The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice.

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

Glucocorticoid analogues: potential therapeutic alternatives for treating inflammatory muscle diseases.

Glucocorticoids (GCs) have been prescribed to treat a variety of diseases, including inflammatory myopathies and Duchenne muscular dystrophy for over 50 years. However, their prescription remains controversial due to the significant side effects associated with the chronic treatment. It is a common belief that the clinical efficacy of GCs is due to their transrepression of pro-inflammatory genes through inhibition of inflammatory transcription factors (i.e. NF-κB, AP-1) whereas the adverse side effects are attributed to the glucocorticoid receptor (GR)-mediated transcription of target genes (transactivation). The past decade has seen an increased interest in the development of GR modulators that maintain the effective anti-inflammatory properties but lack the GR-dependent transcriptional response as a safe alternative to traditional GCs. Many of these analogues or "dissociative" compounds show potential promise in in vitro studies but fail to reach human clinical trials. In this review, we discuss molecular effects of currently prescribed GCs on skeletal muscle and also discuss the current state of development of GC analogues as alternative therapeutics for inflammatory muscle diseases.

Inhibition of iNOS expression and NO production by anti-inflammatory steroids. Reversal by histone deacetylase inhibitors.

In inflammation, nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) induced by bacterial products and cytokines, and NO acts as a regulatory and pro-inflammatory mediator. Glucocorticoids are powerful anti-inflammatory agents that inhibit the expression of iNOS and various other inflammatory factors. Histone deacetylation has been recently described as a novel mechanism how glucocorticoids down-regulate transcriptional activation of some inflammatory genes. The aim of the present study was to investigate the effects of inhibitors of histone deacetylation on the suppressive effects of glucocorticoids on NO production and iNOS expression. Dexamethasone and a dissociated glucocorticoid RU24858 inhibited NO production, and iNOS protein and mRNA expression in macrophages exposed to bacterial lipopolysaccharide (LPS). In the presence of a glucocorticoid receptor (GR) antagonist mifepristone, dexamethasone and RU24858 had no effect on NO production. The role of histone deacetylation in the glucocorticoid effect was studied by using three structurally different inhibitors of histone deacetylases (HDACs): trichostatin A, apicidin and MC1293. HDAC inhibitors reversed the effects of dexamethasone and RU24858 on iNOS expression and NO production. Stably transfected A549/8 cells containing luciferase gene under the control of human iNOS promoter were used in promoter-activity studies. iNOS promoter activity induced by IL-1beta was inhibited by dexamethasone and the inhibitory effect was reversed by HDAC inhibitor trichostatin A. The results suggest that glucocorticoids inhibit iNOS expression and NO production by a GR-mediated and GRE-independent manner through histone deacetylation and transcriptional silencing.

Analysis of the dissociated steroid RU24858 does not exclude a role for inducible genes in the anti-inflammatory actions of glucocorticoids.

Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol 11:1245-1255, 1997) repress interleukin (IL)-1beta-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor kappaB (NF-kappaB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-kappaB-dependent transrepression ( approximately 40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1beta-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepressionbased screens for anti-inflammatory "dissociated" GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.

Effects of dissociated glucocorticoids on OPG and RANKL in osteoblastic cells.

Glucocorticoids are effective anti-inflammatory and immunosuppressive agents, but their use is often associated with debilitating side effects such as glucocorticoid-induced osteoporosis. Newly developed glucocorticoid analogues such as the so-called dissociated glucocorticoids are potent immunosuppressants and have the potential for fewer side effects. The effects of these new analogues on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells have not been studied. OPG and RANKL are osteoblast-derived proteins pivotal to the regulation of bone mass. RANKL stimulates bone resorption by increasing osteoclast differentiation, activation and survival. OPG is the decoy receptor for RANKL and thus inhibits bone resorption. Here, we show that dexamethasone, prednisolone, deflazacort and the dissociated glucocorticoids, RU24858, RU40066, RU24782, AL438-F1 and ZK216348 significantly inhibit OPG production in two human osteoblastic cell lines (MG63 and hFOB). The potency for OPG inhibition was ligand and cell-type specific. In both cell types, dexamethasone and prednisolone were the most potent ligands inhibiting OPG production with IC(50)s of approximately 0.1 nM and 10 nM respectively. In MG63 cells, deflazacort and the RU compounds were the next most potent ligands followed by AL438-F1 and ZK216348. In hFOB cells, however, the RU compounds were the least potent ligands with an IC(50) 74 times higher than in MG63 cells. In contrast, the level of maximum inhibition or effectiveness of OPG inhibition did not vary between cell types but did vary according to the ligand. Dexamethasone, prednisolone, deflazacort and the RU compounds all inhibited OPG production by a maximum of approximately 70-80%, whereas AL438-F1 and ZK 216348 inhibited OPG production by a maximum of only 40-50% at 1 microM. All of the dissociated glucocorticoids and deflazacort were poor stimulators of RANKL gene expression stimulating by only approximately 1-3-fold compared to 7-fold by prednisolone. These data demonstrate that deflazacort and the dissociated glucocorticoids are weak stimulators of the RANKL:OPG ratio compared to prednisolone. Therefore, these compounds have the potential to cause less bone loss than that seen with prednisolone, though this was not investigated here.

Inhibition of tristetraprolin expression by dexamethasone in activated macrophages.

Tristetraprolin (TTP) is a factor that regulates mRNA stability and the expression of certain inflammatory genes. In the present study, we found that TTP expression was increased in macrophages exposed to bacterial lipopolysaccharide (LPS). Dexamethasone and dissociated steroid RU24858 inhibited LPS-induced TTP protein and mRNA expression and the inhibitory effect was reversed by a glucocorticoid receptor antagonist mifepristone. Histone deacetylase inhibitors trichostatin A (TSA) and apicidin reduced the inhibitory effect of dexamethasone and RU24858 on TTP expression, but the glucocorticoids did not alter TTP mRNA half-life. These results suggest that anti-inflammatory steroids reduce TTP expression in activated macrophages by a glucocorticoid response element (GRE)-independent mechanism, possibly through histone deacetylation and transcriptional silencing.

Cell type-dependent divergence of transactivation by glucocorticoid receptor ligand.

The glucocorticoid receptor regulates gene expression mainly by two mechanisms; transactivation and trans-repression. A ligand with strong transrepression and weak transactivation activity is predicted to be a beneficial agent with potent anti-inflammatory activity and minor adverse effects. Recently, the profile of a synthetic steroid, RU24858, has been reported to fulfill this condition in vitro, but others have reported no dissociation between the anti-inflammatory activity and side effects in vivo. To gain further information on the profile of this compound, we evaluated its transactivation ability using a reporter gene analysis both in vitro and in vivo. In the in vitro analysis, RU24858 demonstrated only a weak transactivation activity in HeLa cells, when compared with prednisolone. In CV-1 cells, however, these two glucocorticoids exhibited equivalent transactivation activities. This behavior is independent of whether the reporter gene is regulated by mouse mammary tumor virus promoter or multiple copies of glucocorticoid response element. When the reporter plasmid was inoculated into mouse abdominal skin using a gene gun, followed by orally administration of glucocorticoids, RU24858 induced significantly higher reporter enzyme activity than prednisolone. These results suggest that the profile of RU24858 is divergent and its transactivation ability is comparable to prednisolone depending on the cell-type both in vitro and in vivo.

Dexamethasone inhibits inducible nitric-oxide synthase expression and nitric oxide production by destabilizing mRNA in lipopolysaccharide-treated macrophages.

Nitric oxide (NO) production through the inducible nitric-oxide synthase (iNOS) pathway is increased in inflammatory diseases and leads to cellular injury. Anti-inflammatory steroids inhibit the expression of various inflammatory genes, including iNOS. In the present study, we investigated the mechanism how dexamethasone decreased NO production in murine J774 macrophages. Dexamethasone (0.1-10 microM) inhibited the production of NO and iNOS protein in a dose-dependent manner in cells stimulated with lipopolysaccharides (LPS). In contrast, in cells treated with a combination of LPS and interferon-gamma (IFN-gamma), dexamethasone did not reduce iNOS expression and NO formation. Dissociated glucocorticoid RU24858 inhibited iNOS expression and NO production to levels comparable with that of dexamethasone, suggesting that the reduced iNOS expression by dexamethasone is not a GRE-mediated event. In further studies, the effect of dexamethasone on iNOS mRNA levels was tested by actinomycin assay. The half-life of iNOS mRNA after LPS treatment was 5 h 40 min, and dexamethasone reduced it to 3 h. The increased degradation of iNOS mRNA was reversed by a protein synthesis inhibitor cycloheximide. iNOS mRNA was more stabile in cells treated with a combination of LPS plus IFN-gamma (half-life = 8 h 20 min), and dexamethasone had a minor effect in these conditions. In conclusion, dexamethasone decreases iNOS-dependent NO production by destabilizing iNOS mRNA in LPS-treated cells by a mechanism that requires de novo protein synthesis. Also, decreased iNOS mRNA and protein expression and NO formation by dexamethasone was not found in cells treated with a combination of LPS plus IFN-gamma, suggesting that the effect of dexamethasone is stimulus-dependent.

Therapeutic benefit of a dissociated glucocorticoid and the relevance of in vitro separation of transrepression from transactivation activity.

Glucocorticoids (GCs) are the mainstay of asthma therapy; however, major side effects limit their therapeutic use. GCs influence the expression of genes either by transactivation or transrepression. The antiinflammatory effects of steroids are thought to be due to transrepression and the side effects, transactivation. Recently, a compound, RU 24858, has been identified that demonstrated dissociation between transactivation and transrepression in vitro. RU 24858 exerts strong AP-1 inhibition (transrepression), but little or no transactivation. We investigated whether this improved in vitro profile results in the maintenance of antiinflammatory activity (evaluated in the Sephadex model of lung edema) with reduced systemic toxicity (evaluated by loss in body weight, thymus involution, and bone turnover) compared with standard GCs. RU 24858 exhibits comparable antiinflammatory activity to the standard steroid, budesonide. However, the systemic changes observed indicate that transactivation events do occur with this GC with similar potency to the standard steroids. In addition, the GCs profiled showed no differentiation on quantitative osteopenia of the femur. These results suggest that in vitro separation of transrepression from transactivation activity does not translate to an increased therapeutic ratio for GCs in vivo or that adverse effects are a consequence of transrepression.

Synthetic glucocorticoids that dissociate transactivation and AP-1 transrepression exhibit antiinflammatory activity in vivo.

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-1beta 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.

Synthesis of steroidal 17 beta-carboxamide derivatives.

Several 17 beta-carboxamide derivatives of natural and fluorinated glucocorticoids have been synthesized. The 17 beta-carboxylic derivatives were obtained by periodic acid oxidation of their side chains. They were then activated by N-hydroxybenzotriazole (HOBT) and coupled to several primary amines. Using this method eleven 17 beta-carboxamide derivatives have been prepared in good yields.