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

Date Published: January 25, 2012

Publisher: Public Library of Science

Author(s): Kerstin C. Reuter, Stefan M. Loitsch, Axel U. Dignass, Dieter Steinhilber, Jürgen Stein, Giovambattista Pani.


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.

Partial Text

Glucocorticoids (GCs) represent one of the most powerful therapeutics available for the treatment of acute inflammation, and are a mainstay of therapy in IBD patients [1], [2]. However, the desirable anti-inflammatory and immunosuppressive properties are often accompanied by severe, and sometimes irreversible, side effects, such as fat redistribution, osteoporosis, growth suppression, diabetes, hypertension and a detrimental effect on tissue repair [3], [4]. The effects of GCs are mediated by the glucocorticoid receptor (GR), which rests inactive in the cytoplasm as a multiprotein complex containing several heat-shock proteins (Hsp), such as Hsp90 and Hsp56, (co-)chaperones and immunophilins [5], [6]. In response to ligand binding, the GR adopts an altered conformation and translocates into the nucleus, where it regulates gene expression via several mechanisms [6], [7]. Directly by binding of a ligand-GR dimer to specific DNA sequences within genes, termed glucocorticoid response element (GRE), or indirectly by interaction of a ligand-GR monomer with transcription factors such as nuclear factor κB (NF-κB), cAMP-responsive-element binding protein (CREB), activator protein (AP)-1 or signal transducers and activators of transcription (STATs) [8]. It has been hypothesised that negative gene-regulation, referred to as trans-repression, accounts for the anti-inflammatory action of GCs, whereas positive regulation, or trans-activation, contributes to some adverse effects [9], [10]. Thus, a promising new therapeutic approach based on the selective modulation of GR action and a new class of synthetic agents, the selective GR agonists (SEGRAs), aims to combine anti-inflammatory action with simultaneous reduction of adverse effects [9][11], [12]. Along with several others, Compound A (CpdA) a plant-derived phenyl aziridine precursor isolated from a Namibian shrub [13] and ZK216348 [14], both non-steroidal in structure but exhibiting a strong preference for GR-binding, have been classified as SEGRAs and found to dissociate between trans-activation and trans-repression, both in vitro and in vivo[14], [15], [16], [17].

Glucocorticoids are highly effective in combating inflammation, and represent a powerful tool in the management of inflammatory response in IBD patients [7], [37]. Unfortunately, their long-term use in particular causes a large number of debilitating side effects, thus restricting their application. Hence, in IBD therapy, there is a need for drugs, which are as effective as common GCs, but have a reduced side effect profile. This demand might be satisfied by the novel class of SEGRAs, following the promising concept of selective modulation of GR action. In the present in vitro study in intestinal epithelial cells, it has been demonstrated that the beneficial anti-inflammatory actions of the GR agonists CpdA and ZK216348 are comparable to those of the steroids’ representative Dex. Strikingly, and in contrast to results observed with Dex, this was found in the absence of intestinal epithelial wound healing inhibition, a classical steroid-associated side effect, thus emphasizing SEGRAs’ potential as possible future IBD therapeutics.