Review

Skin Pharmacol Physiol 2005;18:103–114 Received: June 1, 2004 DOI: 10.1159/000084907 Accepted after revision: October 19, 2004

Glucocorticoids for Human Skin: New Aspects of the Mechanism of Action

a a a b c M. Schäfer-Korting B. Kleuser M. Ahmed H.-D. Höltje H.C. Korting

a Pharmakologie und Toxikologie, Institut für Pharmazie, Freie Universität Berlin, Berlin , b Pharmazeutische Chemie, Institut für Pharmazie, Heinrich-Heine-Universität, Düsseldorf , c Klinik und Poliklinik für Dermatologie und Allergologie, Ludwig-Maximilians-Universität, München , Deutschland

Key Words Introduction Genomic effects receptor Non-genomic effects Cellular signalling , the most potent physiologic glucocorticoid, Skin affects almost all physiologic systems of the organism. Cortisol is synthesised and released from the adrenal cor- tex under hypothalamic control in a pulsatile and circa- Abstract dian way. Importantly, under stress the adrenals secrete Topical glucocorticoids have always been considered cortisol on top of this rhythm, which facilitates recovery. fi rst-line drugs for infl ammatory diseases of the skin and In case of immune challenge, cortisol protects the organ- bronchial system. Applied systemically, glucocorticoids ism against damaging infl ammatory responses. Due to are used for severe infl ammatory and immunological the multiple targets of action, cortisol and more impor- diseases and the inhibition of transplant rejection. Ow- tantly synthetic derivatives are fi rst-line drugs in the ing to the progress in molecular pharmacology, the treatment of immunologic and infl ammatory diseases, as knowledge of the mechanism of action has increased for example infl ammatory bowel disease, arthritis and during the last years. Besides distinct genomic targets, systemic lupus erythematosus. Moreover, glucocorticoids which are due to the activation of specifi c cytoplasmatic are effective in the treatment of some neoplastic diseases, receptors resulting in the (trans-) activation or (trans-) e.g. acute lymphatic leukemia of children. repression of target genes, there are non-genomic ef- In many infl ammatory skin diseases [1] as well as in fects on the basis of the interference with membrane-as- persistent bronchial asthma [2] , topical glucocorticoids sociated receptors as well as with membrane lipids. In are a mainstay of therapy, as unwanted effects are less fact, various glucocorticoids appear to differ with respect prominent compared with systemic treatment. With re- to the relative infl uence on these targets. Thus, the ex- spect to the skin, antiproliferative, apoptotic or antiapop- tended knowledge of glucocorticoid-induced cellular totic and also vasoconstrictive effects may contribute to signalling should allow the design and development of the desired anti-infl ammatory and immunosuppressive even more specifi cally acting drugs – as it has been ob- effects, yet may also result in unwanted side effects in tained with other , e.g. estrogens for osteoporo- other diseases. Moreover, it has been learnt that glucocor- sis prevention. ticoids do not only differ with respect to potency but also Copyright © 2005 S. Karger AG, Basel in the benefi t/risk ratio [3], which is linked to a selective infl uence on cytokine synthesis [4, 5] . While this was fi rst

© 2005 S. Karger AG, Basel Prof. Dr. M. Schäfer-Korting 1660–5527/05/0183–0103$22.00/0 Institut für Pharmazie (Pharmakologie und Toxikologie) Fax +41 61 306 12 34 Freie Universität Berlin, Königin-Luise-Strasse 2–4 E-Mail [email protected] Accessible online at: DE–14195 Berlin (Deutschland) www.karger.com www.karger.com/spp Tel. +49 30 838 53283, Fax +49 30 838 54399, E-Mail [email protected] reported concerning topical glucocorticoid use in derma- By inducing cellular adhesion molecules, proinfl am- tology, recently this has also been acknowledged with re- matory cytokines (e.g., TNF , IL-1) increase epidermal spect to asthma therapy [6–10]. The understanding of this chemokine formation and T cell localisation. CCL17 and separation increases since several new aspects of the glu- CCL22, chemokines with two highly conserved adjacent cocorticoid signalling pathways were revealed during the cysteine residues formed by IL-4 and IL-13 stimulation, last years, expanding our knowledge on the mechanism recruit T cells to skin and lung. Keratinocytes from pa- of drug action. Various aspects of glucocorticoid action tients with atopic dermatitis express also other Th attrac- have been reviewed previously emphasising comparison tant chemokines such as RANTES, MCP-1 and – most of glucocorticoids and other immunomodulators [11], importantly – CCL27 more abundantly [23, 24]. non-genomic glucocorticoid effects [12–14], cross-talk Most importantly, there is an intense cross-talk of the with cytokines and transcription factors [15–18], gluco- various signalling pathways described, and maximum ac- corticoid-mediated apoptosis [19], receptor isoforms [20] tivation of a cell means the activation of more than a but also effects in asthma [9, 21] . Here, we describe the single pathway. Moreover, enzymes from the MAP ki- multifaceted aspects of the mechanism of glucocorticoid nase pathway may also interact with the NF- B cascade action which are currently known and their translation [15, 16]. into the therapy of infl ammatory skin diseases. By inhibiting the formation of infl ammatory cyto- kines, glucocorticoids interfere with the activation of a variety of immunologic cells. Glucocorticoids inhibit the Infl uence on the Cytokine Network activation of dendritic cells which normally stimulate Th2 cells and in the lung also B cells. Moreover, eosino- An imbalance of helper T cells in atopic eczema and phil and T cell apoptosis increases. Yet, glucocorticoids asthma results in an increased baseline mediator forma- also infl uence other cells such as keratinocytes, fi broblasts tion [22] . Consequently, alveolar macrophages of asthma and alveolar cells. Besides genomic effects, non-genomic patients and keratinocytes of patients with atopic derma- targets appear to contribute to glucocorticoid effects. titis release interleukin-1 (IL-1), tumour necrosis factor- (TNF- ) and granulocyte-macrophage colony-stimulat- ing factor (GM-CSF) at an increased rate [21, 23–25]. By Genomic Glucocorticoid Effects stimulating cytokine receptors on the cell surface, IL-1 and TNF- induce a cellular signal cascade which acti- Receptor Structure vates transcription factors such as signal transducers and In general, hormones induce their effects by activators of transduction (STATs) or the nuclear factor binding to intracellular receptors (glucocorticoid recep- B (NF- B) which is released from the complex with its tor, GR or hGR ; mineralocorticoid receptor, MR; pro- inhibitory protein I B . Another relevant pathway of gesterone receptor, PR; androgen receptor, AR; estrogen IL-1 and TNF- is the activation of the mitogen-activat- receptor, ER; fi g. 1 ) which are closely interrelated and ed protein (MAP) kinase, and thus the activation of acti- form a superfamily together with the receptors for thyroid vator protein 1 (AP-1) which also results in the activation hormone, retinoids, vitamin D but also the peroxisome of nuclear factor of activated T cells (NFAT) containing proliferator activated receptor. Today, various isoforms AP-1 as an integral part. AP-1 and NF- B activate re- of these receptors are known. These proteins are charac- sponse genes by binding to their promotor region, recruit- terised by an N-terminal domain activating gene expres- ment of co-activators and basal transcription factors. His- sion, a central DNA-binding moiety and a C-terminal tone acetylation by activated histone acetyl transferases ligand-binding domain. results in a more relaxed chromatin environment which The highly conserved central DNA-binding domain of enables the access of RNA polymerase to the coding re- about 70 residues contains two loops which are stabilised gion of the target gene. These are genes coding for cyto- by four cysteine residues each complexing to zinc ions kines (IL-1, IL-2, IL-4, IL-6, IL-13, TNF- ), chemotaxis (zinc fi ngers). The N-terminal zinc fi nger discriminates proteins (e.g., GM-CSF), cellular adhesion molecules, i.e. between the different response elements and therefore is intercellular adhesion molecule (ICAM-1), vascular cell important for the selectivity of glucocorticoid effects. The adhesion molecule (VCAM-1), but also enzymes, i.e. DNA-binding domain is fl anked by an N-terminal trans- phospholipase A 2 , inducible forms of cyclooxygenase activating moiety ( 1) which after receptor binding en- (COX-2) and NO synthase (iNOS). hances the transcription of target genes. The ligand-bind-

104 Skin Pharmacol Physiol 2005;18:103–114 Schäfer-Korting/Kleuser /Ahmed /Höltje /

Korting Fig. 1. Human gene, mRNA splice variants and receptor pro- teins (hGR and hGR ); modifi ed from [20].

Fig. 2. Glucocorticoid receptor binding as determined experimentally [30, 81] . BM = ; BMP = betamethasone di- propionate; BMV = betamethasone 17-val- erate; BU = ; CBP = 17-propionate; DEX = ; MOM = ; MPD = methylpred- nisolone; PC = ; PD = pred- nisolone; TAA = acetonide.

ing domain is built up from 12 -helices, the segment 2 acid and arginine of ER which interact with the phenolic of the liganded receptor, which is part of helix 12, favours group of the estrogens [26, 27]. dimerisation and enhances transactivation but also inter- Recently, the precise structure of the ligand-binding actions with co-activators [26]. The closest homology is domain of GR was revealed by the crystallisation of this found within the group of the 3-oxo-steroid receptors domain [28] and independently by molecular modeling which besides GR are the MR, PR and AR. The ligand- starting from the well-known ligand-binding domain of binding domains contain a central arginine and a gluta- PR [29, 30]. The C-terminal 12 helices of the GR fold mine complexing the 3-keto group, while it is glutaminic into a three-layer sandwich which embeds the hydropho-

New Aspects of Glucocorticoid Action Skin Pharmacol Physiol 2005;18:103–114 105 Table 1. Glucocorticoid receptor binding predicted from quantitative structure activity calculations and experi- mentally determined [30, 81, 82], IC50 values for genomic and non-genomic effects in A549 cells [7] and relative activity in rheumatic diseases [13]

Glucocorticoid Binding Genomic effects Non-genomic effects

calculated determined growth COX-2 PGE2 AA/ antirheumatic inhibition PLA2 activity

Dexamethasone 7.87 8.02 < 8.30 7.00 7.70 7.70 1.2 8.03 Methylprednisolone 7.39 7.43 < 8.30 NS 8.00 8.00 1.0 Fluticasone NR 9.3 <12.00 9.30 9.30 NS ND Mometasone 8.97 8.79 <12.00 10.00 11.00 NS ND 9.12 Budesonide 8.481 9.00 <10.30 9.00 9.00 NS ND Beclomethasone dipropionate 8.701 ND <12 9.12 9.12 NS ND Beclomethasone 7.861 ND < 9.60 6.70 7.70 7.70 ND Prednylidene 7.521 ND

ND = Not determined; NR = reliable results not to be expected; NS = not signifi cant. 1 Recalculated as described by [30].

bic pocket for the ligand. In contrast to other nuclear re- Receptor Activation, Response Elements, ceptors, there is a rather wide side pocket which allows Transactivation and Transrepression access of many steroid esters favoured for the topical Glucocorticoid binding induces hyperphosphoryla- treatment of infl ammatory skin diseases and bronchial tion, exchange of immunophilins (FKB52 replaces asthma. Results of ligand docking experiments to predict FKB51) and recruitment of the transport protein dynein glucocorticoid activity conformed to steroid binding de- which allows nuclear translocation of the receptor. In the termined experimentally. As depicted in fi gure 2, there is nucleus, ligand-activated GRs release hsp90 and change a close correlation of GR binding predicted by quantita- structure [18] which allows the formation of a unique ho- tive structure activity calculations and the binding data modimer characterised by a -sheet made up from the determined experimentally. Extending the set of 17 ste- dimerisation domains of both monomers [28]. GR di- roids [30] by another seven (table 1 ) the only exception is mers as transcription factors bind to specifi c DNA-bind- fl uticasone propionate, calculation of binding data is ex- ing elements (glucocorticoid response elements; GREs) cluded since this agent is the only carbothiolate drug. and infl uence the expression of target genes which har- Non-stimulated GRs form a complex with various re- bour the respective response elements in their promotor ceptor-associated proteins (RAPs) which mask the li- region (simple GRE, transactivation; fi g. 3 a). Simple gand-binding domain. RAPs are chaperones (heat shock GREs are imperfect palindromic 15-base-pair sequences proteins hsp90 and hsp70) which ensure correct folding which are recognised by the zinc fi ngers of the DNA-bind- of the receptor protein and co-chaperones such as the im- ing domains of activated receptors. Gene inducibility in- munophilin FKB51 [18], a FK506 (tacrolimus)-binding creases with the numbers of response elements in the vi- protein. cinity of the polymerase-binding moiety. Composite GREs allow additional binding for non-receptor factors

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Korting Fig. 3. Models describing glucocorticoid in- teractions with DNA and transcription fac- tors (modifi ed from [18]; for details see text).

[18]. Glucocorticoid-induced transactivation is seen, e.g. above which then become inactivated either by sequestra- with genes related to gluconeogenesis, arterial blood pres- tion (fi g. 3 c), masking of transactivation domains (fi g. 3 d) sure and intraocular tension which harbour GREs as does or steric hindrance of the basal transcription machinery the gene encoding the 2 -adrenoceptor. (fi g. 3 e). Residues of the GR DNA and ligand-binding Besides inducing gene expression by binding of a ho- domains interact with AP-1, the DNA-binding domain is modimer to a GRE, glucocorticoids may also inhibit gene involved in the complex formation with NF- B. expression either by the binding of liganded monomers Helix 12 of the activated GR allows co-activator and/ to a promotor containing negative GREs (nGREs; fi g. 3 b) or co-repressor interaction [28] , including the steroid re- or by the sequestration or inhibition of basal transcription ceptor co-activator (SRC)-1 and the cAMP-responsive el- factors (fi g. 3 c–e). nGREs differ in structure from classic ement-binding protein (CREB)-binding protein (CBP). GREs and were fi rst described for the proopiomelanocor- Increasing histone acetylation co-activators (fi g. 3 f) weak- tic gene [21] and then for others, e.g. the IL-1 gene [18] . en histone-chromatine interactions which enables poly- In epidermal cells, the synthesis of basal-cell-specifi c (K5, merase to initiate transcription, while the opposite holds K14) and disease-associated keratins (K6, K16, K17) is true with histone deacetylase. By binding to co-factors, under the control of four nGREs [31] . Another form of glucocorticoids reverse these effects on gene expression. inhibited gene expression has been known even longer. In addition, glucocorticoids may infl uence gene expres- This is due to the complex formation of liganded GR sion by histone methylation and phosphorylation (for re- monomers and the basal transcription factors described view, see [9] ).

New Aspects of Glucocorticoid Action Skin Pharmacol Physiol 2005;18:103–114 107 Table 2. Glucocorticoid activation and suppression of genes relevant for the Inhibition of Gene Transcription factor effi cacy in infl ammatory skin diseases and skin atrophy induction (from [18]) Cytokines IL-2 NF-AT, AP-1, NF- B IL-6 NF-B, AP-1 TNF- GM-CSF,, IL-1 NF-B IFN- AP-1 Enzymes iNOS, COX-2 NF-B collagenase AP-1 Adhesion molecules ICAM-1, E-selectin, VCAM-1 NF-B Skin proliferation keratin GR monomer (nGRE) collagenase AP-1

Another genomic effect includes the increase in gluco- high amounts in almost all cell types including keratino- corticoid-inducible leucin zipper and thus AP-1 [18] and cytes and fi broblasts [41], endothelial cells of blood ves- NF- B formation in macrophages [32] . Inhibition of lym- sels and the airway epithelium as well as the epithelium phocyte and monocyte activation by the NF- B pathway of bronchial vessels [21]. (fi g. 3 g) is due to an enhanced transcription of the I B Breslin et al. [42] identifi ed two more exons upstream gene [21, 33, 34] which, however, is not the case with mu- of exon 1, which can translate into exon 1 of the hGR rine fi broblasts, endothelial cell lines [35] and human ke- protein; these are therefore named exons 1A and 1B, ratinocytes [5]. Table 2 summarises signalling pathways while exon 1, known for a longer period now, is called which are infl uenced by glucocorticoids. exon 1C. Moreover, alternative translation initiation of Moreover, glucocorticoids interfere with the forma- exon 1 (1C) results in the formation of a B isoform of tion of infl ammatory proteins also at the post-transcrip- hGR which consists of 751 residues [43] . Currently, the tional level by mRNA destabilisation as described for, tissue distribution and possible differences in function of e.g., TNF- , IL-1, IL-2, IL-6, COX-2 and iNOS [18] . the isoforms differing in the transactivating domain await to be discovered. Glucocorticoid Antagonists Another broadly distributed isoform, named hGR , is As with other nuclear receptors, GR antagonists have generated from a splice variant of exon 9 of the GR gene been developed. Most frequently used for in vitro and and is found in human tissues (e.g., lung, thymus, lympho- animal experiments is RU486 () which in- cytes, brain, heart, muscle), though it is present at a lower hibits dexamethasone-induced transactivation, while it rate as compared with hGR (1: 40–2,800) [20] . Both iso- acts as a partial agonist with respect to AP-1 and NF- B forms are identical until amino acid 727, then 15 different suppression [36–38] . RU486 also induces immunophilin residues follow in hGR . Due to the truncated C-terminal exchange, dynein recruitment and nuclear translocation domain, hGR does not bind ligands any more and is [39]. In man, RU486 suppresses the symptoms and signs transcriptionally inactive itself. Early investigations sug- of hypercortisolism and dexamethasone-induced pitu- gested a negative effect of hGR on hGR effects [44, 45] . itary inhibition and skin blanching [40]. Most interest- This, however, may be related to some tissues or cell states ingly, the glucocorticoid antagonist ZK98299 acts in a only [20] . A protective effect of hGR against dexameth- more specifi c manner as the liganded GR does not inter- asone induced apoptosis of human neutrophils [46] and a fere with NF- B activity [36, 37]. This distinct interac- correlation of hGR expression, and glucocorticoid resis- tion offers the horizon to more selective GR ligands. tance were described [47–49]. Yet, the relevance of hGR for asthma is still under debate [9, 49, 50] . Following co- Receptor Isoforms transfection of hGR and hGR genes, transactivation Several isoforms of the human GR can be differenti- declined by 40% in COS-7 cells and to a lesser extent in ated. Only in rare cases, however, point mutations or the lung carcinoma cell line A549. Most interestingly, splice variants may result in diseases, e.g. rare forms of overexpression of either isoform inhibited transrepression the glucocorticoid resistance syndrome [20]. The most – even in the absence of dexamethasone stimulation which important form is the broadly distributed -form (hGR ; might be due to complex formation of even the non-ligan- fi g. 1 ) containing 777 amino acids which is expressed in ded GR and transcription factors [50] .

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Korting hGR mRNA also clearly dominates in primary cul- glucocorticoid response is infl uenced by the cross-talk of tures of human keratinocytes (328-fold) and fi broblasts nuclear receptor signalling pathways, since the liganded (710-fold) over hGR mRNA. Increasing hGR over GR does not only form a homodimer, but also heterodi- hGR fi vefold by cotransfection of COS-7 cells did not mers with the other nuclear receptors and can compete reduce the binding of glucocorticoids and RU486 signifi - with those for co-factors [18]. Also receptor-associated cantly [41] . In skin biopsies, hGR is upregulated during immunophilins may induce a cross-talk with other signal- tuberculin response [48]. All these ratios, however, have ling cascades as to be derived from studies in synovial fi - to be interpreted with caution since mRNA ratios must broblasts [54] and the lung adenocarcinoma-derived A549

not translate 1: 1 to the protein level and the quantifi ca- cell [8] stimulated with dexamethasone and FK506. tion of the protein is related to antibody specifi city. Recently, tyrosine nitration has been described as a Another truncated splice variant (GR- ; exon 8) de- mechanism to enhance GR hsp90 dissociation and anti- tected in tumour cells or the rather ubiquitously expressed infl ammatory activity. Nitration, including the impor- hGR- do not seem to be of major clinical relevance [20] . tant Tyr735 [29], was induced by a GR ligand carrying a Yet, this may be different with two other recently de- nitration domain [55]. This observation may stimulate scribed polymorphisms. An S651F mutation of hGR the search for new and possibly more specifi c drugs. was identifi ed in immortalised lymphocytes isolated from atopic Japanese, resulting in the formation of a less stable Glucocorticoid Resistance and therefore less active GR protein. Stability of mRNA Whether hGR is of relevance with respect to gluco- was even more reduced if adenine was inserted into posi- corticoid resistance which occurs, e.g. in up to 10% of tion 2314 of hGR gene. Detected with lymphocytes tak- asthmatic subjects but also in other chronic infl amma- en from patients suffering from lupus nephritis, this poly- tory diseases, currently remains open. Alternatively, a re- morphism may reduce the effi cacy of glucocorticoid ther- duced number of activated GR within the nucleus, a lack apy [51]. of interaction with the basal transcription process, or a Concerning tissue specifi city, another GR polymor- hyperactive MAP kinase pathway may be the reason for phism deserves to be mentioned, which is due to alterna- this steroid resistance [15] . It can only be speculated tive splicing of human GR gene by two forms of the DNA whether one of these mechanisms may be of relevance restriction enzyme BclI. While this does not change the also in glucocorticoid tachyphylaxia, which was a subject coding region of the GR gene, it may infl uence the regu- of major concern in the early days of topical glucocorti- lating region [18]. Most interestingly, the large variant of coid treatment of skin disease [56] . the restriction enzyme was linked to increased sensitivity to budesonide-induced skin blanching, while dexametha- sone more effi ciently inhibited lysozyme release from leu- Non-Genomic Glucocorticoid Effects kocytes from subjects with the small allele [52] . Once more, this hints at tissue-specifi c effects of various gluco- While it takes at least 30 min for the genomic effects corticoids. to become obvious in a cell culture experiment, it takes In the context of GR isoforms, also the GR dim mouse only seconds or a few minutes to detect non-genomic ef- has to be mentioned, which is transfected with a GR iso- fects. As with other steroids, non-genomic targets were form unable for dimer formation and thus transactiva- identifi ed also with glucocorticoids [12–14] . Membrane- tion, yet inducing transrepression as the wild type does. associated receptors appear to be a specifi c isoform or a Therefore, in contrast to the GR knockout mice, the modifi cation of the classical GR. Steroid effects on mem- GR dim mouse grows until adulthood [53]. brane-associated receptors encompass the activation of MAP kinase, adenylate cyclase and protein kinase C [18] . Modulation of Responsiveness Glucocorticoids inhibit the recruitment of signalling fac- Besides GR isoforms and the numbers and locations tors of activated epithelial growth factor receptors and of GREs in the promotor, post-translational processes thus phospholipase A 2 and arachidonic acid release [57] . regulate glucocorticoid responsiveness. Hyperphosphor- Investigations of various glucocorticoids in A549 cells al- ylation of serins in the 1 region of the liganded GR en- lowed to separate three types of glucocorticoids (table 1 ; hances the response to steroids by favouring ligand bind- fi g. 3). The conventional steroids dexamethasone, hydro- ing, RAP binding and nuclear traffi cking, while GR , and beclomethasone dephosphorylation reduces transactivation. Moreover, induced both genomic (COX-2 expression, prostaglandin

New Aspects of Glucocorticoid Action Skin Pharmacol Physiol 2005;18:103–114 109 E release and inhibition of cell growth) and non-genomic dexamethasone induced a drastical reduction of the fi bril effects (phospholipase A 2 activation and arachidonic acid type III collagen and – less – type I collagen. Moreover, release). The latter was proven by its sensitivity to the this drug decreased collagenases which belong to the ma- SRC-1 inhibitor PP2 and insensitivity to the transloca- trix metalloproteinases (MMP) and tissue inhibitors of tion inhibitor geldanamycin. In contrast, methylprednis- the metalloproteinases TIMP-1 and TIMP-2 [61] . In fact, olone exhibited only the non-genomic effect, while the collagenase (MMP-13) produced by basal keratinocytes newly developed steroids fl uticasone 17-propionate, mo- is under AP-1 control and the formation is downregu- metasone and budesonide but also beclomethasone di- lated by dexamethasone treatment of both wild-type and dim propionate did not induce phospholipase A 2 activity, yet the GR mice [33] . Since type I and type III collagens selectively inhibited COX-2 expression, prostaglandin E primarily determine the tensile strength of the skin [62] , release, and cell growth [7]. In fact, these steroids are re- this deterioration explains well striae formation which ported to induce less side effects in asthma therapy as occurs in hypercortisolism and may follow long-term top- compared with conventional steroids. These data clearly ical treatment with glucocorticoids [63] . Moreover, the indicate that a separation of glucocorticoid effects is pos- effect on K5 and K14 explains epidermal thinning during sible. Since RU486 inhibited genomic and non-genomic long-term glucocorticoid treatment, inhibition of K6 and effects, the latter should be induced by the stimulation of K16 the interference with wound healing. The additional membrane-associated GR. effect on keratin synthesis via AP-1 inhibition may in- If applied at very high concentrations ( crease glucocorticoid effects, since besides nGREs these 1200 mg), which are used for, e.g., shock therapy, gluco- keratin gene promotors also contain an AP-1 response site corticoids can also induce non-specifi c non-genomic ef- [31]. fects, possibly due to a physical dissolution of the steroid Glucocorticoids induce cytostatic and cytotoxic ef- molecule within the cell membrane. This results in a par- fects in a cell-specifi c manner [62]. While apoptosis in- tial uncoupling of oxidative phosphorylation, mitochon- duction is a well-known effect of glucocorticoids interfer- drial proton leak and a decrease of cellular entry of cal- ing with NF- B in lymphocytes, and the inhibition of cium ions which may explain that high glucocorticoid immune cells should contribute to its immunosuppres- doses are generally needed for acute exacerbations of im- sive and anti-infl ammatory effects in eczema and psoria- munological diseases. Relative glucocorticoid potencies sis, skin thinning following glucocorticoids is not due to for non-specifi c non-genomic effects [58] clearly differ an increased apoptotic rate. In fact, despite an inhibition from potency for genomic effects (table 1). With respect of NF- B activity, dexamethasone protected fi broblasts to the topical treatment of skin disease or bronchial asth- against the TNF , UV irradiation or ceramide-induced ma, local drug concentrations high enough to induce non- apoptosis by stimulating sphingosine 1-phosphate (S1P) specifi c non-genomic drug actions currently cannot be formation. While NF- B inhibitors did not abolish the excluded. antiapoptotic effect, the sphingosine kinase inhibitor N,N-dimethylsphingosine did so. This effect is cell spe- cifi c as it was not seen with keratinocytes [64] . Although Glucocorticoid Effects on the Skin we previously demonstrated that vitamin D 3 and the transforming growth factor- stimulate fi broblast prolif- Despite of the recent introduction of calcineurin in- eration via an increased sphingosine phosphorylation hibitors into the topical treatment of eczema, glucocorti- [65, 66] and S1P then activates Smad proteins [67] , the coids still are fi rst-line drugs, especially with respect to S1P-induced proliferation is obviously overrun by the an- the acute treatment of the more severe forms [1]. Yet, tiproliferative effect of glucocorticoids due to the inhibi- topical glucocorticoids are also indicated for other infl am- tion of IL-1 and IL-6 release [5] . matory skin diseases, as for example psoriasis. This is due to the inhibition of the formation of infl ammatory pro- teins released from keratinocytes, fi broblasts and leuko- Glucocorticoids: Benefi t/Risk Ratio in Eczema cytes infi ltrating the skin. Unwanted effects most feared Therapy are skin thinning which is due to the inhibition of keratin synthesis of epidermal cells [31] and, more importantly, Since target diseases for glucocorticoid treatment are to an impaired proliferation of fi broblasts [5, 59, 60] and of chronic or relapsing nature, drugs effi cacious and safe disturbed collagen metabolism in the dermis. In the rat, in the long-run are of highest relevance. The unraveling

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Korting of signalling processes offers new perspectives for safer refl ect glucocorticoid effects in vivo. In fact, glucocorti- therapeutic strategies. Antiproliferative effects of topical coids less atrophogenic to the skin (mometasone furoate, glucocorticoids, however, do not parallel steroid potency fl uticasone propionate) strongly inhibit the proliferation in man [68, 69] or cell culture [5] . When looking into the of A549 cells (table 1) [7] . Detecting skin-specifi c effects molecular reason for the partial separation of wanted and asks for studies in vivo or at least in primary skin cell unwanted effects obtained so far, one has to take the cultures. Doing so, however, does not eliminate the ‘time’ multi-faceted mechanism of action into account. factor, since cell culture experiments are run for some Comparing hGR binding of 17 topical glucocorti- hours, while the treatment of skin diseases lasts for days coids widely varying in potency (prednisolone to clobeta- or even weeks which allows for adaptative processes. sole 17-propionate) did not allow to detect an infl uence Therefore, it is to be expected that in vivo experiments of receptor affi nity on the benefi t/risk ratio. In fact, bind- will improve further our understanding of glucocorticoid- ing of less atrophogenic steroids fi tted well into the spec- related differences in wanted and unwanted effects. trum of the more toxic agents [30] . Moreover, calculated receptor binding of six glucocorticoids studied for their infl uence on A549 cells are well in accordance with previ- Future Developments ous data (table 1 , fi g. 2 ). Yet as with asthma, the differ- ences in the benefi t/risk ratio in eczema may result from Glucocorticoid research is an ongoing process for more differences in transrepression, transactivation and spe- than 50 years, and its intensity is currently increasing. cifi c non-genomic effects [7] undistinguished by receptor New congeners of a somewhat improved benefi t/risk ratio binding. Interestingly, low glucocorticoid concentrations have been developed [63] , more selective ones are to be appear to induce transrepression more potently than expected. Current research aims at designing drugs induc- transactivation [9]. The infl uence on NF- B transrepres- ing selective transrepression [71, 72] or enhancing bind- sion, however, did not refl ect the potential in skin dis- ing by nitrotyrosine formation [55] . Increasing target se- eases. Indeed, activity only slightly lectivity at the molecular level, however, may bear the exceeded the effect [38]. Differentiating risk of a major loss in effi cacy. steroids with a favourable effect on transrepression in vi- An alternative for a safer eczema therapy therefore tro [70, 71], however, failed in the animal experiment encompasses drug carrier systems for epidermal glucocor- [72]. Therefore, additional targets appear of relevance. ticoid targeting [74, 75]. Moreover, combined treatment Mometasone and the relatively equipotent fl uticasone with tretinoin should reduce skin atrophy [76, 77], and propionate [73] both belong to the group of glucocorti- ointment preparations containing a glucocorticoid plus a coids with an improved benefi t/risk ratio [69] . At lower vitamin D analogue may improve psoriasis therapy by concentrations, they bind preferentially to GR mono- reducing side effects [78]. Studies in rheumatoid syno- mers, and induce dimer formation only at higher concen- vial [54] and murine skin fi broblasts [79] also indicate a trations [6]. In addition, a separation of budesonide and synergistic effect of tacrolimus and low-dose dexametha- dexamethasone effects has been described in subjects ex- sone which may translate into clinical improvement. Due pressing different GR isoforms [52] . to the still measurable potential for skin atrophy of glu- Therefore, the specifi c interaction of a glucocorticoid cocorticoids and the lower potency and the risk of photo- with the GR may infl uence the structure of the receptor carcinogenesis with tacrolimus and pimecrolimus [80] , a surface and thus its interaction with other proteins and combination therapy that is safe and effective in long- GREs. In fact, recently differing requirements of GR tar- term treatment of severe cases would be welcome. gets for 1 and 2 structures were reported [62], and full and partial GR agonists differing in D ring structure in- duce different receptor conformational changes [29]. In- Acknowledgement terestingly, prednicarbate, which proved less atrophogen- ic than betamethasone 17-valerate and also less than mo- The authors gratefully acknowledge fi nancial support from the Deutsche Forschungsgemeinschaft (FG 463) and the Alexander- metasone [69], turned out as a partial agonist with the von-Humboldt-Stiftung (personal grant to M.A.). N564A mutant of hGR [30] . Since glucocorticoid effects depend on the cell type, cell culture experiments, especially if performed in cell lines or using protein overexpression, may not exactly

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