Synthetic Glucocorticoids ThatDissociate Transactivation andAP-1 Transrepression ExhibitAntiinflammatory Activity in Vivo

Beatrice M. Vayssiere*, Sonia Dupont†, Agnes Choquart,Francis Petit, Teresa Garcia, Christian Marchandeau,Hinrich Gronemeyer, and Michele Resche-Rigon

Roussel UCLAF (B.M.V., S.D., A.C., F.P., T.G., C.M., M.R.-R.)93235 Romainville Cedex, FranceInstitut de Genetique et de Biologie Moleculaire et Cellulaire (H.G.)IGBMC-BP. 16367404 Illkirch Cedex, C.U. de Strasbourg, France

Some of the most potent antiinflammatory andimmunosuppressive agents are synthetic glu-cocorticoids. However, major side effects se-verely limit their therapeutic use. The develop-ment of improved glucocorticoid-based drugswill require the separation of beneficial from del-eterious effects. One possibility toward this goalis to try to dissociate two main activities of glu-cocorticoids, i.e. transactivation and transre-pression. Screening of a library of compoundsusing transactivation and AP-1 transrepressionmodels in transiently transfected cells identifieddissociated glucocorticoids, which exert strongAP-1 inhibition but little or no transactivation.Importantly, despite high ligand binding affinity,the prototypic dissociated compound, RU24858,acted as a weak agonist and did not efficientlyantagonize dexamethasone-induced transcrip-tion in transfected cells. Similar results wereobtained in hepatic HTC cells for the transacti-vation of the endogenous tyrosine amino trans-ferase gene (TAT), which encodes one of theenzymes involved in the glucocorticoid-depen-dent stimulation of neoglucogenesis. To investi-gate whether dissociated glucocorticoidsretained the antiinflammatory and immunosup-pressive potential of classic glucocorticoids,several in vitro and in vivo models were used.Indeed, secretion of the proinflammatory lym-phokine interleukin-1! was severely inhibited bydissociated glucocorticoids in human monocyticTHP 1 cells. Moreover, in two in vivo models,these compounds exerted an antiinflammatoryand immunosuppressive activity as potent asthat of the classic glucocorticoid prednisolone.

These results may lead to an improvement ofantiinflammatory and immunosuppressive thera-pies and provide a novel concept for drug dis-covery. (Molecular Endocrinology 11: 1245–1255,1997)

INTRODUCTION

The glucocorticoid receptor (GR) is a member of thesuperfamily of nuclear receptors which, upon bindingto their cognate ligands, act as positive and negativeregulators of target gene transcription. In the case ofpositive regulation, they transactivate through cis-act-ing palindromic glucocorticoid response elements(GREs), located in the promoter region of responsivegenes (for reviews see Refs. 1–5).

Apart from acting as transactivators, several nuclearreceptors, including GR, are able to negatively regu-late transcription. For GR, two distinct mechanismshave been described. One implies a competition be-tween GR and other transcription factors for binding totheir cognate DNA elements (6, 7). The second, gen-erally referred to as transrepression, is much less wellunderstood. It refers to the original observation thattransactivation by AP-1 was impaired in the presenceof glucocorticoids. Reciprocally, glucocorticoid actionwas inhibited by AP-1 (Refs. 8–12; reviewed in Ref.13). Although evidence for direct interaction betweenGR and AP-1 was provided from in vitro studies indi-cating that GR and AP-1 mutually interfere with eachother’s DNA- binding ability (8, 9, 12), genomic foot-printing experiments demonstrated that in the pres-ence of glucocorticoids, AP-1 remains bound to thecollagenase promoter in vivo (14). Together with theobservation of cell- and promoter-specific transre-pression, these results suggested that AP-1 and GRinteract rather indirectly, possibly through other tran-

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scriptional factors (Ref. 15; reviewed in Ref. 13). Mu-tational studies have demonstrated that both GRDNA- and ligand-binding domains (LBDs) are requiredfor AP-1 transrepression (9–12, 15, 16). Furthermore,by mutating individual amino acids of the DNA-bindingdomain of GR, it could be demonstrated that transac-tivation and transrepression are two separable func-tions (17). A similar conclusion was drawn for retinoicacid receptors since certain synthetic retinoids elicitedAP-1 inhibition without significantly transactivatingcognate reporter genes (18–20).

Glucocorticoids can also repress members of theNF-!B-Rel transcription factor family (21–23). In addi-tion to a cross-coupling mechanism of inhibition be-tween NF-!B and the GR, induction of transcription ofthe gene encoding I!B" has also been recently dem-onstrated in lymphocytes and monocytes (24, 25).However, induction of I!B" expression by glucocorti-coids seems to be cell type-restricted since it cannotaccount for inhibition of NF-!B activity in endothelialcells (26).

Glucocorticoids are highly potent antiinflammatoryand immunosuppressive agents, due to their pleiotro-pic effects on the expression/activity of multiple im-munomodulators and their ability to induce apoptosisin lymphocytes (Ref. 27 and references therein). How-ever, their therapeutic use is limited by severe sideeffects, especially during long-term treatment (seeDiscussion). Whether the dissociation of glucocorti-coid-dependent transactivation and transrepressionmay provide the possibility to separate (some of the)negative side effects from the beneficial antiinflamma-tory action of classic glucocorticoids is still not clear.Therefore, we have systematically compared thetransactivation and transrepression abilities of variousglucocorticoid agonists and antagonists and screeneda series of synthetic compounds to detect those ex-hibiting dissociated characteristics (i.e. mainly trans-activating or AP-1 transrepressing). Here we report theidentification of a novel class of synthetic glucocorti-coids that inhibit transcription of the collagenase pro-moter in transfected Hela cells, while only weakly ac-tivating GRE-based reporter genes. Moreover, weshow that these ligands are potent inhibitors of inter-leukin-1# (IL-1#) secretion in activated monocytes,while they are unable to induce significant tyrosineamino transferase (TAT) activity in rat HTC hepatomacells. Finally, we provide evidence that these dissoci-ated glucocorticoids act as antiinflammatory and im-munosuppressive drugs in vivo.

RESULTS

Identification of Dissociated Glucocorticoids

More than 200 synthetic glucocorticoids, includingseveral clinically important GR ligands as referencecompounds, were subjected to a transient transfec-tion-based screening system to compare their poten-

cies for GR-mediated transactivation and AP-1transrepression (for details see Materials and Meth-ods). Briefly, ligand dose-response curves were estab-lished for transactivation and AP-1 transrepression bythe endogenous HeLa cell GR from the stimulation ofthe GRE5-tk-CAT, and the inhibition of the c-Jun-ac-tivated collagenase promoter-CAT reporter genes, re-spectively. As expected, dexamethasone (Dex) andprednisolone were found to be both strong transacti-vators and strong AP-1 inhibitors (Fig. 1); we refer tothis class of glucocorticoids as “symmetrical” com-pounds. All tested glucocorticoids currently used inmedical therapies as antiinflammatory agents werefound to belong to this class (data available uponrequest). Each compound was classified relative to themaximal transactivation and AP-1 transrepression ob-tained with Dex (100%) and characterized by its rela-tive activation and repression activities at a given con-centration (Table I, and data not shown).

Three of the analogs tested were not, or only weakly,able to confer transactivation ability on GR, whereasits AP-1 transrepression activity was fully retained oronly marginally reduced in the presence of these glu-cocorticoids (hereafter collectively referred to as “dis-sociated” glucocorticoids). Their structures are pre-sented in Fig. 2. For example, 1 $M RU24858 activatedtranscription from the GRE5-tk-CAT reporter only 25%as efficiently as the same concentration of Dex(Fig. 1A), but was similarly potent (85%) in repressingAP-1-induced transcription from the collagenase pro-moter (Fig. 1B). Note that the IC50 of RU24858 forAP-1 transrepression was 20 nM, close to that of pred-nisolone (Fig. 1B). Two other compounds, RU24782and RU40066, exhibited similar dissociated transacti-vation and AP-1 transrepression characteristics (seeTable 1 and Fig. 1; note that the results given in Table1 correspond to 100 nM of the indicated ligand). In vitrocompetition assays with GR-bound [3H]RU28362, apure glucocorticoid agonist (28), or [3H]Dex revealedthat RU24858 and RU24782 bound to GR with a sim-ilar relative affinity as Dex, while RU40066 displayed a3.7-fold lower affinity (Table 1 and data not shown). Inview of their low agonistic potential and high affinity toGR, we expected the three dissociated glucocorti-coids to act as efficient antagonists of Dex-inducedtransactivation. Indeed, RU40066, at 100 and 1000nM, efficiently antagonized both 10 nM Dex-inducedGRE-tk-CAT (transient transfection) and TAT (non-transfected HTC cells) activities (Fig. 3). Surprisingly,however, a 100-fold excess of RU24858 was unable toantagonize the same Dex-induced activities (Fig. 3),although in vitro it exhibited an even higher affinity toGR than Dex (Table 1). In these experiments RU24782was able to partially antagonize the Dex-induced ac-tivities. As in vitro binding assays, RU24858,RU24782, and RU40066 were shown to compete with[3H]RU28362; we also measured transcriptional acti-vation of the GRE-tk-CAT reporter and induction of theTAT activity in Hela and HTC cells, respectively, ex-posed to 5 or 10 nM RU28362 with increasing amounts

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of the dissociated compounds. Again, RU40066 wasable to antagonize RU28362-induced transactivationand RU24782 displayed partial antagonist activity,whereas a 100- or 500-fold excess of RU24858 wasunable to antagonize RU28362-induced activity (datanot shown). Thus, apparently dissociated glucocorti-coids with a prevalent potency for AP-1 repression canbe designed which are nearly devoid of both agonisticand antagonistic activities.

Divergent Potencies of DissociatedGlucocorticoids in TAT Induction and Inhibition ofIL-1! Secretion

Transcription of the TAT gene, one of the enzymesinvolved in the glucocorticoid-dependent stimulationof neoglucogenesis, is induced by glucocorticoids invitro and in vivo, due to the presence of GREs in theTAT gene promoter (29). To assess the activity of thevarious dissociated glucocorticoids identified as de-scribed above with an endogenous GR target gene, ratHTC cells were treated with the synthetic glucocorti-coids, and the resulting TAT activity was measured. A10-fold induction was observed in cells treated withDex at 1 $M (100% in Fig. 4A; and data not shown).Prednisolone (1 $M) induced TAT to 93% of the stim-ulation observed with 1 $M Dex (Fig. 4A). Importantly,only a very weak induction of TAT activity was ob-served in HTC cells treated with the different dissoci-ated glucocorticoids, in keeping with the transactiva-tion results obtained in Hela cells transientlytransfected with GRE5-tk-CAT (Figs. 1A and 4A). Forexample, at 1 $M, RU24858 and RU24782 inducedTAT activity to 11% and 17%, respectively, of thestimulation seen with 1 $M Dex. RU40066, like theantagonist RU486, did not significantly induce TATactivity (Fig. 4A).

IL-1# plays a central role in the regulation of inflam-matory responses (for review see Ref. 30 and refstherein). Its induction is repressed by glucocorticoidsat both the transcriptional and posttranscriptional level(31–33). To investigate whether dissociated glucocor-ticoids would also exibit IL-1#-repressing activity, theireffect on IL-1# secretion was measured in THP1 cells,a monocytic cell line that responds to lipopolysaccha-ride (LPS) stimulation by secreting high amounts ofIL-# and tumor necrosis factor-" (TNF"). Dex, which at1 $M inhibited 75% of LPS-induced IL-1# secretion(not shown), was taken as the reference compound(!100% in Fig. 4B). Note that the same IC50 values (2nM) were observed for the Dex-dependent inhibition ofLPS-induced IL-1# secretion from THP1 cells and thec-Jun-induced collagenase promoter activity in tran-siently transfected Hela cells (Fig. 1, and data notshown). At 1 $M, the dissociated glucocorticoidsRU24782 and RU24858 inhibited IL-1# expression70% and 95%, respectively, of the maximum inhibitionobtained with Dex (Fig. 4B). RU40066 was inactive atdoses up to 10 nM and displayed moderate activity(65%) at high concentrations only, with an IC50 of 0.3

Fig. 1. Effect of Glucocorticoids on GRE-tk-CAT Transacti-vation and AP-1 Transrepression in HeLa Cells

Transactivation assays (A). Hela cells were cotransfectedwith the GRE5-tk-CAT reporter (1 $g), the polyII#Gal internalcontrol vector (1 $g), and 3 $g of the empty vector BluescriptKS and treated with the various compounds as indicated.CAT activity is relative to the #-galactosidase activity origi-nating from the internal control as described in Materials andMethods. Activation is given relative to the maximalGRE5-tk-CAT activity obtained with 10!6 M Dex (100%).Transrepression assays (B). For AP-1 transrepression, cellswere transfected with the Coll(!517/"63)CAT reporter (3$g), together with pSVc-Jun (250 ng), polyII#Gal (1 $g), and1 $g Bluescript KS. The cells were treated with the GRligands as indicated. The Coll(!517/"63)CAT reporter wasonly weakly active in absence of pSVc-Jun; cotransfection of250 ng pSVc-Jun resulted in a 20-fold induction of transcrip-tion (data not shown). Repression of the Coll(!517/"63)CATreporter in the presence of 10!7 M Dex was taken as !100%.Positive (transactivation) and negative (AP-1 transrepression)effects of the glucocorticoids are represented as positive andnegative values, respectively. The results presented aremeans # SEM of at least three independent experiments.

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$M (Fig. 4B, and data not shown). As was the case forDex, the IC50 values for inhibition of IL-1# secretion byprednisolone, RU24858, and RU24782 were veryclose to those determined for AP-1 transrepression intransiently transfected Hela cells (the following IC50

values were obtained: prednisolone, 6 nM; RU24858,30 nM; RU24782, $20 nM; compare with Fig. 1B). It isnoteworthy that similar results were obtained with thedissociated glucocorticoids for the inhibition of LPS-actived IL-1# secretion from peripheral blood mono-nuclear cells (data not shown).

Dissociated Glucocorticoids DisplayAntiinflammatory and Thymolytic Activity in Vivo

Glucocorticoids are major tools in the therapy of in-flammatory disorders. Encouraged by the observationthat expression of IL-1#, an immunomodulator at thetop of the inflammatory cascade, was affected bydissociated glucocorticoids, we investigated whetherthese compounds could also exibit antiinflammatoryactivity in vivo. Two classic animal models were se-lected. In the “cotton pellet granuloma” test (34) thecompounds were given orally to rats using Dex and

prednisolone as reference compounds. RU24858 andRU24782 displayed a similar antiinflammatory activityas shown by the inhibition of granuloma formation; thecorresponding ED50 values were estimated to 7 mg/kgand 5 mg/kg, respectively (Table 2). Dexamethasone,prednisolone, RU24858, and RU24782 inhibited gran-uloma formation up to 80%. Prednisolone exhibitedmaximal efficiency above 5 mg/kg, whereas the activ-ity of RU24782 in this assay plateaued off above dosesof 10 mg/kg (Fig. 5). Although these compounds were50 to 70 times less active than Dex, their activitieswere very similar to that of prednisolone (ED50 of 2.5mg/kg; Table 2); in this test system RU40066 wasinactive at the dose of 10 mg/kg, but was not tested athigher doses.

The “croton oil-induced ear edema” model (35)was the second test selected to study the antiin-flammatory activity of the dissociated glucocorti-coids in vivo and was performed in mice. In thismodel, the antiinflammatory activity of compoundsis determined by their ability to prevent local inflam-mation induced by croton oil. Topically appliedRU24858 and RU24782 displayed very strong anti-inflammatory activities with ED50 values of 2 $g/ear

Fig. 2. Structure of the Dissociated Glucocorticoids AnalogsThe structure of Dex is also shown for comparison.

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and 10 $g/ear, respectively. Note that RU24858 wasonly 2-fold less efficient than Dex and 2-fold moreefficient than prednisolone (Table 2). RU40066 dis-played a significant, albeit low activity when appliedtopically (Table 2).

Notably, RU486, which displayed some transrepres-sion activity with the Coll-CAT (but not TRE-tk-CAT;Table 1) reporter, was completely inactive in the cottonpellet granuloma and croton oil-induced ear edemamodels at concentrations up to 100 mg/kg (Table 2).Finally, two further animal models, the “mouse zymo-san paw edema” (36) and “rat carrageenin pawedema” (37), confirmed that the antiinflammatory po-tency of RU24858 was, albeit lower than that of Dex,similar to that of prednisolone (data not shown).

As the immunosuppressive activity of classic glu-cocorticoids is linked to their ability to induce lympho-cyte apoptosis, we measured thymolytic activity ofdissociated glucocorticoids in the same rats that wereused for the cotton-pellet granuloma test. The dosesat which a reduction of thymus weights could be ob-served were generally lower than those needed forinhibiting granuloma formation (Table 2). RU24782 andRU24858 displayed the same activity (ED50 estimatedto 2.5 mg/kg) which was, as in the granuloma test,near to that of prednisolone (1.6 mg/kg). Doses of 5mg/kg of RU24858 or RU24782 induced a thymusweight reduction of 81% and 77%, respectively, com-pared with control animals. Notably, even at 1 mg/kg,these glucocorticoids exhibited significant activity(25% reduction of thymus weight, P $ 0.05), whereasthey were inactive at these doses in the granuloma test(data not shown). As expected, Dex was highly activein the thymolysis test (with an ED50 below 0.05 mg/kg)since at this lowest dose tested, the decrease of thy-mus weight was 60% (data not shown). As previouslyobserved in the cotton-pellet granuloma, RU40066

was inactive at the dose tested (10 mg/kg). Takentogether, in several in vivo animal models, the disso-ciated glucocorticoids RU24858 and RU24782, whichare weak activators of positively regulated GR targetgenes, displayed consistently a strong antiinflam-matory action that was similar, or even superior, tothat of the classic antiinflammatory glucocorticoidprednisolone.

DISCUSSION

A Novel Class of Dissociated Glucocorticoids

Three classes of glucocorticoids have been distin-guished by their ability to induce/inhibit certain func-tions of GR: agonists, and type I and type II antago-nists (2). Agonists may induce the activation functionAF-2 in the ligand-binding domain (LBD) with varyingefficiencies (38), while in the presence of type I antag-onists, such as RU486 (39–41), GR AF-2 will be inac-tive (38, 42). Type I antagonists do not impair theactivity of the activation function AF-1, located in theN-terminal region A/B of GR (42, 43) and may act ascell-specific agonists, since it has been shown in thecase of the estrogen receptor that its AF-1 acts in acell-specific manner in the presence of the type I an-tagonist hydroxytamoxifen (44). In contrast, type IIantiglucocorticoids, such as RU43044, are defined bytheir ability to impair DNA binding of GR and, thus, donot induce any significant transcriptional activity of GRtarget genes (43).

With the aim of better understanding how glucocor-ticoids enable GR to transrepress AP-1 activity (seeIntroduction for references), we have compared AP-1inhibition in the presence of the three above types ofligands and, moreover, screened a library of glu-

Table 1. GR Binding, Transactivation, and AP-1 Antagonism by Glucocorticoids

Compounds(100 nM)

Transactivation(%) GRE-tk-CAT

Transrepression(%) RBA % (Dex % 100)

Hela-GRColl-CAT TRE-tk-CAT

Dexamethasone 89.48 # 2.26 96.63 # 1.78 97.55 # 1.55 100Prednisolone 79.37 # 1.73 81.41 # 18.96 63.9 # 11.04 NDRU24782 35.21 # 2.24 57.61 # 2.46 82.55 # 6.24 85 # 6RU24858 20.76 # 4.33 71.59 # 1.82 82.85 # 0.12 128 # 15RU40066 13.35 # 4.73 77.6 # 6.42 70.43 # 5.08 27 # 3RU486 4.18 # 3.8 48.19 # 1.68 Inactive NDRU43044 Inactive Inactive Inactive ND

The transactivation potential of the endogenous HeLa cell GR in the presence of various ligands (100 nM) was determined fromtransient transfection experiments with the GRE5-tk-CAT reporter; for the determination of AP-1 repression, HeLa cells weretransfected with the Coll(!517/"63)CAT or the TRE5-tk-CAT reporters and identically exposed to 100 nM of the syntheticglucocorticoids. PolyII#Gal was used as an internal control in all transfection experiments to normalize for variations in thetransfection efficiencies. The results are given as percentage of activation/inhibition, relative to maximal activation/inhibitionobtained with 100 nM Dex in each experiment. The results indicated for Dex treatment are means # SEM of 15 differentexperiments, each standardized to 100 nM (note that these mean values do therefore not reveal exactly 100% activation/inhibition). In vitro binding experiments were performed with the endogenous GR extracted from Hela cells. Note that similarresults have been obtained with human GR expressed in SF9 insect cells from recombinant baculoviruses (data not shown). RBA,Relative binding affinity.

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cocortcoids to find ligands that would inhibit AP-1activity without significantly inducing agonistic activity(termed “dissociated” glucocorticoids). Confirming theoriginal data of Jonat et al. (9), we observed that intransfected HeLa cells the type I antiglucocorticoidRU38486 was unable to repress AP-1-induced trans-

activation of a TRE5-tk-CAT reporter (Table 1), and,indeed, could reverse the effect of the agonist Dex(data not shown). With a collagenase promoter-basedreporter, some RU38486-dependent AP-1 repressionwas observed but the antagonist still reversed therepression induced by 10 nM Dex (Fig. 6). No transre-pressing ability was conferred onto GR in the presenceof the type II antagonist RU43044 with any of the twoAP-1 reporters (Table 1). Note that in the above casestransrepression was via the endogenous GR; tran-siently expressed exogenous GR can apparently fur-

Fig. 3. The Dissociated Glucocorticoid RU24858 Does NotAntagonize Dex-Induced Transactivation in Intact Cells

A, HeLa cells, transfected with the GRE5-tk-CAT reporter(and the internal control, see Materials and Methods), wereexposed to Dex alone or 10 nM Dex with increasing amountsof the indicated dissociated glucocorticoids. The normalizedCAT activity induced by the endogenous HeLa cell GR in thepresence of 1 $M Dex was taken as 100% induction. B,Analysis of tyrosine amino transferase (TAT) activity (see Ma-terials and Methods for details) originating from transactiva-tion of the endogenous TAT gene by the endogenous GR inHTC cells under identical conditions as in panel A. The en-zymatic activity measured in non-ligand-treated control cellswas subtracted, and the results were expressed relative tothe maximal activity measured in cells treated with 1 $M Dex(100%). The results shown are the means # SEM of at leastthree independent experiments.

Fig. 4. Dissociated Glucocorticoids Are Weak Transactiva-tors of Tyrosine Amino Transferase (TAT) in Cultured LiverCells but Potent Inhibitors of LPS-Induced IL-1# Secretion inthe Human Monocytic Cell Line THP 1

A, HTC cells were treated with 1 $M of either Dex (DEX),prednisolone (PRED), RU24782, RU24858, RU40066, orRU38486 (RU486). TAT activities were determined as de-scribed in Materials and Methods and expressed relative tothe activity seen with 1 $M Dex (100%). Note that half-maximal activation of TAT induction was observed at Dex andprednisolone concentrations, which resulted also in half-maximal stimulation of transactivation from the GRE5-tk-CATreporter gene in transfected Hela cells, i.e. 9 nM, and 40 nM,respectively (see Fig. 1). B, THP 1 cells, activated with 5$g/ml LPS, were treated for 18 h with 1 $M of the indicatedcompounds. IL-1# was measured in 50 $l of the culturesupernatant, using the ELISA kit from R&D Systems. IL1#level was undetectable in untreated control cell supernatants.LPS-treated cells secreted 1–3 ng/ml of IL-1#. Inhibition ofIL-1# secretion in the presence of 1 $M Dex was taken as!100%. The results presented are means # SEM of at leastthree independent experiments.

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ther increase the AP-1 inhibition (17) but leads to non-physiological GR concentrations. In conclusion,neither type I nor type II antiglucocorticoids have thecharacteristics of dissociated glucocorticoids and,thus, have only limited promise for use as antiinflam-matory agents (see below).

Screening of a library of glucocorticoids yieldedthree molecules (RU24782, RU24858, and RU40066)that exhibited the dissociated characteristics. All ofthem exerted residual agonistic activity (9% to 35% ofthat of Dex) but induced 58% to 83% of the anti-AP-1activity seen with Dex (Table 1). Note in this contextthat prednisolone, widely used as antiinflammatoryglucocorticoid, showed a similar anti-AP-1 activity intransfected cells (Table 1). The most interesting ofthese compounds is RU24858, since it exemplifies anovel class of glucocorticoids: despite a binding affin-ity that is even higher than that of Dex, RU24858 doesnot significantly antagonize Dex-induced transcription(Fig. 3). However, in in vitro binding assays RU24858does efficiently compete with radiolabeled Dex (andother glucocorticoid agonists) for binding to the GR.This implies that, in contrast to classic glucocorticoids,the binding affinity of RU24858 to transcriptionally en-gaged GR in vivo is lower than that measured in vitro.Importantly, however, this lower in vivo binding affinitydoes not significantly decrease the ability of the GR-RU24858 complex to repress AP-1 activity. We con-clude from these data that AP-1 repression involvesvery specific ligand-induced structural alterations ofGR.

How can RU24858 bind GR with different affinitiesin vitro and in vivo? Transcriptional interference/squelching studies have suggested that transcrip-tional intermediary factors (TIFs, also termed medi-

ators, coactivators, bridging factors) mediate theligand-dependent activation function AF-2 of steroidreceptors to the transcriptional machinery (45–47).

Fig. 5. Dissociated Glucocorticoids Display in Vivo Antiinflammatory Activity in the Cotton Pellet Granuloma ModelTwo cotton pellets (10 mg each) were inserted subcutaneously into the upper dorsal area of female Wistar rats. Test

compounds were administered orally, once a day, for 4 days. Eight animals were included for each treatment. Twenty-four hoursafter the last administration the pellet dry weight was determined (see Materials and Methods). The activities of the varioussynthetic glucocorticoids were expressed as percent inhibition of granuloma weight increase. **, P $ 0.01.

Table 2. In Vivo Activities of Dissociated GlucocorticoidsCotton-pellet Granuloma

(rat) (oral route) ED50(mg/kg)

Croton Oil-InducedEar Edema (mouse)

(topical administration)ED50 ($g/ear)Granuloma Thymolysis

Dexamethasone 0.1 $0.05 1Prednisolone &2.5 1.6 4RU24782 &5.0 2.5 10RU24858 &7 2.5 2RU40066 IN 10 IN 10 '100 (!22%)RU486 IN 100 IN 100 ND

The antiinflammatory activities of the different compoundswere evaluated in two animal models, the cotton-pellet gran-uloma test performed with female Wistar rats and the crotonoil-induced ear edema test with mice (for details and refer-ences, see Materials and Methods). In the first model thecompounds were given orally once a day during 4 days. Theantiinflammatory potential of the compounds was evaluatedby their ability to prevent granuloma formation and expressedas the compound doses at which the granuloma formationwas decreased by 50% (ED50, given in milligrams/kg) esti-mated from dose-response curves. In the same test, thethymus was removed, and thymolysis was measured. Theglucocorticoid doses needed to obtain 50% reduction of thethymus weight (ED50) are given in milligrams/kg. In the crotonoil-induced ear edema model the compounds were topicallyapplied during 6 h. The ED50 values are expressed in micro-grams/ear. Cases where no ED50 could be determined butweak effects were clearly measured at the maximal concen-tration (x) tested, are indicated as ED50 'x. IN (x), inactive (x,maximal concentration tested); ND, not determined.

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Putative TIFs have been recently isolated and char-acterized on the basis of their ligand-dependentinteraction with several nuclear receptors (48–54).We propose that the interaction between the LBDand TIF(s) (or any other factor that interacts with theGR LBD in vivo) may differentially affect the bindingof Dex and RU24858, resulting in a decreased affin-ity of RU24858, but not Dex, to the GR-TIF complex.However, noncomplexed GRs, or a set of GR com-plexes distinct from those involved in transactiva-tion, may bind RU24858 with high affinity and giverise to the strong AP-1 inhibition observed in thepresence of this compound. Note that there is aprecedent for an altered affinity of glucocorticoidsupon GR complex formation, since different affini-ties have been reported for the GR and the GR-hsp90 complex (55, 56).

Taken together, the above results suggest that wehave identified a novel class of glucocorticoids that areneither efficient agonists nor antagonists in vivo, butcan very efficiently induce GR-dependent AP-1repression.

In Contrast to Classic Antiglucocorticoids,Dissociated Glucocorticoids Are Strong Inhibitorsof LPS-Induced IL-1! Secretion

Glucocorticoids are powerful antiinflammatory agents,most likely due to their ability to block the expressionof multiple cytokines. Inhibition of cytokine expression

has been reported to occur at the transcriptional levelfor IL-1 to IL-6, IL-8, TNF", colony-stimulating factor(CSF)-1/macrophage (M)-CSF, granulocyte macro-phage (GM)-CSF, and %-interferon (IL-1 expression isblocked at other levels as well; TNF and GM-CSFexpression may be blocked through degradation oftheir mRNAs; for reviews see Refs. 27, 57–60, andreferences therein). Most of these genes require tran-scription factors, such as AP-1, NF-!B, or NF-AT, fortheir expression, suggesting that glucocorticoids exerttheir antiinflammatory functions by negatively interfer-ing with the activity of (some of) these factors. Disso-ciated glucocorticoids, such as RU24858, were aspotent inhibitors of IL-1# secretion as prednisolone inLPS-stimulated cultured human monocytes, whereasclassic antiglucocorticoids with no (RU43044) orpromoter-dependent AP-1-transrepression activities(RU486) inhibited IL-1# secretion only weakly (RU486)or not at all (RU43044) in this assay (Fig. 4 and data notshown).

In Vivo Dissociated Glucocorticoids AreAntiinflammatory Agents as Potent asPrednisolone

To investigate whether dissociated glucocorticoidscould be active as antiinflammatory drugs in vivo,we studied two established animal models, the cot-ton pellet granuloma model, in which the glucocor-ticoid is given orally to rats, and the croton oil-induced ear edema model, in which the drug isapplied topically to mice (see Materials and Methodsfor details and references). Importantly, in these invivo models, RU24858 was as active as pred-nisolone (Fig. 5 and Table 2). In the croton oil-induced ear edema model, which relies on phorbolester-induced skin inflammation, RU24858 waseven 2-fold more active than prednisolone, andRU24782 was about half as active as prednisolone.It is unclear why RU40066 was inactive; at present,no data on the metabolism and stability in vivo ofthis compound are available. Importantly, in keepingwith their inability to inhibit IL-1# secretion, the clas-sic antagonists RU486 and RU43044 were com-pletely inactive in these in vivo tests (Table 2 anddata not shown).

The immunosuppressant potential of glucocorti-coids relates to their ability to induce T cell apopto-sis and to inhibit cytokine gene expression. Theseactivities are therapeutically exploited in organtransplantation and in treatment of leukemias. Wecompared, in the cotton pellet granuloma model, theextent of thymolysis induced by classic (anti)glu-cocorticoids and the novel class of dissociated glu-cocorticoids. Again we observed a high activity ofRU24858 and RU24782, similar to that of pred-nisolone, while antiglucocorticoids were inactive(Table 2 and data not shown).

Fig. 6. The Classic Antiglucocorticoid RU38486 InducesOnly Weak AP-1 Transrepression with the Endogenous HelaCell GR and Antagonizes the Effect of High Dex Concentra-tions

Hela cells were transfected as described in the legend toFig. 1B. AP-1 transrepression of c-Jun-induced Coll(!517/"63)CAT transcription was measured after treating the cellswith increasing concentrations of Dex or RU38486 (RU486)alone, or with 10 nM Dex and increasing concentrations ofRU486.

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Dissociated Glucocorticoids May be Novel Toolsto Improve Antiinflammatory GlucocorticoidTherapy

We describe here a novel class of dissociated glu-cocorticoids, distinct from the various previously re-ported agonists and antagonists. These compoundsefficiently transrepress AP-1 activity, while only mar-ginally activating glucocorticoid target genes. The dis-sociated glucocorticoids have maintained the antiin-flammatory and, according to our as yet limiting data,also the thymolytic capacity of classical glucocorticoidagonists. Moreover, its prototypic member RU24858is as active, or, depending on the system, even moreactive, than prednisolone in standard in vivo models.Hypothalamic-pituitary-adrenal axis insufficiency, os-teoporosis, diabetes, steroid myopathy, and infectiousand neuropsychiatric complications limit the therapeu-tic use of classic glucocorticoid agonists (61). At leastsome of these complications are likely to derive fromthe agonistic activities of the classic glucocorticoids,and it has not escaped our attention that our glucocor-ticoid analogs, provided that the dissociation of trans-activation and transrepression is maintained in targetorgans, have the potential to constitute a new class ofantiinflammatory glucocorticoids with significantly re-duced side effects. Studies are under way to confirmthis hypothesis.

MATERIALS AND METHODS

Plasmids

GRE5-tk-CAT, TRE5-tk-CAT reporters, and pSVc-Jun havebeen described (15, 62). The polyII#Gal used as internalcontrol was constructed by inserting the HindIII-BamHI frag-ment of pCH110 (Pharmacia, Piscataway, NJ) containing thecoding sequence of the #-galactosidase gene into theHindIII-BglII sites of pG4MpolyII (63). The plasmid Coll(-517/"63)CAT was a kind gift of C. Wasylyk.

Cell Culture and Transfection

Hela cells were cultured in DMEM supplemented with 10%FCS and grown at 37 C and 5% CO2. Transient transfectionassays for transactivation or AP-1 transrepression were car-ried out using the calcium phosphate coprecipitation proce-dure. Briefly, 4 ( 105 cells were seeded 24 h before trans-fection in six-well plates containing DMEM supplementedwith 5% charcoal/dextran-treated serum. For transactivationassays, HeLa cells were transfected with 1 $g GRE5-tk-CATreporter, 1 $g polyII#Gal, and 3 $g Bluescript KS (Strat-agene, La Jolla, CA). For transrepression assays, HeLa cellswere transfected with 3 $g of the Coll(!517/"63)CAT re-porter, 1 $g polyII#Gal, 250 ng pSVc-Jun expression plas-mid, and 1 $g Bluescript KS. Sixteen hours later, cells wererinsed with Earle’s Balanced Salt Solution (EBSS), the me-dium was replaced, and the hormones were added as indi-cated. After 24 h, cells were washed with ice-cold EBSS andresuspended in 250 $l of lysis buffer provided with the chlor-amphenicol acetyltransferase enzyme-linked immunosorbentassay (CAT ELISA) kit (Boehringer Mannheim, Indianapolis,IN). The amount of CAT enzyme was quantified according to

the manufacturer’s instructions. Results were normalized ac-cording to the #-galactosidase activity originating from co-transfected polyII#Gal and plotted relative to the transacti-vation seen with 1 $M Dex (%100%) or relative to the AP-1transrepression exerted by 1 $M Dex (%!100%). SEMs werecalculated from a minimum of three independent assays percompound and per concentration.

Determination of Relative Binding Affinities

Relative binding affinities were determined by incubatingHeLa cell cytosol for 24 h at 0 C with either [3H]RU28362 (28)or [3H]Dex with or without different concentrations of com-petitor steroids. Bound and free ligands were separated bythe dextran-coated charcol method (38). The relative bindingaffinity of Dex was taken as reference (100%). Similar resultswere obtained with crude SF9 cell extracts containing highlevels of human GR expressed from recombinantbaculoviruses.

TAT Assay

HTC cells were cultured in DMEM supplemented with 10%FCS and kept at 37 C and 5% CO2. For steroid treatment 106

cells per well were seeded into six-well plates. After 24 h, atconfluency, the medium was replaced by DMEM withoutserum, and hormones were added for 18 h. TAT activity wasmodified from Ref. 64 to be measured in 96-well plates. Theendogenous enzymatic activity measured in control cells wassubtracted, and the results were expressed relative to themaximal activity measured in cells treated with 10!6 M Dex.

Assay of IL-1! Secretion

The monocytic cell line THP1 (ATCC: TIB202) was cultured inRPMI 1640 supplemented with 10% FCS and grown at 37 Cand 5% CO2. IL-1# secretion was induced by treating thecells with 5 $g/ml LPS (Sigma, St. Louis, MO); glucocorti-coids were added simultaneously. After 18 h, supernatantswere collected and IL-1# was quantitated using the humanIL-1# ELISA kit from R&D Systems (Minneapolis, MN).

Antiinflammatory Activity in the Cotton-PelletGranuloma Test

The cotton-pellet granuloma assay was performed as de-scribed previously (34). Two cotton pellets (10 mg each) wereinserted subcutaneously into the upper dorsal area of femaleWistar rats (weight range 90–100 g, Iffa Credo, France). Testcompounds were administered orally, once a day, for 4 days.Twenty-four hours after the last treatment, the animal waskilled and the pellet, along with the surrounding granuloma,was carefully dissected from the animal and its dry weightwas determined. For this, granuloma and pellet were heatedat 60 C overnight. By subtracting the initial weight of thepellet, the dry weight of the granuloma was determined. Theactivities of the various synthetic glucocorticoids were ex-pressed as ED50 values (dose causing a reduction of granu-loma weight by 50%).

Thymolytic Activity

The thymolytic activity of the synthetic compounds was de-termined by weighing the thymus of the rats treated in thegranuloma test. The ED50 values correspond to the glucocor-ticoid doses inducing a 50% decrease in thymus weights.

Topical Antiinflammatory Effect on the Croton Oil-Induced Ear Edema

This test was carried out as described previously (35) ongroups of eight male OF1 mice weighing 18–22 g (Iffa Credo,

Dissociated Glucocorticoids Are Antiinflammatory in Vivo 1253

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L’Arbresle, France). The edema was induced on one ear bythe application of a solution of croton oil (2% vol/vol) inpyridine-water-ether 4:1:14.6 (by volume). Animals werekilled 6 h later, and the ears were removed and weighed.Edema was determined from the difference in weight be-tween the irritant-treated and the contralateral ear. The com-pounds to be tested were dissolved in the croton oil solutionand topically applied on the ear. ED50 values correspond tothe doses reducing the control edema by 50%.

Acknowledgments

We thank Dr. Michel Pons (Montpellier, France) for the kindgift of HTC cells. We are grateful to the staff members of theendocrinology department, and especially to Dr. MartineGaillard and Dr. Andre Ulmann for their help and support. Dr.F. Fassy is acknowledged for her help and advice in settingup the TAT assay. We are grateful to O. Krebs for her con-tribution in the set-up of the CAT ELISA assay. Evelyne Ce-rede and Francoise Bouchoux are acknowledged for theiressential contribution in binding experiments. Finally, wewish to thank Dr. Neerja Bhatnagar for compound synthesisand Elisa Francesconi and Francoise Perrier for their excel-lent technical assistance.

Received November 27, 1996. Revision received May 14,1997. Accepted May 19, 1997.

Address requests for reprints to: Michele Resche-Rigon,ROUSSEL UCLAF, 102 route de Noisy, 93235 RomainvilleCedex France.

*Present address: INSERM U 248, Institut Curie, 26 rued’Ulm, 75231 Paris Cedex 05.

† S. Dupont is currently working at Institut de Genetique etde Biologie Moleculaire et Cellulaire.

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