Selective Non-Steroidal Glucocorticoid Receptor
Agonists Attenuate Inflammation but Do Not Impair
Intestinal Epithelial Cell Restitution In Vitro
Kerstin C. Reuter1, Stefan M. Loitsch1, Axel U. Dignass2, Dieter Steinhilber1, Ju ¨rgen Stein1,3,4*
1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt/Main, Campus Riedberg, Frankfurt/Main, Germany, 2Department of Medicine I, Markus Hospital,
Frankfurt/Main, Germany, 3Department of Internal Medicine, Elisabethen Hospital, Frankfurt/Main, Germany, 4Crohn Colitis Centrum Frankfurt, Frankfurt/Main, Germany
Introduction: 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.
Methods: 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-kB/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-b- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter
gene assay, Western blot and qPCR analysis.
Results: Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and
ZK216348 effectively inhibited NF-kB 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-b-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.
Conclusion: 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.
Citation: Reuter KC, Loitsch SM, Dignass AU, Steinhilber D, Stein J (2012) Selective Non-Steroidal Glucocorticoid Receptor Agonists Attenuate Inflammation but
Do Not Impair Intestinal Epithelial Cell Restitution In Vitro. PLoS ONE 7(1): e29756. doi:10.1371/journal.pone.0029756
Editor: Giovambattista Pani, Catholic University Medical School, Italy
Received May 16, 2011; Accepted December 5, 2011; Published January 25, 2012
Copyright: ? 2012 Reuter et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a graduate scholarship grant from the Deutsche Forschungsgemeinschaft (DFG, GRK757) to KCR. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
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 immuno-
philins [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 transcrip-
tion factors such as nuclear factor kB (NF-kB), cAMP-responsive-
element binding protein (CREB), activator protein (AP)-1 or signal
transducers and activators of transcription (STATs) . 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  [11,12]. Along with several others,
Compound A (CpdA) a plant-derived phenyl aziridine precursor
isolated from a Namibian shrub  and ZK216348 , both
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e29756
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].
In the context of IBD, one of the major consequences of GC
use, is the inhibition of intestinal wound healing [18,19,20], which
limits their therapeutic application considerably, despite their
excellent anti-inflammatory action. The mucosal lining of the
intestine consists of fast-renewing epithelial cells which function as
a barrier between the luminal environment and the mucosal
immune system. In the course of IBD, damage and impairment of
the intestinal epithelial surface are frequently observed, and
dysfunction of the epithelial barrier results in systemic penetration
of detrimental substances, leading to a generalised immune
response and chronic intestinal inflammation [21,22]. Normally,
after injury, tissue integrity is restored by a rapid, organised series
of cellular events in which where inflammation, cell migration and
proliferation, the production of connective tissue ground sub-
stances, angiogenesis and wound contraction are orchestrated by
biochemical substances [20,23]. Furthermore, over the past
decade, a complex network of regulatory peptides (chemokines,
cytokines, growth factors, enzymes and extra-cellular matrix
molecules) have been found to be expressed by, and to produce
functional effects among, different cell populations in the mucosa
[22,24,25,26] contributing to the preservation of the intestinal
barrier following injury. Several of these regulatory peptides, such
as transforming growth factor (TGF)-b, epidermal growth factor
(EGF), tumor necrosis factor (TNF)-a, hepatocyte growth factor
(HGF) and insuline-like growth factors (IGF) I and II have been
identified as strong healing factors, and thus play an important role
in intestinal healing [22,27,28]. GCs’ inhibition of tissue repair is
currently attributed to their modulation of the wound repair
processes, i.e. migration, proliferation and differentiation of
epithelial cells, but in addition, to their ability to influence the
expression and respective signalling pathways of a broad number
of these regulatory peptides [3,19,20,29].
Prerequisites of disease remission in IBD are repair of the
damaged epithelium and the absence of inflammation, so that
normal homeostasis of the host is restored. Therefore, in the
present study, the anti-inflammatory actions of the novel SEGRAs
CpdA and ZK216348 in comparison to the common GC Dex
were investigated in intestinal epithelial cell lines. Furthermore,
with the aid of an in vitro wound healing model, their influences on
intestinal epithelial wound repair, a key process impaired under
GC treatment in IBD, and on the TGF-b and EGF/ERK1/2/
MAPK signalling pathway, were studied to reveal potential causes
for differences in intestinal wound healing under GC versus
Effects of CpdA and ZK216348 on nuclear translocation
GCs easily diffuse through the cell membrane to interact with
the GR, thereby inducing its activation and subsequent nuclear
translocation. To study the impact of SEGRAs on nuclear
translocation of GR in colon cells, the localisation of the GR
was investigated using immunofluorescence analysis. Immuno-
staining of non-GR-transfected Caco-2 cells revealed that both
CpdA and ZK216348 induced GR nuclear translocation similar to
that induced by Dex (Figure 1A and B). This was further
confirmed by Western blot analysis, where in the presence of Dex
and SEGRAs, the GR was predominantly localised in the nucleus
of Caco-2/GR cells, indicating Dex- and SEGRA-binding to, and
activation of, the GR. As described earlier for LNCaP-GR cells
, nuclear import in the presence of CpdA was found to be
reduced compared to Dex - an effect related rather to the
substance CpdA than to the class of SEGRAs in general, as
ZK216348 induced GR shuttling comparable to Dex. Further-
more, the reduced translocation in the presence of CpdA is not cell
line specific, as the same observation was made in HeLa cell lysates
(Figure 1C and D). Thus, CpdA and ZK216348 can be confirmed
to be functional GR agonists in Caco-2 cells.
Effects of CpdA and ZK216348 on GR-mediated
transcriptional suppression (trans-repression)
After activation and translocation of the GR to the nucleus, the
GR-mediated mechanism of trans-repression is constituted by
inhibition of activation of various transcription factors and
accounts for the beneficial anti-inflammatory effects of GCs.
Therefore the effect of Dex, CpdA and ZK216348 was evaluated
on the activity and expression of NF-kB, a key regulator of
inflammation, in intestinal epithelial cells. EMSA analysis with a
NF-kB consensus oligonucleotide revealed the binding of a TNF-
a/IL-1b-inducible complex in nuclear extracts of IEC-6 cells after
30 min of cytokine treatment. This complex contained mainly the
p65 subunit and less p50 subunit, as it was strongly supershifted by
anti-p65-, and less strongly by anti-p50-specific antibody.
Treatment with TNF-a/IL-1b plus Dex, CpdA or ZK216348
repressed the DNA-binding activity of NF-kB concentration-
dependently, indicating an inhibitory effect of Dex and SEGRAs
on transcription factor activity (Figure 2A). To exclude the
possibility of cell line-specific effects, the activation of NF-kB in
nuclear extracts of TNF-a-/IL-1b-stimulated Caco-2 cells was
determined using the TransAM NF-kB p65 kit. Upon cytokine
stimulation, a strong induction of p65 activation was observed,
which was significantly suppressed by Dex and both SEGRAs
Next, it was investigated whether the Dex- and SEGRA-
induced changes in NF-kB-DNA binding were associated with a
reduced nuclear translocation of NF-kB. Cytokine treatment of
Caco-2 cells was followed by the appearance of p65 protein within
the nuclear extracts, whereas Dex and SEGRA treatment
concentration-dependently decreased nuclear p65 expression.
Furthermore, in contrast to results seen with TNF-a/IL-1b
treatment alone, the presence of cytosolic IkB-a (inhibitor protein
of NF-kB) was preserved in Dex, CpdA and ZK216348 treatment,
suggesting that the decrease in nuclear p65 protein apparent with
Dex and SEGRA is paralleled by an attenuated degradation of
IkB-a (Figure 2C). The expression of cytokines involved in the
inflammatory process has been shown to be inhibited by GCs via
suppression of the activity of various transcription factors.
Interleukin-8 (IL-8) represents a classically NF-kB-regulated
cytokine, and it was thus evaluated whether SEGRAs were able
to suppress TNF-a-/IL-1b-stimulated IL-8 secretion. Indeed,
similar to those treated with Dex, Caco-2/GR cells stimulated
with TNF-a/IL-1b showed a concentration-dependent decrease of
cytokine-induced IL-8 secretion when treated with CpdA or
ZK216348 (Figure 2D). These data indicate that, following
inhibition of NF-kB activation and nuclear translocation, the
tested SEGRAs exert anti-inflammatory action comparable to that
Effect of CpdA and ZK216348 on GR-mediated
transcriptional activation (trans-activation)
In contrast to trans-repression, the mechanism of trans-
activation of target gene expression by the GR-ligand complex is
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org2 January 2012 | Volume 7 | Issue 1 | e29756
was measured by transcription factor assay for p65. Bars indicate
mean 6 S.E.M., n=3, *P#0.05, **P#0.01, ***P#0.001 relative
to vehicle or TNF-a, respectively. Concentration-dependent
trans-repression and –activation effects of CpdA and
ZK216348: Clearly, each of the three GR-agonists has a different
potency and therefore the pGRE-reporter gene assay was used to
test their trans-activation activity over a wider range of
concentrations. Cell treatment with higher concentrations of
Dex resulted in dose-dependent acceleration of relative luciferase
activity (Figure S1A). This was also observed for ZK216348
treatment (Figure S1A), which one could already suspect from the
data obtained and pictured in Figure 2 E. No induction of
luciferase activity was observed at higher CpdA concentrations
(Figure S1A), a result most likely attributable to CpdA’s
cytotoxicity and apoptosis inducing properties above 20 mM
(Figure 3). Both firefly and Renilla values were much lower in
concentrations .20 mM, so that after normalization, CpdA would
appear to have less trans-activation potential than Dex or
ZK216348 (Figure S1A). Similarly, employing the TransAMH
p65 Kit for NF-kB activation after cytokine stimulation in Dex- or
SEGRA-treated Caco-2 cells, no potentiating effect of CpdA could
be shown for evidently cytotoxic or apoptotic concentrations.
However, the treatments of cells with increasing concentrations of
ZK216348 lead to an increased inhibition of NF-kB activity
(Figure S1B). In summary, ZK216348 and especially CpdA, with
its additional apoptotic potential, possess a narrow therapeutic
window in which they act as ‘‘dissociating’’ GR ligands, i.e.
suppressing inflammatory effects without displaying GR-mediated
We thank Dr. H. Scha ¨cke, Bayer Pharma AG, Berlin for supplying us with
ZK216348 Dr. Ellen Preuß, Jutta Mu ¨ller, Roswitha Mu ¨ller for their
excellent technical assistance, Prof. Dr. Eberhardt and Prof. Dr.
Kippenberger for helpful discussion, support and providing access to
technical equipment and techniques and Janet Collins for carefully proof-
reading the manuscript.
Conceived and designed the experiments: KCR SML JS. Performed the
experiments: KCR SML. Analyzed the data: KCR SML. Contributed
reagents/materials/analysis tools: DS. Wrote the paper: KCR DS JS.
Critically reading and corrections of the manuscript: AUD.
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