Selective Non-Steroidal Glucocorticoid Receptor Agonists Attenuate Inflammation but Do Not Impair Intestinal Epithelial Cell Restitution In Vitro

Article (PDF Available)inPLoS ONE 7(1):e29756 · January 2012with14 Reads
DOI: 10.1371/journal.pone.0029756 · Source: PubMed
Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile. The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis. Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348. Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.

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    • "Mapracorat (Schacke et al., 2009) and ZK 216348 (Schacke et al., 2004) have shown reduced skin atrophy compared to classical GCs after long-term topical treatment, yet the pathways or exact targets involved were not documented. Related, ZK 216348 and CpdA also do not inhibit intestinal epithelial cell restitution in vitro (Reuter et al., 2012b). In vivo studies of CpdA on mice indicated that CpdA did not stimulate protein degradation and had an ameliorative effect on intermediate markers of muscle dystrophy, compared to GCs (Huynh et al., 2013 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Glucocorticoids remain the frontline treatment for inflammatory disorders, yet represent a double-edged sword with beneficial therapeutic actions alongside adverse effects, mainly in metabolic regulation. Considerable efforts were made to improve this balance by attempting to amplify therapeutic beneficial anti-inflammatory actions and to minimize adverse metabolic actions. Most attention has focused on the development of novel compounds favoring the transrepressing actions of the glucocorticoid receptor, assumed to be important for anti-inflammatory actions, over the transactivating actions, assumed to underpin the undesirable actions. These compounds are classified as selective glucocorticoid receptor agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). The latter class is able to modulate the activity of a GR agonist and/or may not classically bind the glucocorticoid receptor ligand-binding pocket, and is collectively denominated SEGRAMs (selective glucocorticoid receptor agonists and modulators). Although this transrepression vs transactivation concept proved to be too simplistic, the developed SEGRAMs were helpful in elucidating various molecular actions of the glucocorticoid receptor, but have also raised many novel questions. We discuss lessons learned from recent mechanistic studies of selective glucocorticoid receptor modulators. This is approached by analyzing recent experimental insights in comparison with knowledge obtained using mutant GR research, thus clarifying the current view on the SEGRAM field. These insights also contribute to our understanding of the processes controlling glucocorticoid-mediated side effects as well as glucocorticoid resistance. Our perspective on non-steroidal SEGRAs and SEGRMs considers remaining opportunities to address research gaps in order to harness the potential for more safe and effective glucocorticoid receptor therapies. Copyright © 2015. Published by Elsevier Inc.
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    • "Stimulation of this promoter with TNFa could be clearly counteracted by Dex and CpdA. In conclusion, despite the impaired ability of CpdA to translocate GR in CT5.3hTERT myofibroblasts, both compounds used are functional and show NFkB-transrepressive properties inL929sA cells, the latter of which corresponds with earlier reports in various cell types [12,13,16,17,20,353637. The effects of Dex and CpdA on NFkB-mediated gene expression in CT5.3hTERT myofibroblasts is investigated more specifically in "
    [Show abstract] [Hide abstract] ABSTRACT: The glucocorticoid receptor functions as a ligand-dependent transcription factor that positively or negatively regulates the transcription of various specific target genes. Not only steroidal glucocorticoids can bind and activate the glucocorticoid receptor, but also the intensively examined non-steroidal selective glucocorticoid receptor modulators can do so, albeit with a select effector profile skewed to glucocorticoid receptor transrepression. Glucocorticoids are widely used to treat inflammatory afflictions, but also as anti-cancer therapies or adjuvants thereof. As the impact of glucocorticoids and selective glucocorticoid receptor modulators has scarcely been researched in this setting, we focused on colon cancer and its stromal environment, in particular the stromal myofibroblasts, which are known to influence cancer cells via paracrine signaling.
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    • "Furthermore, elevated Hsp70 levels can negatively affect the release of the pro-inflammatory high mobility group box 1 (HMGB1) [38]. Given the mechanistic resemblance between heat shock and CpdA, with respect to their effects on cytokine gene expression, IκBα degradation and NF-κB p65 nuclear translocation (Figure 2, Figure S2) in various cell systems [39], [40], [49], [50], we were not surprised to detect that CpdA can elicit a concentration-dependent, yet transient, rise in Hsp70-coding mRNA levels and promoter activity (Figure 4, 8, Figure S4) in human A549 and PC-3 cells and murine L929sA cells. These findings could be confirmed in human MCF7 breast cancer cells (Figure S5) and skins of BALB/c mice (Figure 4C), indicating species-, cell- and tissue independence of this phenomenon. "
    [Show abstract] [Hide abstract] ABSTRACT: Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-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 IκBα degradation and NF-κB 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.
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