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

Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-kappa B-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated I kappa B alpha degradation and NF-kappa B p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells

Studying the therapeutic benefit of a glucocorticoid receptor phytomodulator in synoviocytes derived from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a chronic inflammatory ailment that is characterized by the hyperproliferative and invasive nature of fibroblast-like synoviocytes (FLS). Although research is advancing and therapies have greatly improved over the past years, current treatment regimens still suffer major drawbacks. This is unfortunately also the case for glucocorticoids (GCs), the most important and frequently used class of inflammatory drugs. Indeed, the usage of glucocorticoids (GCs) is overshadowed not only by the occurrence of severe side effects, such as osteoporosis, but also by the occurrence of resistance to the therapeutic effects of GCs, thereby limiting the treatment options of patients with RA. The main goal of GC/glucocorticoid receptor (GR) research thus remains to find new GR modulators that provide the same anti-inflammatory power, yet with reduced toxicity. A milestone in the history of identifying new, improved glucocorticoid receptor (GR) ligands, coincided with the recognition that activation (metabolic/unwanted effects) and repression (anti-inflammatory effects) of gene expression by GR could be addressed separately. Consequently, the objectives of this thesis were to unravel the therapeutic potential of Compound A (CpdA), a dissociative GR modulator, for the treatment of RA. In addition, it was questioned whether the anti-inflammatory effect of CpdA is associated with an improved side effect profile in comparison with currently available synthetic GCs, using the strong GR agonist dexamethasone (DEX) as a paradigm. In this work, the possible advantage of CpdA over DEX with regard to glucocorticoid-induced osteoporosis (GIO) and acquired resistance to the therapeutic effects of GCs was brought into special focus. As it is well known that cell lines and primary cells differ in their response to pro-inflammatory cytokines, a particularly important aspect of this research was the use of fibroblasts, isolated from the inflamed synovium of RA-patients, as a physiologically relevant ex vivo cell system to be able to unravel the molecular mechanisms by which CpdA exerts its effects

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

Objective. To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR)-activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-kappa B activation pathway in rheumatoid arthritis (RA) synovial cells. Methods. Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor alpha (TNF alpha)-induced cytokine gene expression of interieukin-1 beta (IL-1 beta) 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-kappa B (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1 beta. 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, I kappa B alpha, phospho-p38, phospho-ERK, and phospho-JNK. Results. Both DEX and CpdA efficiently inhibited IL-1 beta gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNF alpha-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent I kappa B alpha degradation. CpdA also displayed profound effects on TNF alpha-induced MAPK activation. The effects of CpdA on TNF alpha-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNF alpha-induced IKK phosphorylation, I kappa B alpha degradation, p65 nuclear translocation, or MAPK activation in RA FLS. Conclusion. DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-kappa B activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (non-genomic), may explain the gene-inhibitory effects of CpdA in RA FLS

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-kappa B 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

CpdA stimulation of the Hsp70 gene promoter occurs via a GR-dependent mechanism.

<p>(A) A549 cells were transfected with siControl or siRNA targeting GR (siGR). Total RNA or total protein extracts were prepared 48h post-transfection. In the left panel, purified mRNA was subjected to RT-qPCR detecting GR gene expression levels, normalized to housekeeping controls cyclophilin and 28S. For the siControl-transfected sample, signal was set at 100%. Data from SiGR-transfected cells were recalculated accordingly. Statistical analysis (unpaired t-test) was performed to show significant difference between siControl and siGR conditions (*** p<0.001). In the right panel, total cell lysates were subjected to Western blot analysis to detect GR protein, with NF-κB p65 as a loading control. (B) In parallel with (A) A549 cells were transfected with siControl or siGR. 41h post transfection, cells were induced with Solv or CpdA (10µM) for 8h. The derived purified mRNA was subjected to RT-qPCR detecting HSPA1A gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S. The condition Solv (siControl) was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (ns not significant; * p<0.05). This experiment is representative for 2 independent experiments. (C) and (D) A549 cells, serum-starved for48h in 0% DMEM, were treated with Solv, Dex (1µM), CpdA (10 µM) for 2h, or exposed to a 43°C heat shock (HS) for 1h. Total cell extracts were subjected to a ChIP assay targeting GR. Ensuing, qPCR signal of immunoprecipitated HSPA1A and GILZ gene promoter fragments is presented relative to input data. Binding to rabbit IgG represents aspecific binding. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference with the Solv condition (ns not significant; *** p<0.001). This experiment is representative for 2 independent experiments.</p