Synthetic Glucocorticoids That Dissociate Transactivation and AP-1 Transrepression Exhibit Antiinflammatory Activity in Vivo

Béatrice M. Vayssière1, Sonia Dupont2, Agnès Choquart, Francis Petit, Teresa Garcia, Christian Marchandeau, Hinrich Gronemeyer and Michèle Resche-Rigon

Roussel UCLAF (B.M.V., S.D., A.C., F.P., T.G., C.M., M.R.-R.) 93235 Romainville Cedex, France
Institut de Génétique et de Biologie Moléculaire et Cellulaire (H.G.) IGBMC-BP. 163 67404 Illkirch Cedex, C.U. de Strasbourg, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Some of the most potent antiinflammatory and immunosuppressive agents are synthetic glucocorticoids. However, major side effects severely limit their therapeutic use. The development of improved glucocorticoid-based drugs will require the separation of beneficial from deleterious effects. One possibility toward this goal is to try to dissociate two main activities of glucocorticoids, i.e. transactivation and transrepression. Screening of a library of compounds using transactivation and AP-1 transrepression models in transiently transfected cells identified dissociated glucocorticoids, which exert strong AP-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 efficiently antagonize dexamethasone-induced transcription in transfected cells. Similar results were obtained in hepatic HTC cells for the transactivation of the endogenous tyrosine amino transferase gene (TAT), which encodes one of the enzymes involved in the glucocorticoid-dependent stimulation of neoglucogenesis. To investigate whether dissociated glucocorticoids retained the antiinflammatory and immunosuppressive potential of classic glucocorticoids, several in vitro and in vivo models were used. Indeed, secretion of the proinflammatory lymphokine interleukin-1ß was severely inhibited by dissociated glucocorticoids in human monocytic THP 1 cells. Moreover, in two in vivo models, these compounds exerted an antiinflammatory and immunosuppressive activity as potent as that of the classic glucocorticoid prednisolone. These results may lead to an improvement of antiinflammatory and immunosuppressive therapies and provide a novel concept for drug discovery.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid receptor (GR) is a member of the superfamily of nuclear receptors which, upon binding to their cognate ligands, act as positive and negative regulators of target gene transcription. In the case of positive regulation, they transactivate through cis-acting palindromic glucocorticoid response elements (GREs), located in the promoter region of responsive genes (for reviews see Refs. 1, 2, 3, 4, 5).

Apart from acting as transactivators, several nuclear receptors, including GR, are able to negatively regulate transcription. For GR, two distinct mechanisms have been described. One implies a competition between GR and other transcription factors for binding to their cognate DNA elements (6, 7). The second, generally referred to as transrepression, is much less well understood. It refers to the original observation that transactivation by AP-1 was impaired in the presence of glucocorticoids. Reciprocally, glucocorticoid action was inhibited by AP-1 (Refs. 8, 9, 10, 11, 12 ; reviewed in Ref. 13). Although evidence for direct interaction between GR and AP-1 was provided from in vitro studies indicating that GR and AP-1 mutually interfere with each other’s DNA- binding ability (8, 9, 12), genomic footprinting experiments demonstrated that in the presence of glucocorticoids, AP-1 remains bound to the collagenase promoter in vivo (14). Together with the observation of cell- and promoter-specific transrepression, these results suggested that AP-1 and GR interact rather indirectly, possibly through other transcriptional factors (Ref. 15 ; reviewed in Ref. 13). Mutational studies have demonstrated that both GR DNA- and ligand-binding domains (LBDs) are required for AP-1 transrepression (9, 10, 11, 12, 15, 16). Furthermore, by mutating individual amino acids of the DNA-binding domain of GR, it could be demonstrated that transactivation and transrepression are two separable functions (17). A similar conclusion was drawn for retinoic acid receptors since certain synthetic retinoids elicited AP-1 inhibition without significantly transactivating cognate reporter genes (18, 19, 20).

Glucocorticoids can also repress members of the NF-{kappa}B-Rel transcription factor family (21, 22, 23). In addition to a cross-coupling mechanism of inhibition between NF-{kappa}B and the GR, induction of transcription of the gene encoding I{kappa}B{alpha} has also been recently demonstrated in lymphocytes and monocytes (24, 25). However, induction of I{kappa}B{alpha} expression by glucocorticoids seems to be cell type-restricted since it cannot account for inhibition of NF-{kappa}B activity in endothelial cells (26).

Glucocorticoids are highly potent antiinflammatory and immunosuppressive agents, due to their pleiotropic effects on the expression/activity of multiple immunomodulators and their ability to induce apoptosis in lymphocytes (Ref. 27 and references therein). However, their therapeutic use is limited by severe side effects, especially during long-term treatment (see Discussion). Whether the dissociation of glucocorticoid-dependent transactivation and transrepression may provide the possibility to separate (some of the) negative side effects from the beneficial antiinflammatory action of classic glucocorticoids is still not clear. Therefore, we have systematically compared the transactivation and transrepression abilities of various glucocorticoid agonists and antagonists and screened a series of synthetic compounds to detect those exhibiting dissociated characteristics (i.e. mainly transactivating or AP-1 transrepressing). Here we report the identification of a novel class of synthetic glucocorticoids that inhibit transcription of the collagenase promoter in transfected Hela cells, while only weakly activating GRE-based reporter genes. Moreover, we show that these ligands are potent inhibitors of interleukin-1ß (IL-1ß) secretion in activated monocytes, while they are unable to induce significant tyrosine amino transferase (TAT) activity in rat HTC hepatoma cells. Finally, we provide evidence that these dissociated glucocorticoids act as antiinflammatory and immunosuppressive drugs in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Dissociated Glucocorticoids
More than 200 synthetic glucocorticoids, including several clinically important GR ligands as reference compounds, were subjected to a transient transfection-based screening system to compare their potencies for GR-mediated transactivation and AP-1 transrepression (for details see Materials and Methods). Briefly, ligand dose-response curves were established for transactivation and AP-1 transrepression by the endogenous HeLa cell GR from the stimulation of the GRE5-tk-CAT, and the inhibition of the c-Jun-activated collagenase promoter-CAT reporter genes, respectively. As expected, dexamethasone (Dex) and prednisolone were found to be both strong transactivators and strong AP-1 inhibitors (Fig. 1Go); we refer to this class of glucocorticoids as "symmetrical" compounds. All tested glucocorticoids currently used in medical therapies as antiinflammatory agents were found to belong to this class (data available upon request). Each compound was classified relative to the maximal transactivation and AP-1 transrepression obtained with Dex (100%) and characterized by its relative activation and repression activities at a given concentration (TableGo I, and data not shown).



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Figure 1. Effect of Glucocorticoids on GRE-tk-CAT Transactivation and AP-1 Transrepression in HeLa Cells

Transactivation assays (A). Hela cells were cotransfected with the GRE5-tk-CAT reporter (1 µg), the polyIIßGal internal control vector (1 µg), and 3 µg of the empty vector Bluescript KS and treated with the various compounds as indicated. CAT activity is relative to the ß-galactosidase activity originating from the internal control as described in Materials and Methods. Activation is given relative to the maximal GRE5-tk-CAT activity obtained with 10-6 M Dex (100%). Transrepression assays (B). For AP-1 transrepression, cells were transfected with the Coll(-517/+63)CAT reporter (3 µg), together with pSVc-Jun (250 ng), polyIIßGal (1 µg), and 1 µg Bluescript KS. The cells were treated with the GR ligands as indicated. The Coll(-517/+63)CAT reporter was only weakly active in absence of pSVc-Jun; cotransfection of 250 ng pSVc-Jun resulted in a 20-fold induction of transcription (data not shown). Repression of the Coll(-517/+63)CAT reporter 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 and negative values, respectively. The results presented are means ± SEM of at least three independent experiments.

 

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

 
Three of the analogs tested were not, or only weakly, able to confer transactivation ability on GR, whereas its AP-1 transrepression activity was fully retained or only marginally reduced in the presence of these glucocorticoids (hereafter collectively referred to as "dissociated" glucocorticoids). Their structures are presented in Fig. 2Go. For example, 1 µM RU24858 activated transcription from the GRE5-tk-CAT reporter only 25% as efficiently as the same concentration of Dex (Fig. 1AGo), but was similarly potent (85%) in repressing AP-1-induced transcription from the collagenase promoter (Fig. 1BGo). Note that the IC50 of RU24858 for AP-1 transrepression was 20 nM, close to that of prednisolone (Fig. 1BGo). Two other compounds, RU24782 and RU40066, exhibited similar dissociated transactivation and AP-1 transrepression characteristics (see Table 1Go and Fig. 1Go; note that the results given in Table 1Go correspond to 100 nM of the indicated ligand). In vitro competition assays with GR-bound [3H]RU28362, a pure glucocorticoid agonist (28), or [3H]Dex revealed that RU24858 and RU24782 bound to GR with a similar relative affinity as Dex, while RU40066 displayed a 3.7-fold lower affinity (Table 1Go and data not shown). In view of their low agonistic potential and high affinity to GR, we expected the three dissociated glucocorticoids to act as efficient antagonists of Dex-induced transactivation. Indeed, RU40066, at 100 and 1000 nM, efficiently antagonized both 10 nM Dex-induced GRE-tk-CAT (transient transfection) and TAT (non-transfected HTC cells) activities (Fig. 3Go). Surprisingly, however, a 100-fold excess of RU24858 was unable to antagonize the same Dex-induced activities (Fig. 3Go), although in vitro it exhibited an even higher affinity to GR than Dex (Table 1Go). In these experiments RU24782 was able to partially antagonize the Dex-induced activities. As in vitro binding assays, RU24858, RU24782, and RU40066 were shown to compete with [3H]RU28362; we also measured transcriptional activation of the GRE-tk-CAT reporter and induction of the TAT activity in Hela and HTC cells, respectively, exposed to 5 or 10 nM RU28362 with increasing amounts of the dissociated compounds. Again, RU40066 was able to antagonize RU28362-induced transactivation and RU24782 displayed partial antagonist activity, whereas a 100- or 500-fold excess of RU24858 was unable to antagonize RU28362-induced activity (data not shown). Thus, apparently dissociated glucocorticoids with a prevalent potency for AP-1 repression can be designed which are nearly devoid of both agonistic and antagonistic activities.



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Figure 2. Structure of the Dissociated Glucocorticoids Analogs

The structure of Dex is also shown for comparison.

 


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Figure 3. The Dissociated Glucocorticoid RU24858 Does Not Antagonize Dex-Induced Transactivation in Intact Cells

A, HeLa cells, transfected with the GRE5-tk-CAT reporter (and the internal control, see Materials and Methods), were exposed to Dex alone or 10 nM Dex with increasing amounts of the indicated dissociated glucocorticoids. The normalized CAT activity induced by the endogenous HeLa cell GR in the presence of 1 µM Dex was taken as 100% induction. B, Analysis of tyrosine amino transferase (TAT) activity (see Materials and Methods for details) originating from transactivation of the endogenous TAT gene by the endogenous GR in HTC cells under identical conditions as in panel A. The enzymatic activity measured in non-ligand-treated control cells was subtracted, and the results were expressed relative to the maximal activity measured in cells treated with 1 µM Dex (100%). The results shown are the means ± SEM of at least three independent experiments.

 
Divergent Potencies of Dissociated Glucocorticoids in TAT Induction and Inhibition of IL-1ß Secretion
Transcription of the TAT gene, one of the enzymes involved in the glucocorticoid-dependent stimulation of neoglucogenesis, is induced by glucocorticoids in vitro and in vivo, due to the presence of GREs in the TAT gene promoter (29). To assess the activity of the various dissociated glucocorticoids identified as described above with an endogenous GR target gene, rat HTC cells were treated with the synthetic glucocorticoids, and the resulting TAT activity was measured. A 10-fold induction was observed in cells treated with Dex at 1 µM (100% in Fig. 4AGo; and data not shown). Prednisolone (1 µM) induced TAT to 93% of the stimulation observed with 1 µM Dex (Fig. 4AGo). Importantly, only a very weak induction of TAT activity was observed in HTC cells treated with the different dissociated glucocorticoids, in keeping with the transactivation results obtained in Hela cells transiently transfected with GRE5-tk-CAT (Figs. 1AGo and 4AGo). For example, at 1 µM, RU24858 and RU24782 induced TAT activity to 11% and 17%, respectively, of the stimulation seen with 1 µM Dex. RU40066, like the antagonist RU486, did not significantly induce TAT activity (Fig. 4AGo).



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Figure 4. Dissociated Glucocorticoids Are Weak Transactivators of Tyrosine Amino Transferase (TAT) in Cultured Liver Cells but Potent Inhibitors of LPS-Induced IL-1ß Secretion in the Human Monocytic Cell Line THP 1

A, HTC cells were treated with 1 µM of either Dex (DEX), prednisolone (PRED), RU24782, RU24858, RU40066, or RU38486 (RU486). TAT activities were determined as described in Materials and Methods and expressed relative to the activity seen with 1 µM Dex (100%). Note that half-maximal activation of TAT induction was observed at Dex and prednisolone concentrations, which resulted also in half-maximal stimulation of transactivation from the GRE5-tk-CAT reporter gene in transfected Hela cells, i.e. 9 nM, and 40 nM, respectively (see Fig. 1Go). B, THP 1 cells, activated with 5 µg/ml LPS, were treated for 18 h with 1 µM of the indicated compounds. IL-1ß was measured in 50 µl of the culture supernatant, using the ELISA kit from R&D Systems. IL1ß level was undetectable in untreated control cell supernatants. LPS-treated cells secreted 1–3 ng/ml of IL-1ß. Inhibition of IL-1ß secretion in the presence of 1 µM Dex was taken as -100%. The results presented are means ± SEM of at least three independent experiments.

 
IL-1ß plays a central role in the regulation of inflammatory responses (for review see Ref. 30 and refs therein). Its induction is repressed by glucocorticoids at both the transcriptional and posttranscriptional level (31, 32, 33). To investigate whether dissociated glucocorticoids would also exibit IL-1ß-repressing activity, their effect on IL-1ß secretion was measured in THP1 cells, a monocytic cell line that responds to lipopolysaccharide (LPS) stimulation by secreting high amounts of IL-ß and tumor necrosis factor-{alpha} (TNF{alpha}). Dex, which at 1 µM inhibited 75% of LPS-induced IL-1ß secretion (not shown), was taken as the reference compound (-100% in Fig. 4BGo). Note that the same IC50 values (2 nM) were observed for the Dex-dependent inhibition of LPS-induced IL-1ß secretion from THP1 cells and the c-Jun-induced collagenase promoter activity in transiently transfected Hela cells (Fig. 1Go, and data not shown). At 1 µM, the dissociated glucocorticoids RU24782 and RU24858 inhibited IL-1ß expression 70% and 95%, respectively, of the maximum inhibition obtained with Dex (Fig. 4BGo). RU40066 was inactive at doses up to 10 nM and displayed moderate activity (65%) at high concentrations only, with an IC50 of 0.3 µM (Fig. 4BGo, and data not shown). As was the case for Dex, the IC50 values for inhibition of IL-1ß secretion by prednisolone, RU24858, and RU24782 were very close to those determined for AP-1 transrepression in transiently transfected Hela cells (the following IC50 values were obtained: prednisolone, 6 nM; RU24858, 30 nM; RU24782, <20 nM; compare with Fig. 1BGo). It is noteworthy that similar results were obtained with the dissociated glucocorticoids for the inhibition of LPS-actived IL-1ß secretion from peripheral blood mononuclear cells (data not shown).

Dissociated Glucocorticoids Display Antiinflammatory and Thymolytic Activity in Vivo
Glucocorticoids are major tools in the therapy of inflammatory disorders. Encouraged by the observation that expression of IL-1ß, an immunomodulator at the top of the inflammatory cascade, was affected by dissociated glucocorticoids, we investigated whether these compounds could also exibit antiinflammatory activity in vivo. Two classic animal models were selected. In the "cotton pellet granuloma" test (34) the compounds were given orally to rats using Dex and prednisolone as reference compounds. RU24858 and RU24782 displayed a similar antiinflammatory activity as shown by the inhibition of granuloma formation; the corresponding ED50 values were estimated to 7 mg/kg and 5 mg/kg, respectively (Table 2Go). Dexamethasone, prednisolone, RU24858, and RU24782 inhibited granuloma formation up to 80%. Prednisolone exhibited maximal efficiency above 5 mg/kg, whereas the activity of RU24782 in this assay plateaued off above doses of 10 mg/kg (Fig. 5Go). Although these compounds were 50 to 70 times less active than Dex, their activities were very similar to that of prednisolone (ED50 of 2.5 mg/kg; Table 2Go); in this test system RU40066 was inactive at the dose of 10 mg/kg, but was not tested at higher doses.


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Table 2. In Vivo Activities of Dissociated Glucocorticoids

 


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Figure 5. Dissociated Glucocorticoids Display in Vivo Antiinflammatory Activity in the Cotton Pellet Granuloma Model

Two 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 hours after the last administration the pellet dry weight was determined (see Materials and Methods). The activities of the various synthetic glucocorticoids were expressed as percent inhibition of granuloma weight increase. **, P < 0.01.

 
The "croton oil-induced ear edema" model (35) was the second test selected to study the antiinflammatory activity of the dissociated glucocorticoids in vivo and was performed in mice. In this model, the antiinflammatory activity of compounds is determined by their ability to prevent local inflammation induced by croton oil. Topically applied RU24858 and RU24782 displayed very strong antiinflammatory activities with ED50 values of 2 µg/ear and 10 µg/ear, respectively. Note that RU24858 was only 2-fold less efficient than Dex and 2-fold more efficient than prednisolone (Table 2Go). RU40066 displayed a significant, albeit low activity when applied topically (Table 2Go).

Notably, RU486, which displayed some transrepression activity with the Coll-CAT (but not TRE-tk-CAT; Table 1Go) reporter, was completely inactive in the cotton pellet granuloma and croton oil-induced ear edema models at concentrations up to 100 mg/kg (Table 2Go). Finally, two further animal models, the "mouse zymosan paw edema" (36) and "rat carrageenin paw edema" (37), confirmed that the antiinflammatory potency of RU24858 was, albeit lower than that of Dex, similar to that of prednisolone (data not shown).

As the immunosuppressive activity of classic glucocorticoids is linked to their ability to induce lymphocyte apoptosis, we measured thymolytic activity of dissociated glucocorticoids in the same rats that were used for the cotton-pellet granuloma test. The doses at which a reduction of thymus weights could be observed were generally lower than those needed for inhibiting granuloma formation (Table 2Go). RU24782 and RU24858 displayed the same activity (ED50 estimated to 2.5 mg/kg) which was, as in the granuloma test, near to that of prednisolone (1.6 mg/kg). Doses of 5 mg/kg of RU24858 or RU24782 induced a thymus weight reduction of 81% and 77%, respectively, compared with control animals. Notably, even at 1 mg/kg, these glucocorticoids exhibited significant activity (25% reduction of thymus weight, P < 0.05), whereas they were inactive at these doses in the granuloma test (data not shown). As expected, Dex was highly active in the thymolysis test (with an ED50 below 0.05 mg/kg) since at this lowest dose tested, the decrease of thymus weight was 60% (data not shown). As previously observed in the cotton-pellet granuloma, RU40066 was inactive at the dose tested (10 mg/kg). Taken together, in several in vivo animal models, the dissociated glucocorticoids RU24858 and RU24782, which are weak activators of positively regulated GR target genes, displayed consistently a strong antiinflammatory action that was similar, or even superior, to that of the classic antiinflammatory glucocorticoid prednisolone.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Novel Class of Dissociated Glucocorticoids
Three classes of glucocorticoids have been distinguished by their ability to induce/inhibit certain functions of GR: agonists, and type I and type II antagonists (2). Agonists may induce the activation function AF-2 in the ligand-binding domain (LBD) with varying efficiencies (38), while in the presence of type I antagonists, such as RU486 (39, 40, 41), GR AF-2 will be inactive (38, 42). Type I antagonists do not impair the activity of the activation function AF-1, located in the N-terminal region A/B of GR (42, 43) and may act as cell-specific agonists, since it has been shown in the case of the estrogen receptor that its AF-1 acts in a cell-specific manner in the presence of the type I antagonist hydroxytamoxifen (44). In contrast, type II antiglucocorticoids, such as RU43044, are defined by their ability to impair DNA binding of GR and, thus, do not induce any significant transcriptional activity of GR target genes (43).

With the aim of better understanding how glucocorticoids enable GR to transrepress AP-1 activity (see Introduction for references), we have compared AP-1 inhibition in the presence of the three above types of ligands and, moreover, screened a library of glucocortcoids to find ligands that would inhibit AP-1 activity without significantly inducing agonistic activity (termed "dissociated" glucocorticoids). Confirming the original data of Jonat et al. (9), we observed that in transfected HeLa cells the type I antiglucocorticoid RU38486 was unable to repress AP-1-induced transactivation of a TRE5-tk-CAT reporter (Table 1Go), and, indeed, could reverse the effect of the agonist Dex (data not shown). With a collagenase promoter-based reporter, some RU38486-dependent AP-1 repression was observed but the antagonist still reversed the repression induced by 10 nM Dex (Fig. 6Go). No transrepressing ability was conferred onto GR in the presence of the type II antagonist RU43044 with any of the two AP-1 reporters (Table 1Go). Note that in the above cases transrepression was via the endogenous GR; transiently expressed exogenous GR can apparently further increase the AP-1 inhibition (17) but leads to nonphysiological GR concentrations. In conclusion, neither type I nor type II antiglucocorticoids have the characteristics of dissociated glucocorticoids and, thus, have only limited promise for use as antiinflammatory agents (see below).



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Figure 6. The Classic Antiglucocorticoid RU38486 Induces Only Weak AP-1 Transrepression with the Endogenous Hela Cell GR and Antagonizes the Effect of High Dex Concentrations

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

 
Screening of a library of glucocorticoids yielded three molecules (RU24782, RU24858, and RU40066) that exhibited the dissociated characteristics. All of them exerted residual agonistic activity (9% to 35% of that of Dex) but induced 58% to 83% of the anti-AP-1 activity seen with Dex (Table 1Go). Note in this context that prednisolone, widely used as antiinflammatory glucocorticoid, showed a similar anti-AP-1 activity in transfected cells (Table 1Go). The most interesting of these compounds is RU24858, since it exemplifies a novel class of glucocorticoids: despite a binding affinity that is even higher than that of Dex, RU24858 does not significantly antagonize Dex-induced transcription (Fig. 3Go). However, in in vitro binding assays RU24858 does efficiently compete with radiolabeled Dex (and other glucocorticoid agonists) for binding to the GR. This implies that, in contrast to classic glucocorticoids, the binding affinity of RU24858 to transcriptionally engaged GR in vivo is lower than that measured in vitro. Importantly, however, this lower in vivo binding affinity does not significantly decrease the ability of the GR-RU24858 complex to repress AP-1 activity. We conclude from these data that AP-1 repression involves very specific ligand-induced structural alterations of GR.

How can RU24858 bind GR with different affinities in vitro and in vivo? Transcriptional interference/squelching studies have suggested that transcriptional intermediary factors (TIFs, also termed mediators, coactivators, bridging factors) mediate the ligand-dependent activation function AF-2 of steroid receptors to the transcriptional machinery (45, 46, 47). Putative TIFs have been recently isolated and characterized on the basis of their ligand-dependent interaction with several nuclear receptors (48, 49, 50, 51, 52, 53, 54). We propose that the interaction between the LBD and TIF(s) (or any other factor that interacts with the GR LBD in vivo) may differentially affect the binding of Dex and RU24858, resulting in a decreased affinity of RU24858, but not Dex, to the GR-TIF complex. However, noncomplexed GRs, or a set of GR complexes distinct from those involved in transactivation, may bind RU24858 with high affinity and give rise to the strong AP-1 inhibition observed in the presence of this compound. Note that there is a precedent for an altered affinity of glucocorticoids upon GR complex formation, since different affinities have been reported for the GR and the GR-hsp90 complex (55, 56).

Taken together, the above results suggest that we have identified a novel class of glucocorticoids that are neither efficient agonists nor antagonists in vivo, but can very efficiently induce GR-dependent AP-1 repression.

In Contrast to Classic Antiglucocorticoids, Dissociated Glucocorticoids Are Strong Inhibitors of LPS-Induced IL-1ß Secretion
Glucocorticoids are powerful antiinflammatory agents, most likely due to their ability to block the expression of multiple cytokines. Inhibition of cytokine expression has been reported to occur at the transcriptional level for IL-1 to IL-6, IL-8, TNF{alpha}, colony-stimulating factor (CSF)-1/macrophage (M)-CSF, granulocyte macrophage (GM)-CSF, and {gamma}-interferon (IL-1 expression is blocked at other levels as well; TNF and GM-CSF expression may be blocked through degradation of their mRNAs; for reviews see Refs. 27, 57, 58, 59, 60 , and references therein). Most of these genes require transcription factors, such as AP-1, NF-{kappa}B, or NF-AT, for their expression, suggesting that glucocorticoids exert their antiinflammatory functions by negatively interfering with the activity of (some of) these factors. Dissociated glucocorticoids, such as RU24858, were as potent inhibitors of IL-1ß secretion as prednisolone in LPS-stimulated cultured human monocytes, whereas classic antiglucocorticoids with no (RU43044) or promoter-dependent AP-1-transrepression activities (RU486) inhibited IL-1ß secretion only weakly (RU486) or not at all (RU43044) in this assay (Fig. 4Go and data not shown).

In Vivo Dissociated Glucocorticoids Are Antiinflammatory Agents as Potent as Prednisolone
To investigate whether dissociated glucocorticoids could be active as antiinflammatory drugs in vivo, we studied two established animal models, the cotton pellet granuloma model, in which the glucocorticoid is given orally to rats, and the croton oil-induced ear edema model, in which the drug is applied topically to mice (see Materials and Methods for details and references). Importantly, in these in vivo models, RU24858 was as active as prednisolone (Fig. 5Go and Table 2Go). In the croton oil-induced ear edema model, which relies on phorbol ester-induced skin inflammation, RU24858 was even 2-fold more active than prednisolone, and RU24782 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 of this compound are available. Importantly, in keeping with their inability to inhibit IL-1ß secretion, the classic antagonists RU486 and RU43044 were completely inactive in these in vivo tests (Table 2Go and data not shown).

The immunosuppressant potential of glucocorticoids relates to their ability to induce T cell apoptosis and to inhibit cytokine gene expression. These activities are therapeutically exploited in organ transplantation and in treatment of leukemias. We compared, in the cotton pellet granuloma model, the extent of thymolysis induced by classic (anti)glucocorticoids and the novel class of dissociated glucocorticoids. Again we observed a high activity of RU24858 and RU24782, similar to that of prednisolone, while antiglucocorticoids were inactive (Table 2Go and data not shown).

Dissociated Glucocorticoids May be Novel Tools to Improve Antiinflammatory Glucocorticoid Therapy
We describe here a novel class of dissociated glucocorticoids, distinct from the various previously reported agonists and antagonists. These compounds efficiently transrepress AP-1 activity, while only marginally activating glucocorticoid target genes. The dissociated glucocorticoids have maintained the antiinflammatory and, according to our as yet limiting data, also the thymolytic capacity of classical glucocorticoid agonists. Moreover, its prototypic member RU24858 is as active, or, depending on the system, even more active, than prednisolone in standard in vivo models. Hypothalamic-pituitary-adrenal axis insufficiency, osteoporosis, diabetes, steroid myopathy, and infectious and neuropsychiatric complications limit the therapeutic use of classic glucocorticoid agonists (61). At least some of these complications are likely to derive from the agonistic activities of the classic glucocorticoids, and it has not escaped our attention that our glucocorticoid analogs, provided that the dissociation of transactivation and transrepression is maintained in target organs, have the potential to constitute a new class of antiinflammatory glucocorticoids with significantly reduced side effects. Studies are under way to confirm this hypothesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
GRE5-tk-CAT, TRE5-tk-CAT reporters, and pSVc-Jun have been described (15, 62). The polyIIßGal used as internal control was constructed by inserting the HindIII-BamHI fragment of pCH110 (Pharmacia, Piscataway, NJ) containing the coding sequence of the ß-galactosidase gene into the HindIII-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 transfection assays for transactivation or AP-1 transrepression were carried out using the calcium phosphate coprecipitation procedure. Briefly, 4 x 105 cells were seeded 24 h before transfection in six-well plates containing DMEM supplemented with 5% charcoal/dextran-treated serum. For transactivation assays, HeLa cells were transfected with 1 µg GRE5-tk-CAT reporter, 1 µg polyIIßGal, and 3 µg Bluescript KS (Stratagene, La Jolla, CA). For transrepression assays, HeLa cells were transfected with 3 µg of the Coll(-517/+63)CAT reporter, 1 µg polyIIßGal, 250 ng pSVc-Jun expression plasmid, and 1 µg Bluescript KS. Sixteen hours later, cells were rinsed with Earle’s Balanced Salt Solution (EBSS), the medium was replaced, and the hormones were added as indicated. After 24 h, cells were washed with ice-cold EBSS and resuspended in 250 µl of lysis buffer provided with the chloramphenicol acetyltransferase enzyme-linked immunosorbent assay (CAT ELISA) kit (Boehringer Mannheim, Indianapolis, IN). The amount of CAT enzyme was quantified according to the manufacturer’s instructions. Results were normalized according to the ß-galactosidase activity originating from cotransfected polyIIßGal and plotted relative to the transactivation seen with 1 µM Dex (=100%) or relative to the AP-1 transrepression exerted by 1 µM Dex (=-100%). SEMs were calculated from a minimum of three independent assays per compound and per concentration.

Determination of Relative Binding Affinities
Relative binding affinities were determined by incubating HeLa cell cytosol for 24 h at 0 C with either [3H]RU28362 (28) or [3H]Dex with or without different concentrations of competitor steroids. Bound and free ligands were separated by the dextran-coated charcol method (38). The relative binding affinity of Dex was taken as reference (100%). Similar results were obtained with crude SF9 cell extracts containing high levels of human GR expressed from recombinant baculoviruses.

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, at confluency, the medium was replaced by DMEM without serum, and hormones were added for 18 h. TAT activity was modified from Ref. 64 to be measured in 96-well plates. The endogenous enzymatic activity measured in control cells was subtracted, and the results were expressed relative to the maximal activity measured in cells treated with 10-6 M Dex.

Assay of IL-1ß Secretion
The monocytic cell line THP1 (ATCC: TIB202) was cultured in RPMI 1640 supplemented with 10% FCS and grown at 37 C and 5% CO2. IL-1ß secretion was induced by treating the cells with 5 µg/ml LPS (Sigma, St. Louis, MO); glucocorticoids were added simultaneously. After 18 h, supernatants were collected and IL-1ß was quantitated using the human IL-1ß ELISA kit from R&D Systems (Minneapolis, MN).

Antiinflammatory Activity in the Cotton-Pellet Granuloma Test
The cotton-pellet granuloma assay was performed as described previously (34). Two cotton pellets (10 mg each) were inserted subcutaneously into the upper dorsal area of female Wistar rats (weight range 90–100 g, Iffa Credo, France). Test compounds were administered orally, once a day, for 4 days. Twenty-four hours after the last treatment, the animal was killed and the pellet, along with the surrounding granuloma, was carefully dissected from the animal and its dry weight was determined. For this, granuloma and pellet were heated at 60 C overnight. By subtracting the initial weight of the pellet, the dry weight of the granuloma was determined. The activities of the various synthetic glucocorticoids were expressed as ED50 values (dose causing a reduction of granuloma weight by 50%).

Thymolytic Activity
The thymolytic activity of the synthetic compounds was determined by weighing the thymus of the rats treated in the granuloma test. The ED50 values correspond to the glucocorticoid 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) on groups of eight male OF1 mice weighing 18–22 g (Iffa Credo, L’Arbresle, France). The edema was induced on one ear by the application of a solution of croton oil (2% vol/vol) in pyridine-water-ether 4:1:14.6 (by volume). Animals were killed 6 h later, and the ears were removed and weighed. Edema was determined from the difference in weight between the irritant-treated and the contralateral ear. The compounds to be tested were dissolved in the croton oil solution and topically applied on the ear. ED50 values correspond to the doses reducing the control edema by 50%.


    ACKNOWLEDGMENTS
 
We thank Dr. Michel Pons (Montpellier, France) for the kind gift of HTC cells. We are grateful to the staff members of the endocrinology department, and especially to Dr. Martine Gaillard and Dr. André Ulmann for their help and support. Dr. F. Fassy is acknowledged for her help and advice in setting up the TAT assay. We are grateful to O. Krebs for her contribution in the set-up of the CAT ELISA assay. Evelyne Cérède and Françoise Bouchoux are acknowledged for their essential contribution in binding experiments. Finally, we wish to thank Dr. Neerja Bhatnagar for compound synthesis and Elisa Francesconi and Françoise Perrier for their excellent technical assistance.


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

1 Present address: INSERM U 248, Institut Curie, 26 rue d’Ulm, 75231 Paris Cedex 05. Back

2 S. Dupont is currently working at Institut de Génétique et de Biologie Moléculaire et Cellulaire. Back

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


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