (554). The former decrease in insulin levels wasattributed to a nGRE element in the insulin promoter aswell as GC-mediated PP2B-dependent apoptosis of insu-lin-secreting cells (550, 551, 572). The latter insulin re-sistance could in part occur via a GC-induced rapid andtransient inhibition of insulin receptor kinase activity andseveral downstream intermediates such as p70 S6K, PKB,3-phosphoinositide-dependent protein kinase (PDK),Fyn, and GSK3 (494, 512). Additionally, GCs could ac-tually enhance JNK MAPK phosphorylation in adipocytes,which was associated with a perturbed insulin receptor-de-pendent signaling resulting in insulin resistance (494). Inthese same cells, an increase in DUSP1 expression was sug-gested to associate with a decrease in cellular glucose uptake(512). However, additional evidence for a role of DUSP1 inGC-induced hyperglycemia is currently lacking.

Lastly, GCs can stimulate the expression of various glu-coneogenetic enzymes in the liver, among which TAT,pyruvate dehydrogenase kinase 4, glucose-6-phosphatase(G6Pase), and pyruvate carboxykinase (PEPCK), culmi-nating in elevated glucose levels (573–579).


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C. Other side effectsGC-induced hypertension is in part caused by a dys-

regulation of Na� homeostasis. As an important factorin this event, GCs can enhance the GRE-regulated tran-scription of the epithelial Na� channel (�ENaC) geneand cell-specifically augment ENaC and Na(�)/H(�)exchanger 3 (NHE3) activity in a SGK1-dependentmanner (554, 580 –588). The GC-activated rise inNHE3 activity would involve a direct SGK1-mediatedNHE3 phosphorylation (588).

In the ENaC modulating mechanism, a SGK1-medi-ated inhibiting phosphorylation of ALL1-fused gene fromchromosome 9 (Af9) of the histone-methylating Dot1a(disruptor of telomeric silencing alternative splice varianta)-Af9 complex can derepress the ENaC promoter, thusfacilitating transcription (589). Furthermore, an aldoste-rone-induced SGK1 can phosphorylate the E3 ubiquitinligase Nedd4-2, which is stabilized by 14-3-3, and can thusdiminish the degradation rate of ENaC by reducing theinteraction affinity between Nedd4-2 and ENaC (590–593). Alternatively, a specific and direct phosphorylationof ENaC by overexpressed SGK1 can directly enhanceENaC activity (594). Moreover, a steroid-mediated rise inGILZ expression and the concomitant inhibition of ERKMAPK signaling was recently linked to an increase ofENaC expression and activity (379, 380, 390). In addi-tion, Na� retention could possibly be enhanced by GCs viaa SGK1-dependent increase in Na�/glucose cotransporterSGLT1 activity via a similar Nedd4-2- based mechanism(595). However, evidence to the specific GR-dependenceof the above mechanisms is currently still lacking.

Furthermore, a GC-induced increase in gastric acid se-cretion, thus contributing to an enhanced risk of gastro-intestinal bleeding and peptic ulcer development, occurs ina PI3K/SGK1-dependent manner of which the precisemechanism remains currently unclear (261, 596).

Lastly, long-term GC therapy can in some cases lead toeye problems, such as cataract. A contributing mechanismin the GC-induced development of cataract is an increasedrate of gluconeogenesis, thus implicating PEPCK andG6Pase regulation (554). Recently, it was discovered thatGCs can enhance GILZ and DUSP1 expression in lensepithelial cells, coinciding with a decrease of Raf, ERK,p38 MAPK, and PKB phosphorylation (597). However,currently a role for this mechanism in cataractogenesis hasnot been firmly established.

VI. Kinase/Phosphatase Regulation inGlucocorticoid Resistance

Patients suffering from GC resistance are refractory to anantiinflammatory GC treatment. Although GC resistance

can be innate, it can also be acquired due to a prolongedGC treatment (201). Here we will shortly discuss differentmechanisms that possibly lie at the basis of GC resistancein inflammation, with special attention for GR phosphor-ylation in this phenomenon.

Foremost, the cause of innate GC resistance lies in amutation of the GR itself, leading to abnormal GR con-centrations, ligand-binding affinity, GR stability, GC-induced nuclear translocation, or GR-cofactor interac-tions (201). Currently, the research field concerning theseveral known GR haplotypes with regard to its specificeffects on GR phosphorylation and GR effects on kinaseand phosphatase regulation remains largely unexplored.

Furthermore, innate and acquired GC resistance wasassociated with a decrease in GR� protein levels via ho-mologous down-regulation, an increase in the protein lev-els of the dominant-negatively acting GR�, a decreasedGR ligand-binding affinity, or GR DNA binding (598–601). Moreover, the sensitivity to GCs could be alignedwith the degree of GC-induced GR nuclear translocation(602). Alternatively, GC resistance has been linked to anelevated expression of FKBP51, an element of the GRchaperone complex (603). At the level of cofactor regu-lation, GC resistance was associated with a reducedbrahma-related gene (Brg1) 1 expression and a PI3K-reg-ulated decrease in HDAC2 activity and expression (365,604–606). However, in some cases GC resistance wasattributed to a failure of GCs to acetylate histone H4 K5and thus transactivate gene expression, rather than to adisturbed GR transrepression mechanism (602). How-ever, the existence of a multidrug resistance membranetransporter, extrudingGCsoutof the cell and thus limitingtheir activity, has also been reported in GC resistance andcan function as a time-restricting control mechanism ofactivated GR (607).

In addition, GC-induced alterations in the GR phos-phorylation status have been associated with acquired GCresistance. As such, GC resistance has also been linked tothe inflammatory status of the diseased tissue via an en-hanced kinase activity of JNK, ERK, and p38 MAPKs; anincreased synthesis and/or activity of the transcription fac-tors NF-�B and AP-1; and increased cytokine production(330, 331, 608–615). In this respect, p38 MAPK-, JNKMAPK-, and GSK3-mediated phosphorylations of GRhave been linked to a decrease in GR transcriptional ac-tivity (321, 330–335) (see Section II.A). Interestingly, ap38 MAPK- and GSK3-mediated phosphorylation of GRcoincided with an attenuated repression of NF-�B-medi-ated gene expression by GCs (321, 330). Conversely, GCresistance was associated with the inability of GCs to de-activate JNK MAPK, which was reflected in elevatedphosphorylated c-Jun and c-fos gene expression in GC


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resistance and coincided with an observed decrease of GR-AP-1 interaction intensity in steroid-resistant asthma pa-tients, when compared with samples from steroid-respon-sive patients (600, 609, 616–618). Moreover, the GCresponse of steroid-resistant patient samples could be re-stored via the addition of MAPK inhibitors (330, 612,613, 619), and thus the MAPK-mediated inhibition of GRfunction appears to be a central player in GC resistance. Ofinterest, GC-resistant T cells, in which the steroid respon-siveness was restored via the addition of IFN, featuredelevated DUSP1 expression levels and concomitantlyan inhibition of p38 MAPK phosphorylation (619). Inpatients with severe asthma, which showed decreasedresponsiveness to GCs, an increased p38 MAPKphosphorylation corresponds to reduced induction ofDUSP1 expression (614). Taken together, GC unre-sponsiveness features a central role for MAPK dysregu-lation and probably also impaired DUSP1 induction.

VII. Future Perspectives in the Combatof Inflammation

In this section, we will discuss new and upcoming GR-based therapeutics and therapeutic strategies, with spe-cial attention to the effect of these ligands on GR phos-phorylation and GR-based kinase and phosphatasemodulations.

A. New glucocorticoid receptor ligandsA long-standing hypothesis driving steroid develop-

ment pharmacology strategies for the past decade was thatthe side effects of GCs are mainly attributed to GR tran-scriptional activation, whereas the antiinflammatory ef-fects of GCs are predominantly mediated via GR transre-pression mechanisms. This viewpoint was supported bythe finding that GRdim, a GR variant with a mutationhampering GR homodimerization, thereby compromisingDNA-binding at some GRE-driven genes, and thus pre-venting the stimulation of a classical GRE, still allows forGC-mediated repression of AP-1- and NF-�B-mediatedproinflammatory gene expression (193, 285, 620). Morerecently, it was demonstrated that this model is too rigidand that GRdim still leads to transactivation of a numberof genes, exemplified by phenyl-N-methyl-transferase.Thus, on a subset of GR-responsive promoters, GRs canform concerted multimers in a manner that is independentof the DBD-dimer interface (506). Indeed, GRdim micestill suffer from some side effects in response to GCs (276),and clearly not all GR-mediated side effects are solely con-trolled by classical GR transactivation mechanisms (621).Of course, a number of side effects are also attributed totransrepression mechanisms, e.g., HPA axis suppression.

Although it is recognized that a few antiinflammatorygenes are under stimulatory control by GCs, the transcrip-tion of these genes sometimes depends on an atypical GRtransactivation mechanism, and their role in the GC-induced antiinflammatory mechanism is not completelyclear (243). Although the model in which GR transrepres-sion mechanisms are associated with antiinflammatory ef-fects and GR transactivation mechanisms are associatedwith undesired effects clearly has to be put into a morenuanced perspective, it is still believed that a selective mod-ulation of GR, resulting in distinct GR mechanisms, couldcontribute to yielding a more beneficial side-effect profile inantiinflammatory therapies (268,285,622). It isparamount,however, to obtain further insights into the “context-depen-dency” phenomenon of GR-mediated regulation. Com-pounds that could activate select GR mechanisms and thusalter GR-mediated gene expression profiles are beingdesignated as dissociated compounds, selective GR ago-nists (SEGRAs) or modulators (SEGRMs) (621–624). Theterminology “SEGRMs” can have the most broad inter-pretation and can thus also include molecules that are GRantagonists, or even molecules that could activate partic-ular functionalities of GR without actually being a ligand.Various compounds—steroidal and nonsteroidal—capa-ble of activating GR transrepression without inducing GRtransactivation mechanism are called dissociated com-pounds (625). However, when the dissociation is not ab-solute, it is semantically more correct to refer to them asSEGRAs or SEGRMs, depending on a reported binding ofthe compound in the ligand-binding pocket (623).

Multiple SEGRAs and SEGRMs have been reported:RU24858, RU24782, and RU40066 (286, 626, 627);A276575 (628); AL-438 (629, 630); compound 25 (631);CpdA (293, 422, 632); ZK216348 (633, 634); LGD-5552(635, 636); and various other compounds (622, 637–643).When one of the above-mentioned compounds stimulatesGR, cellular effects can mechanistically differ from clas-sical GCs in affinity for the GR LBD, GR GRE DNA bind-ing, dimerization, cofactor promoter occupancy, histonemodifications at targeted promoters, but also GR phos-phorylation—all resulting in a divergent gene expressionprofile (293, 422, 622, 623, 629, 636).

Currently, GR phosphorylation-related research hasonly been conducted with CpdA. As a dissociative GRmodulator, CpdA [i.e., 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride] can efficiently repressthe transcription of inflammatory genes via diminishingNF-�B p65 DNA binding and the overall NF-�B p65transactivation potential and promoting nuclear export ofthe transcription-facilitating kinase MSK1 (293, 420).Conversely, CpdA cannot stimulate GRE-mediated genepromoter activation (293, 422). Interestingly, unlike clas-


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sical GCs, CpdA does not induce DUSP1 human or mousereporter genes, nor can it elevate DUSP1 protein levels invarious cell lines (I. M. E. Beck, unpublished results). Ofspecial interest, CpdA instigates a differential phosphor-ylation profile of GR in comparison to classical GCs be-cause it did not induce a hyperphosphorylation of hGRS211 (293). Although the N-terminal domain of GR is notnecessary for CpdA- or classical GC-mediated trans-repression of NF-�B-regulated gene expression, the lack ofhGR S211 phosphorylation in CpdA-stimulated cellscould possibly explain the deficiency in GR recruitment toand transactivation of GRE-regulated promoters (293).Taking into account that this phosphorylation site is con-sidered crucial in the GR transactivation process (320,327), this leads to a question about whether all dissociatedGR modulators share this feature. GR phosphorylationhas also been implicated in ligand-mediated GR down-regulation (320, 325, 345, 346). In this respect, it is in-teresting to note that CpdA does not evoke homologousGR down-regulation in fibroblast-like synovial cells,which is reflected in a diminished antiinflammatory ther-apy resistance in long-term treatment protocols (632).Currently, the mechanism lying at the basis of this CpdA-mediated GR preservation and thus the possible involve-ment of differential GR phosphorylation is unknown.Taken together, additional research into the phosphory-lation profiles of GRs liganded with SEGRMS, could openup new and interesting perspectives in SEGRM research.

With respect to side effects, we can note that SEGRMsthat cannot induce the GRE-regulated PEPCK andG6Pase, such as CpdA, typically do not induce hypergly-cemia and hyperinsulinemia (293, 422). Also, AL-438 andZK216348 cannot instigate hyperglycemia (629, 633).However, further investigations comparing the side-effectprofile of classical GCs vs. the SEGRMs in a more elab-orate manner and research into the in vivo antiinflamma-tory potential of these SEGRMs remain a future challengein which special attention for SEGRM-induced effects onkinases and phosphatases could shed a new light on themechanisms of these GR modulators.

B. Combination therapiesConsidering the detrimental side-effect profile evoked

by a long-term GC therapy, research efforts additionallyfocused on possible strategies to lower therapeutic GCconcentrations while maintaining a similar antiinflamma-tory potential. Mechanistically, the idea of combining twoor more different antiinflammatory compounds would al-low lower dosages of each, while maintaining the antiin-flammatory profile and attenuating possible side effects ofthese therapeutics (644).

The highly efficient antiinflammatory combinationof long-acting �2 agonists (LABAs) with GCs is already

clinically used for the treatment of asthma and COPD(645, 646). Interestingly, LABAs can stimulate phos-phorylation of hGR at, e.g., S211 and stimulate its nu-clear translocation (647– 649). Moreover, combiningLABAs with GCs leads to a more pronounced inhibitionof ERK MAPK, JNK MAPK, and I�B� phosphoryla-tions (650) and increased expression levels of the anti-inflammatory DUSP1, whereas the GC-induced expres-sion of GILZ or TTP was not increased by the additionof LABAs (648, 651).

Additionally, the antiinflammatory effects of combin-ing Cdk, MAPK, or MSK inhibitors with GCs has beenassessed, all of which resulted in an enhanced antiinflam-matory treatment profile (330, 331, 456, 652). Althoughthese results appear promising, additional immunologicalresearch remains necessary to investigate the immunolog-ical and clinical aspects of these combination therapies.

Although it may seem temptingly simple to combineGCs with inhibitors targeted at inflammatory process-in-volved kinases to maximize the inflammatory effect, thisreview shows that because of the widespread crosstalk ofkinases and phosphatases with all levels of GR signaling,caution is in order. Therefore, research into topical appli-cation or intermittent therapy strategies for GCs and ki-nase inhibitors, surpassing general systemic side effects;and more specific ligands for GR, such as the SEGRMs;and more specific kinase inhibitors and phosphatase mod-ulators could broaden the array of available therapeuticagents. From this review, it is apparent that further re-search into the effects of kinases and phosphatases onto orby GC-mediated signaling could conduce the developmentof various new drug targets in the combat against inflam-mation and the control of GC-mediated side effects andGC resistance. In the treatment of chronic diseases, a moreconstrained therapeutic window is required than in acuteafflictions, allowing for a short-term treatment. Prefer-ence should go to the targeting of kinases or phosphatasesdownstream or at the end of a signaling cascade, insteadof targeting kinases at the top of the cascade. However,because of their actions in multiple pathways and be-cause of a known difficulty in specific targeting, thedecision to target kinases/phosphatases has to be ap-proached with extreme caution, and the risk to off-tar-get effects should be investigated and carefully weighed.As a prerequisite, the in-depth knowledge of the stereo-dynamic structure of the active enzymatic pocket of thekinase and phosphatase and pharmaceutical design willhave to be broadened in the future.

C. MicroRNA-specific modulation of GRThe GC response varies among individuals, as well as

within tissues from the same individual, depending on pre-receptor stage, ligand metabolism, GR polymorphisms,


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selective expression of GR subtypes (197, 199, 653–659),kinase/phosphatase-dependent posttranslational modifi-cations of the GR (as discussed in the current manuscript),the cofactor environment (531, 660, 661), and cell type-specific hormone receptor crosstalk with aryl hydrocar-bon receptor, MR, and peroxisome proliferator-activatedreceptor (PPAR) (662–664).

MicroRNAs (miRNAs) are single-stranded small andnoncoding RNA molecules that can regulate gene expres-sion. miRNAs turn off target gene expression within cellsby binding complementary regions in mRNA transcripts,consequently affecting mRNA translation. The possibilityexists that various kinases/phosphatases may impact onmiRNA-dependent regulation of GR function, involved inthe control of developmental, tissue-specific, and individ-ual-specific GC responses in human health and disease.Interestingly, miR-18 and miR-124a reduced GR-medi-ated transactivation in addition to decreasing GR proteinlevels and GC responsiveness in a cell type-specific fashion(665). Another target of miR-124a includes the mRNA ofa small CTD phosphatase, an antineuronal factor ex-pressed in nonneuronal tissues, playing a role during em-bryonic development (666).

Because GR ligands themselves can also changemiRNA transcription profiles, miRNA control of GR andinflammatory stress-sensitive kinases and phosphataseswill become a hot area of further research in GR stressbiology and immune functionality (152, 667–670).

D. Epigenetic approachesEnvironmental exposure to low concentrations of hor-

mones, air pollution, or toxicants can have heritable epi-genetic DNA methylation effects in animals and humansand persistently change inflammatory stress responseslater in life, or even in next generations (671–677). Inter-estingly, life events occurring during the prenatal, neona-tal, and perinatal period have programming effects on theHPA axis, brain neurotransmitter systems, and cognitiveabilities of the offspring and long-term effects on the be-havioral and neuroendocrine response to stressors (678–680). Of special note, maternal stress during gestation,including maternal GCs, may predispose to immune-re-lated pathologies later in life or in offspring (681, 682). Inmale and female rats exposed to prenatal restraint stress,these effects include a long-lasting hyperactivation of theHPA response associated with an altered circadian rhythmof corticosterone secretion (682). More particularly, themethylation status of the hGR (NR3C1) gene promoter innewborns is sensitive to prenatal maternal mood and maystimulate a potential epigenetic process that links antena-tal maternal mood and altered HPA stress reactivity dur-ing infancy (683). DNA methylation of CpG dinucleotidesis generally associated with epigenetic silencing of tran-

scription and is heritable through cell division. MultipleCpG sequences are rare in mammalian genomes, but fre-quently occur near the transcriptional start site of activegenes, with clusters of CpGs (CpG islands) being hypo-methylated.TheGRCpGislandcontains sevenalternativefirst exons, and their promoters show highly variableDNA methylation patterns between individuals. As such,methylation may orchestrate alternative first exon usageand silencing and may control tissue-specific expression ofGR. The observed heterogeneity may reflect epigeneticmechanisms for fine-tuning GR activity, programmed byearly life environment and events (684). Of particular in-terest, various epigenetic (co)factors, including HDACs,HATs, polycomb proteins, methyl CpG binding proteins[methyl CpG binding protein 2 (MeCP2), methyl CpGbinding domain protein (MBD)], and even DNA methyl-transferases themselves are regulated by kinase/phospha-tase complexes during inflammatory stress (661,685–696). Reciprocally, lack of MeCP2 in a mouse modelof Rett syndrome increases various GC-regulated genes,including the SGK1 (697). It remains to be further inves-tigated whether kinase/phosphatase-dependent changesin DNA methylation at the GR gene promoter or GR tar-get genes are cyclical or persistent (698, 699). Conse-quently, combination therapy of GCs, kinase/phospha-tase inhibitors, and epigenetic drugs may hold promises tomodulate GR activity in a time-dependent, cell-specific, orgene-specific way.

VIII. Conclusions

GR activity is regulated by kinases and phosphatases at mul-tiple levels. Alternatively, both kinases and phosphatases aresubjected to regulation by the GR. The phosphomodulationof GR can affect its ligand affinity, DNA binding, cofactorrecruitment, cellular localization, half-life and recycling,overall posttranslational modification profile, and ulti-mately transactivational and transrepressional properties.Because these receptors exist in a dynamic regulation, cul-minating in different receptor functions at different times,GRs cannot be considered as a uniform population, andresearchers should be beware of this complication. In turn,GC-activated GRs can alter the expression level, activity,half-life, localization, and specific interactions of variouskinases and the expression and activity of various phos-phatases. In conclusion, the regulation and implications ofGR phosphorylation and the effects of GR on variousphosphorylation and dephosphorylation events in theframework of inflammatory models forms an intricateweb. Because the current use of kinase inhibitors in bothresearch and therapy has markedly increased in the field ofinflammatory disease control, this web of regulations


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should be taken into account when interpreting results ofkinase inhibitors with regard to the antiinflammatorymechanism of GCs.

Current scientific knowledge may lead to the identifi-cation of new kinase or phosphatase drug targets and thedesign of novel combination therapies that target GR aswell as its associated kinases/phosphatases to promote theantiinflammatory efficacy, reduce side effects, tackle GCresistance, and ultimately culminate in a better therapeuticprofile. Recently, kinases/phosphatases have also enteredthe field of epigenetic and miRNA-dependent regulation ofGR function and are, as such, also involved in the control ofdevelopmental, tissue-specific, and individual-specific diver-sity in the GC pathway in human health and disease. How-ever, further research, both at the basic and clinical levels,is still required to understand and deepen knowledgeabout the specificity and intertwinement of GR-, kinase-,and phosphatase-mediated events.

In conclusion, as the plethora of GR-regulated effectson and by kinase and phosphatase activities unfolds, thein-depth understanding of the antiinflammatory mecha-nism of GCs opens up new perspectives in manipulatingthis process therapeutically in a focused manner.

Acknowledgments

Address all correspondence and requests for reprints to: Karolien DeBosscher, Laboratory of Eukaryotic Gene Expression and Signal Trans-duction, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. E-mail:[email protected].

This work was financially supported by grants from InteruniversityAttraction Poles 6/18 (to G.H.), a Concerted Research Actions GOAfrom Ghent University (to G.H.), and U.S. National Institutes of HealthGrant CA020535 (to K.R.Y.). I.M.E.B., W.V.B., L.V., and K.D.B. are post-doctoral fellows of the Research Foundation-Flanders (FWO–Vlaanderen).

Disclosure Summary: I.M.E.B., W.V.B., L.V., G.H., and K.D.B. havenothing to declare. K.R.Y. is a consultant for Merck and Co. andSangamo Biosciences.

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