Guy Haegeman

A plant-derived ligand favoring monomeric glucocorticoid receptor conformation with impaired transactivation potential attenuates collagen-induced arthritis

The glucocorticoid receptor (GR) is a transcription factor regulating its target genes either positively, through direct binding to the promoter of target genes, or negatively by the interference with the activity of transcription factors involved in proinflammatory gene expression. The well-known adverse effects of glucocorticoids are believed to be mainly caused by their GR-mediated gene-activating properties. Although dimerization of GR is thought to be essential for gene-activating properties, no compound has yet been described which selectively imposes GR monomer formation and interference with other transcription factors. In the present study, we report on a GR-binding, plant-derived compound with marked dissociative properties in rheumatoid arthritis fibroblast-like synoviocytes, which are important effector cells in inflammation and matrix degradation in rheumatoid arthritis. In addition, these findings could be extended in vivo in murine collagen-induced arthritis, in which joint inflammation was markedly inhibited without inducing hyperinsulinemia. Therefore, we conclude that GR monomers are sufficient for inhibition of inflammation in vivo.

A Dissociated Glucocorticoid Receptor Modulator Reduces Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4–induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.

An anti-inflammatory selective glucocorticoid receptor modulator preserves osteoblast differentiation

Glucocorticoids (GCs) are in widespread use to treat inflammatory bone diseases, such as rheumatoid arthritis (RA). Their anti‐inflammatory efficacy, however, is accompanied by deleterious effects on bone, leading to GC‐induced osteoporosis (GIO). These effects include up‐regulation of the receptor activator of NF‐κB ligand/osteoprotegerin (RANKL/OPG) ratio to promote bone‐resorbing osteoclasts and include inhibition of bone‐forming osteoblasts. We previously identified suppression of osteoblast differentiation by the monomer glucocorticoid receptor (GR) via the inhibition of Il11 expression as a crucial mechanism for GIO. Here we show that the GR‐modulating substance compound A (CpdA), which does not induce GR dimerization, still suppresses proinflammatory cytokines in fibroblast‐like synovial cells from patients with RA and in osteoblasts. In contrast to the full GR agonist dexamethasone, it does not unfavorably alter the RANKL/OPG ratio and does not affect Il11 expression and subsequent STAT3 phosphorylation in these cells. Notably, while dexamethasone inhibits osteoblast differentiation, CpdA does not affect osteoblast differentiation in vitro and in vivo. We describe here for the first time that selective GR modulators can act against inflammation, while not impairing osteoblast differentiation.—Rauch, A., Gossye, V., Bracke, D., Gevaert, E., Jacques, P., Van Beneden, K., Vandooren, B., Rauner, M., Hofbauer, L. C., Haegeman, G., Elewaut, D., Tuckermann, J. P., De Bosscher, K. An anti‐inflammatory selective glucocorticoid receptor modulator preserves osteoblast differentiation. FASEB J. 25, 1323–1332 (2011). www.fasebj.org

Differential mechanism of NF‐κB inhibition by two glucocorticoid receptor modulators in rheumatoid arthritis synovial fibroblasts

OBJECTIVEnTo investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR)-activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-kappaB activation pathway in rheumatoid arthritis (RA) synovial cells.nnnMETHODSnQuantitative polymerase chain reaction was performed to detect the tumor necrosis factor alpha (TNFalpha)-induced cytokine gene expression of interleukin-1beta (IL-1beta) and to investigate the effects of DEX and CpdA in RA fibroblast-like synoviocytes (FLS) transfected with small interfering RNA (siRNA) against GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-kappaB (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1beta. Signaling pathways were studied via Western blotting of siGR-transfected cells, compared with nontransfected and nontargeting siRNA-transfected control cells, to detect the regulation of phospho-IKK, IkappaBalpha, phospho-p38, phospho-ERK, and phospho-JNK.nnnRESULTSnBoth DEX and CpdA efficiently inhibited IL-1beta gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNFalpha-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent IkappaBalpha degradation. CpdA also displayed profound effects on TNFalpha-induced MAPK activation. The effects of CpdA on TNFalpha-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNFalpha-induced IKK phosphorylation, IkappaBalpha degradation, p65 nuclear translocation, or MAPK activation in RA FLS.nnnCONCLUSIONnDEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-kappaB activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of CpdA in RA FLS.

Withaferin A inhibits NF-kappaB activation by targeting cysteine 179 in IKKβ

The transcription factor NF-κB is one of the main players involved in inflammatory responses during which NF-κB becomes rapidly activated. However to maintain homeostasis, this NF-κB activation profile is only transient. Nevertheless deregulation of NF-κB activity is often observed and can lead to chronic inflammatory diseases as well as cancer. Therefore various research projects focus on the development of therapeutics that target the NF-κB activation pathway. One such compound is Withaferin A from the Ayurvedic plant Withania somnifera. Several reports already described the NF-κB inhibiting, anti-inflammatory capacity of WA, either in vitro as well as in vivo. However the underlying molecular mechanism remains largely unknown. In this paper we demonstrate a direct interaction of WA with the IKK-complex, more specifically with IKKβ, a kinase which is indispensable for the nuclear translocation of NF-κB. Hereby WA directly inhibits IKK catalytic activity. By mutation of Cys179 in IKKβ we could demonstrate loss of interaction between IKKβ and WA indicating that WA exerts its anti-inflammatory effects by targeting the crucial Cys179 residue located in the catalytic site of IKKβ. Upon docking of WA to a IKKβ homology structure model, WA was found to fit nicely into the groove of IKKβ where it can form hydrogen bond to stabilize its interaction with Cys179.

Antiinflammatory Properties of a Plant-Derived Nonsteroidal, Dissociated Glucocorticoid Receptor Modulator in Experimental Autoimmune Encephalomyelitis

Compound A (CpdA), a plant-derived phenyl aziridine precursor, was recently characterized as a fully dissociated nonsteroidal antiinflammatory agent, acting via activation of the glucocorticoid receptor, thereby down-modulating nuclear factor-kappaB-mediated transactivation, but not supporting glucocorticoid response element-driven gene expression. The present study demonstrates the effectiveness of CpdA in inhibiting the disease progress in experimental autoimmune encephalomyelitis (EAE), a well-characterized animal model of multiple sclerosis. CpdA treatment of mice, both early and at the peak of the disease, markedly suppressed the clinical symptoms of EAE induced by myelin oligodendrocyte glycoprotein peptide immunization. Attenuation of the clinical symptoms of EAE by CpdA was accompanied by reduced leukocyte infiltration in the spinal cord, reduced expression of inflammatory cytokines and chemokines, and reduced neuronal damage and demyelination. In vivo CpdA therapy suppressed the encephalogenicity of myelin oligodendrocyte glycoprotein peptide-specific T cells. Moreover, CpdA was able to inhibit TNF- and lipopolysaccharide-induced nuclear factor-kappaB activation in primary microglial cells in vitro, in a differential mechanistic manner as compared with dexamethasone. Finally, in EAE mice the therapeutic effect of CpdA, in contrast to that of dexamethasone, occurred in the absence of hyperinsulinemia and in the absence of a suppressive effect on the hypothalamic-pituitary-adrenal axis. Based on these results, we propose CpdA as a compound with promising antiinflammatory characteristics useful for therapeutic intervention in multiple sclerosis and other neuroinflammatory diseases.

Selective modulation of the glucocorticoid receptor can distinguish between transrepression of NF-κB and AP-1

Glucocorticoids (GCs) block inflammation via interference of the liganded glucocorticoid receptor (GR) with the activity of pro-inflammatory transcription factors NF-κB and AP-1, a mechanism known as transrepression. This mechanism is believed to involve the activity of GR monomers. Here, we explored how the GR monomer-favoring Compound A (CpdA) affects AP-1 activation and activity. Our results demonstrate that non-steroidal CpdA, unlike classic steroidal GCs, blocks NF-κB- but not AP-1-driven gene expression. CpdA rather sustains AP-1-driven gene expression, a result which could mechanistically be explained by the failure of CpdA to block upstream JNK kinase activation and concomitantly also phosphorylation of c-Jun. In concordance and in contrast to DEX, CpdA maintained the expression of the activated AP-1 target gene c-jun, as well as the production of the c-Jun protein. As for the underlying mechanism, GR is a necessary intermediate in the CpdA-mediated gene expression of AP-1-regulated genes, but seems to be superfluous to CpdA-mediated JNK phosphorylation prolongation. The latter phenomenon concurs with the inability of CpdA to stimulate DUSP1 gene expression. ChIP analysis demonstrates that DEX-activated GR, but not CpdA-activated GR, is recruited to AP-1-driven promoters. Furthermore, in mice we observed that CpdA instigates a strong enhancement of TNF-induced AP-1-driven gene expression. Finally, we demonstrate that this phenomenon coincides with an increased sensitivity towards TNF lethality, and implicate again a role for JNK2. In conclusion, our data support the hypothesis that a ligand-induced differential conformation of GR yields a different transcription factor cross-talk profile.

A plant-derived glucocorticoid receptor modulator attenuates inflammation without provoking ligand-induced resistance

Background: Acquired resistance to glucocorticoids constitutes a major clinical challenge, often overlooked in the search for improved alternatives to classic steroids. We sought to unravel how two glucocorticoid receptor-activating compounds, dexamethasone and Compound A, influence glucocorticoid receptor levels and how this can be correlated to their gene regulatory potential. Methods: Compound A and dexamethasone were applied in a short-term and long-term treatment protocol. By quantitative PCR analysis in fibroblast-like synoviocytes (FLS) the gene regulatory potential of both compounds in the two experimental conditions was analysed. A parallel Western blot assay revealed the glucocorticoid receptor protein levels in both conditions (ex vivo). In addition, this study examined the effect of systemic administration of dexamethasone and Compound A, in concentrations effective to inhibit collagen-induced arthritis, in DBA/1 mice on glucocorticoid receptor levels (in vivo). Results: Compound A does not induce a homologous downregulation of glucocorticoid receptor in vivo and ex vivo, thereby retaining its anti-inflammatory effects after prolonged treatment in FLS. This is in sharp contrast to dexamethasone, showing a direct link between prolonged dexamethasone treatment, decreasing glucocorticoid receptor levels, and the abolishment of inflammatory gene repression in FLS. It was also observed that the acquired low receptor levels after prolonged dexamethasone treatment are still sufficient to sustain the transactivation of endogenous glucocorticoid-responsive element-driven genes in FLS, a mechanism partly held accountable for the metabolic side-effects. Conclusion: Compound A is less likely to evoke therapy resistance, as it does not lead to homologous glucocorticoid receptor downregulation, which is in contrast to classic glucocorticoids.

Effects of the Selective Glucocorticoid Receptor Modulator Compound A on Bone Metabolism and Inflammation in Male Mice With Collagen-Induced Arthritis

Glucocorticoids (GCs) are potent drugs to treat rheumatoid arthritis but exert adverse skeletal effects. Compound A (CpdA) is a selective GC receptor modulator with an improved risk/benefit profile in mouse models of inflammation and bone loss. Here we tested whether CpdA also exerts bone-sparing effects under proinflammatory circumstances using the collagen-induced arthritis model, a murine model of rheumatoid arthritis. CpdA decreased disease activity, paw swelling, and the paw temperature by 43%, 12%, and 7%, respectively, but was less potent than dexamethasone (DEX), which reduced these parameters by 72%, 22%, and 10%, respectively. Moreover, T cells isolated from CpdA- and DEX-treated animals were less active based on proliferation rates after challenge with type II collagen and produced smaller amounts of interferon-γ and TNF as compared with T cells from PBS-treated mice. Histological assessment of the joints confirmed the weaker potency of CpdA as compared with DEX in preventing infiltration of inflammatory cells, induction of osteoclastogenesis, and destruction of articular cartilage. Due to the lack of GC-susceptible arthritis models, we were not able to fully address the bone-sparing potential of CpdA in inflammatory conditions. Nevertheless, the bone formation marker procollagen type 1 N-terminal peptide, a surrogate marker for GC-mediated suppression of bone formation, was significantly decreased by DEX in arthritic mice but not by CpdA. Our data indicate that CpdA moderately suppresses inflammation, whereas the concurrent effects on bone remain unknown. In light of its narrow therapeutic range, CpdA may be more useful as a molecular tool for dissecting GC actions rather than a therapeutic agent.

Compound A, a Dissociated Glucocorticoid Receptor Modulator, Inhibits T-bet (Th1) and Induces GATA-3 (Th2) Activity in Immune Cells

Background Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively. Results Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively. Conclusions Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.