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: email@example.com
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
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achieved by binding to DNA consensus sequences (GC response
elements (GREs)). This is thought to be a contributing factor in
the numerous side effects of GCs, as the expression of proteins
involved in metabolic processes is increased by this mechanism.
Therefore, with the aid of a reporter gene assay, the ability of
SEGRAs to induce luciferase activity of a GRE-driven promoter
construct (pGRE-Luc) transiently transfected to Caco-2/GR cells
was studied. Treatment with Dex resulted in an eight-fold
increase in luciferase activity, which was completely abolished by
pre-treatment with RU-486, a non-selective GR and PR
antagonist. This strong induction was not observed either of the
SEGRAs. Although, interestingly, CpdA revealed no effect in
tested concentrations, ZK216348 treatment led to the concen-
tration-dependent acceleration of luciferase expression. This
could be explained by the binding affinity of ZK216348 to other
nuclear receptors, such as progesterone (PR) or mineralcorticoid
receptor (MR) , capable of inducing luciferase response by
binding to the GRE motif. This assumption is underscored by the
fact that the ZK216348-effect is absent in the presence of RU-
486 (Figure 2E). Moreover, in Caco-2/GR cells treated with
CpdA or ZK216348, the expression of MAPK phosphatase
(MKP-1) and Annexin-1, which expression is known to be
initiated by the GR-mediated trans-activation mechanism, was
found to be significantly reduced compared to Dex (Figure 6D
and E). These results suggest that in the presence of the GR
ligands, CpdA and ZK216348, the induction of gene transcrip-
tion via the trans-activation mechanism is less pronounced than
Figure 1. Effect of SEGRAs on GR binding and nuclear translocation. (A) Immunofluorescence analysis for GR location in Caco-2 cells. Cells
were treated for 3 h with Dex [1 mM], CpdA or ZK216348 [10 mM]. DAPI staining was used for visualisation of the cell nuclei. (B) 100 cells were
randomly chosen, analysed and the percentage of nuclear GR calculated. Western blot analysis for translocation of GR in (C) Caco-2/GR and (D) HeLa
cell lysates after 3 h cultivation with Dex or SEGRAs. One representative blot of three is shown. Densitometric analysis of GR is normalised to b-actin
(cytoplasmic extract) or lamin (nuclear extract), respectively. Bars represent mean 6 S.E.M., n=3, *P,0.05; **P,0.01 relative to vehicle.
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Figure 2. Effect of SEGRAs on transcriptional activity of GR. Cells were pre-treated with Dex [0.1–1 mM], CpdA or ZK216348 [1–10 mM] for 1 h
before cultivation in the co-presence or absence of TNF-a/IL-1b [0.5 nM] and harvested for cytoplasmic and nuclear protein extract preparations. (A)
The activity of NF-kB in nuclear extracts of IEC-6 cells was analysed by EMSA using an end-labelled NF-kB consensus oligonucleotide. The EMSA is
representative for two experiments giving similar results. (B) NF-kB activity in nuclear extracts of Caco-2 cells was measured by transcription factor
assay for p65. (C) Western blot analysis for nuclear p65, or cytoplasmic Ik-Ba expression in TNF-a-/IL-1b-stimulated Caco-2 cell extracts. One
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Apoptotic and cytotoxic effects of CpdA and ZK216348
in Caco-2 and IEC-6 cells
Apoptosis is a central mechanism for the maintenance of
homeostasis and it has already been shown that GCs have a
regulative function in the apoptotic mechanisms of certain cell
types . Furthermore, the findings of Wuest et al.  and our
in vivo experiments in the mouse model of acute TNBS-mediated
colitis (unpublished data) have shown CpdA to have a narrow
therapeutic window. The proven profound toxicity in higher doses
was attributed to the formation of highly reactive breakdown
products (aziridines) of CpdA and from these following subsequent
apoptotic and cytotoxic potential. For this reason, the apoptotic
effects of CpdA and ZK216348 were assessed in vitro in the
intestinal epithelial cell lines used for our experimental setup. After
24 h treatment of IEC-6 cells with Dex, a concentration-
dependent increase of apoptosis could be confirmed by FACS
analysis (Figure 3A). The treatment of cells treated with
ZK216348 at concentrations of 1–20 mM showed no effect on
cell apoptosis; while CpdA concentrations $15 mM induced cell
death (Figure 3B). The results of cytotoxicity testing in Caco-2 cells
representative blot of three is shown. (D) IL-8 content of cell culture supernatants was quantified by ELISA. Caco-2/GR cells were pre-treated with Dex
[0.1–1 mM], CpdA or ZK216348 [1–10 mM] for 1 h before cultivation for 16–24 h in the co-presence or absence of TNF-a/IL-1b [0.5 nM]. Bars indicate
mean 6 S.E.M., n=4, *P#0.05; **P#0.01; ***P#0.001 relative to TNF-a/IL-1b treatment. (E) Relative Luciferase Activity of Caco-2/GR cells transfected
with the glucocorticoid response element (GRE)-driven luciferase construct (pGRE-luc) and pSV-40 Renilla after 24 h of treatment with or without Dex
[1 mM], CpdA or ZK216348 [1–10 mM] or co-presence of the GR agonist RU-486 [10 mM] given as 1 h pre-treatment. Bars indicate mean 6 S.E.M., n=3,
***P#0.001 relative to vehicle,###P#0.001 relative to Dex- or ZK216348-treatment, respectively.
Figure 3. Effect of SEGRAs on cell apoptosis. Apoptotic effects of (A) Dex- [0.1–10 mM] or (B) CpdA- and ZK216348- [1–20 mM] treatment on IEC-
6 cells was evaluated after 24 h by Annexin-/7-AAD staining and flow cytometric analysis. (C) Cytotoxicity of CpdA or ZK216348 [1–25 mM] treatment
on Caco-2 cells was tested by Cytotoxicity Detection (LDH release) kit. Bars indicate mean 6 S.E.M., n=3, *P#0.05, **P#0.01, ***P#0.001 relative to
vehicle. (D) Activation of Caspase-3 in Caco-2 cells after 24 h of incubation with Dex, CpdA and ZK216348 [1–25 mM]. Results are expressed as a
percentage of control. Bars indicate mean 6 S.E.M., n=3, *P#0.05, **P#0.01, ***P#0.001 relative to vehicle.
SEGRAs and Intestinal Epithelial Repair
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were in line with the FACS analysis for IEC-6 cells, where LDH
activity in presence of increasing CpdA or ZK216348 concentra-
tions was not elevated until SEGRA concentrations of $15 mM
(Figure 3C). Similarly, the effector Caspase-3 was activated 24 h
after stimulation with CpdA concentrations higher than 15 mM,
while neither CpdA ,15 mM nor increasing Dex or ZK216348
concentrations showed significant changes in Caspase-3 activation
(Figure 3D). Taken together, evidence points to a narrow
therapeutic window of CpdA and its pro-apoptotic and cytotoxic
effects in higher concentrations complicates the evaluation and
comparison of GR-mediated trans-activation and trans-repression
effects in a wider range of concentrations (see Figure S1).
Nevertheless, in Caco-2/GR cells after exposure to 10 mM CpdA
or ZK216348, the concentration in which anti-inflammatory
effects comparable to the common GC Dex are observed, no
significant apoptosis induction, cytotoxicity or increased Caspase-3
activity compared to non-treated cells occur, indicating that the
relevant effective SEGRA concentrations utilized in our experi-
ments were not affecting cell viability of intestinal epithelial cells.
Effects of CpdA and ZK216348 on intestinal epithelial cell
GCs have been shown to impair intestinal wound healing, thus
preventing the full restoration of normal host homeostasis
[18,19,20]. Usually, after superficial injury, the first step in
mucosal healing involves the migration of epithelial cells across the
mucosal defect to the wound area, a process termed restitution.
Migration is followed by proliferation, to increase the cell pool
available for wound resurfacing. To determine the effects of Dex,
CpdA and ZK216348 on intestinal epithelial cell migration, a
well-established in vitro wound healing model was performed,
utilising the non-transformed rat small-intestinal epithelium cell
line (IEC-6). After 24 h, the presence of Dex significantly impeded
the migration of IEC-6 cells into the wounded area in a
concentration-dependent manner, compared to control cells
cultured in medium alone (Figure 4A). In contrast to Dex, CpdA
and ZK216348 did not inhibit wound closure, (Figure 4B, 4C and
4E). Additional evidence that SEGRAs do not exert a negative
influence on cell migration was gained using the skin derived
keratinocyte cell line HaCaT was utilised in the same in vitro
wound healing model. Results were similar to those attained using
IEC-6 cells, indicating that, while Dex, concentration-dependently
inhibits wound closure, this is not the case with CpdA or
ZK216348 (Table 1).
Effects of CpdA and ZK216348 on intestinal epithelial cell
After it was shown that Dex modulates intestinal epithelial
migration, whereas SEGRAs do not, the effects of the three
substances on epithelial cell proliferation, the subsequent step to
migration in wound healing, was studied. After 24 h, Dex and
ZK216348 showed no significant effect on BrdU-incorporation
into DNA of IEC-6 cells at concentrations between 0.1 mM and
20 mM. While this was also true of CpdA in concentrations
between 0.1–10 mM (Figure 4D) a decrease in cell proliferation
was observed above 10 mM, which is in accordance with its
examined apoptotic and cytotoxic effects in concentrations higher
than 15 mM (see Figure 3). Nevertheless, it can be ruled out that
the beneficial effects of CpdA, at least below 15 mM, and of
ZK216348, seen in the in vitro wound healing assay, are due to
substance influences on cell proliferation. Hence, within these in
vitro assays, the novel SEGRAs displayed no negative influence on
intestinal epithelial cell restitution and proliferation, a fact which,
in combination with their anti-inflammatory properties, might give
them the advantage over common synthetic GCs, as, along with
the attenuation of inflammation, mucosal healing is defined as an
important goal of IBD management .
Effects of CpdA and ZK216348 on TGF-b - mediated
intestinal epithelial cell restitution
Several cytokines and chemokines expressed in the intestinal
mucosa promote epithelial restitution after injury through
increased production of bioactive TGF-b1 in epithelial cells. To
check if Dex or SEGRAs modulate intestinal epithelial migration
by TGF-b, or its pathway, the migration properties of IEC-6 cells
in the presence or absence of exogenously-added TGF-b or
SB431542, a selective inhibitor of activin receptor-like kinase
(ALK) receptors, was investigated. As expected, both TGF-b alone
and TGF-b in combination with CpdA or ZK216348 increased
IEC-6 wound closure compared to vehicle treatment. Interesting-
ly, TGF-b in the presence of Dex could only partially reverse the
inhibitory effect of Dex on intestinal epithelial migration
(Figure 5A). The blockade of TGF-b receptor brought about by
the addition of SB431542 significantly decreased cell migration
into the wounded area and further intensified the already
inhibitory effect of Dex. In the co-presence of CpdA or
ZK216348, however, no difference in epithelial cell migration
was found in comparison to that seen in SB431543 treatment
alone, suggesting a different modulation of the TGF-b-dependent
promotion of cell migration (Figure 5B).
The binding of TGF-b to its receptor initiates the intracellular
signalling machine via Smad proteins, and transcription is started
by the binding of the smad3/4 complex to smad binding elements
(SBE) in the promoter of TGF-b-sensitive genes. Indeed in
HEK293T cells transiently transfected with a reporter gene
construct carrying four SBEs (SBE4-Luc) the addition of TGF-b
induced luciferase activity up to 5-fold, while neither the addition
of Dex nor CpdA or ZK216348 showed any effect compared to
vehicle control (Figure 5C). Although TGF-b peptide levels and
mRNA expression (Figure 5D and E) were decreased after Dex-
treatment, CpdA and ZK216348 revealed no modulatory effects,
hinting toward a TGF-b-independent mechanism for unrestricted
wound closure in the presence of SEGRAs.
Effects of CpdA and ZK216348 on EGF/ERK1/2/MAPK
pathway-mediated intestinal epithelial cell restitution
In addition to TGF-b, the extracellular stimulus EGF is also
known as a strong enhancer of epithelial cell restitution [24,33,34]
and triggers the ERK1/2/MAPK pathway, which is linked to cell
migration and proliferation. Thus, the migration properties of
IEC-6 cells in the presence or absence of exogenously added EGF
and PD98059, a selective inhibitor of MEK1 phosphorylation
upstream of ERK1/2, were examined, to see if Dex or the
SEGRAs CpdA and ZK216348 modulate intestinal epithelial
migration by this pathway. Indeed, EGF alone and in combination
with CpdA and ZK216348, increased wound closure significantly.
If cells were incubated with Dex in combination with EGF, EGF
was able to compensate the inhibitory effect of Dex on intestinal
epithelial migration that was observed with Dex alone (Figure 6A).
Furthermore, the blockade of ERK1/2 phosphorylation by
addition of PD98059 diminished wound closure, alone or in the
co-presence of Dex, CpdA and ZK216348, underscoring the
importance of this pathway in epithelial repair (Figure 6B).
GCs have been shown to interfere with the ERK1/2/MAPK
pathway and amongst other a decreased ERK1/2 phosphoryla-
tion was observed. Western blot analysis of the ERK1/2
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phosphorylation status of Caco-2/GR cells after treatment with
Dex or SEGRA indeed revealed reduced ERK1/2 phosphoryla-
tion when cells were exposed to Dex, but not CpdA or ZK216348
(Figure 6C). It has been shown that this ERK1/2 phosphorylation
is blocked by MKP-1, whose expression is typically initiated by the
trans-activation mechanism of GCs [35,36]. In comparison to
vehicle-treated Caco-2/GR cells, Dex-treated cells revealed an up-
regulation in MKP-1 mRNA levels, while CpdA and ZK216348,
as SEGRAs, showed no significant induction of MKP-1 expression
(Figure 6D). The same was true for expression of Annexin-1
mRNA and protein (Figure 6D and E), likewise a well-known
trans-activated gene and competitor for the phosphorylation of
Figure 4. Effect of SEGRAs on intestinal epithelial cell restitution and proliferation. IEC-6 cells were wounded and cultured in the presence
of (A) Dex [0.1–1 mM] or (B) CpdA or (C) ZK216348 [1–20 mM] for 24 h. Cell migration was assessed using an in vitro migration assay. Bars indicate
mean values of remaining wounded area 6 S.E.M., n=3, *P#0.05, **P#0.01 relative to control. (D) BrdU incorporation assay was used for
determination of IEC-6 cell proliferation after 24 h incubation with Dex or SEGRAs [1–20 mM]. Results indicate mean 6 S.E.M., n=3, ***P#0.001
relative to vehicle. (E) Representative experiments illustrating the effects of Dex and SEGRAs on intestinal epithelial cell restitution. Cntr (0 h)
represents cells immediately after wounding, picture cntr (24 h) and others show wounds 24 h after cultivation with Dex, CpdA or ZK216348. Dotted
line indicates original margin of wound (Magnification 6100).
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EGF receptor (EGFR), which in turn was blocked in the presence
of Dex but not SEGRAs (Figure 6F). Addition of the GR
antagonist RU-486 reversed the Dex-mediated induction of
Annexin-1, therefore indicating the GR-dependency of the
increase of Annexin-1 expression (Figure 6E). These data suggest
that the difference in the inhibitory effects of Dex and the
SEGRAs CpdA and ZK216348 on wound closure might be a
result of their different interference with the EGF/ERK1/2/
MAPK pathway, again pointing towards a clear advantage for
SEGRAs in IBD treatment.
Glucocorticoids are highly effective in combating inflamma-
tion, 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.
The efficacy of GCs in diminishing inflammation, results from
the pleiotropic effects of their receptor (the activated GR) on
multiple signalling pathways. Within this study, the SEGRAs
CpdA and ZK216348 were confirmed as functional GR agonists
in intestinal cells, as GR activation and transmigration to the cell
nucleus occurred following application of CpdA and ZK216348.
After translocation, several models for transcriptional modulation
by the GR have been presented. It is widely accepted that the
trans-repressive mechanism is responsible for the beneficial anti-
inflammatory effect of GCs, while their side effects are thought to
result from the transcriptional activation of genes. Indeed, within
this study CpdA and ZK216348 have shown desirable trans-
repressive, and therefore anti-inflammatory, action in intestinal
epithelial cells by inhibiting the activity of NF-kB, a transcription
factor associated with numerous pro-inflammatory genes in IBD
. Moreover, the expression of the NF-kB-driven cytokine IL-8
in TNF-a-/IL-1b-stimulated cells was significantly reduced and
the anti-inflammatory action of the novel SEGRAs, CpdA and
ZK216348 found to be comparable to that of Dex. Furthermore,
both SEGRAs induced trans-activation of GRE-containing
promoter-reporter constructs to a lesser extent than Dex, and
expression of the typically GRE-driven genes, MKP-1 and
Annexin-1, was induced neither by CpdA nor ZK216348, leading
to the conclusion, that in intestinal epithelial cells also, CpdA and
ZK216348 show dissociative properties attributed to the class of
Previous studies applying CpdA and ZK216348 in vitro and in
vivo focused on several side effects associated with GC-treatment
such as GC resistance  or hyperglycaemia [13,14,15,16,40]. In
addition to these side effects, GCs are known inhibitors of wound
healing [20,29,41], which considerably limits their therapeutic
application in various inflammatory conditions. The mechanism
behind the inhibitory effect of GCs on tissue repair is mainly
subscribed to the ability of GCs to regulate the expression of
several key proteins at the wound site , but the modulation of a
range of other physiological processes such as metabolism,
migration, cell proliferation and differentiation also plays an
important role [18,19,41].
In the context of IBD, where the continuity of the mucosal
epithelial surface constitutes a key requisite for combating the
inappropriate and ongoing activation of the immune system
[20,42], impaired tissue repair under GC therapy represents a very
drastic side effect. In the present study, a dose-dependent
inhibition of intestinal epithelial restitution by Dex was observed,
consistent with results described for budesonide and prednisolone
[18,19]. Strikingly, neither CpdA nor ZK216348 showed negative
effects on intestinal epithelial migration or proliferation. Thus, a
beneficial effect for SEGRAs in wound healing can be stated. This
is further supported by the absence of inhibitory effects on
migratory capacity of the skin-derived keratinocyte cell line
HaCaT. Moreover, the present results confirm the conclusion
drawn by Grose et al., who studied the role of endogenous GCs in
wound repair in mice harbouring the DNA-binding-defective
mutant version of the GR (GRdim), and observed improved wound
healing compared to wild type mice. While unfamiliar with the
class of SEGRAs, the authors suggested the use of more specific
GCs as advantageous therapeutic modalities for wound healing
Table 1. Effect of SEGRAs on HaCaT cell restitution.
Dex wound closureCpdA wound closureZK216348wound closure
[mM][%][mM] [%][mM] [%]
- 65.8962.64- 66.062.38- 63.8262.31
0.0161.6363.030.167.0664.15 0.158. 9364.00
0.05 52.2562.74 *1 64.4162.171 59.9363.16
0.146.2665.08 **565.1362.975 60.9264.58
0.5 38.1062.79 ***10 61.2163.4810 62.1464.34
1 27.8061.64 *** 2064.9462.36 2057.3665.07
HaCaT cells were wounded and cultured in the presence of Dex, CpdA or ZK216348 for 24 h. Cell migration was assessed using an in vitro migration assay. Data indicate
mean values of remaining wounded area 6 S.E.M., n=3,
***P#0.001 relative to control.
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org8 January 2012 | Volume 7 | Issue 1 | e29756
Further experiments were conducted to identify the differen-
tially modulated mechanism of GCs and SEGRAs in intestinal
wound healing. A central role has been highlighted for the wound
healing promoting factor TGF-b, as many of the regulatory
peptides have been demonstrated to enhance epithelial restitution
through a TGF-b-dependent mechanism. Additionally, the
improved wound healing seen in GRdimmice, could be linked to
enhanced fibroblast secretion of TGF-b . It therefore seems
conceivable that the unrestricted intestinal wound closure
observed under treatment with the SEGRAs CpdA and
ZK216348 might be due to a different modulation of TGF-b or
its signalling pathway. However, in line with previous studies using
the GCs prednisolone and budesonide, addition of TGF-b only
partially reversed the Dex-mediated inhibition of cell restitution,
and other than an inhibitory effect of Dex on TGF-b levels in IEC-
6 cells, no significant changes in the TGF-b signalling pathway for
CpdA or ZK216348 could be found.
This implicates the involvement of a non-Smad TGFb-induced
signalling mechanism in epithelial wound closure that comple-
ments the canonical Smad pathway and might be differently
modulated by GCs and SEGRAs. With respect to mucosal
healing, one of the regulatory peptides, EGF, likewise a strong
enhancer of epithelial cell restitution [24,33,34], and its signalling
via the EGF/ERK1/2/MAPK pathway, are of particular interest,
Figure 5. Effect of SEGRAs on TGF-b - mediated intestinal epithelial cell restitution. Wounded IEC-6 cells were cultured for 24 h in the
presence of Dex, CpdA or ZK216348 with or without co-presence of (A) TGF-b or (B) selective inhibitor of activin receptor-like kinase (ALK) receptor
SB431542. Cell migration was assessed using an in vitro migration assay. Bars indicate mean values of remaining wounded area 6 S.E.M., n=3,
*P#0.05, **P#0.01, ***P#0.001 relative to vehicle,###P#0.001 relative to Dex (C) Relative luciferase activity of HEK293T cells transfected with the
reporter gene construct pSBE4-luc construct after 24 h incubation with TGF-b (5 ng/ml), Dex or SEGRAs. IEC-6 cells were treated with Dex, CpdA or
ZK216348 for the indicated time periods. (D) TGF-b peptide levels in cell culture supernatants were determined by ELISA. (E) TGF-b mRNA expression
was monitored by qPCR and normalised against b-actin. Bars represent mean 6 S.E.M., n=3, *P#0.05 relative to vehicle.
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org9 January 2012 | Volume 7 | Issue 1 | e29756
as its downstream targets are involved in cellular events such as cell
motility and proliferation [28,44], and the influence of GCs upon
the EGF/ERK1/2/MAPK pathway is also well documented
[45,46]. Indeed, addition of exogenous EGF in combination with
Dex led to a complete restoration of the Dex-mediated decrease of
restitution in the utilised in vitro wound healing model. Also, in
correlation with the ERK1/2 and EGFR phosphorylation status
expression of MKP-1 and Annexin-1 was significantly induced by
Figure 6. Effect of SEGRAs on EGF/ERK1/2/MAPK pathway - mediated intestinal epithelial restitution. Wounded IEC-6 cells were
cultured for 24 h in the presence of Dex [1 mM], CpdA or ZK216348 [10 mM] with or without co-presence of (A) EGF (5 nM) or (B) specific inhibitor of
MEK1/2 phosphorylation PD98059 [50 mM]. Cell migration was assessed using an in vitro migration assay. Bars indicate mean values of remaining
wounded area 6 S.E.M., n=3, *P#0.05, **P#0.01, ***P#0.001 relative to vehicle,###P#0.001 relative to Dex. (C) Western blot analysis for pERK1/2
and ERK2 in Caco-2/GR cell lysates treated with Dex [1 mM], CpdA or ZK216348 [10 mM] for 1 h. One representative blot of three is shown and
densitometric analysis of pERK1/2 is normalised to ERK2. (D) MKP-1 or Annexin-1 mRNA expression in Caco-2/GR cells after 6 h of treatment was
monitored by qPCR and normalized against b-Actin. Western blot analysis for (E) Annexin-1 or (F) EGF-R protein expression in Caco-2/GR cells treated
for 24 h or 1 h respectively with Dex or SEGRAs in the presence or absence of RU-486 [10 mM]. One representative blot of three is shown. Bars
indicate mean 6 S.E.M., n=3, *P#0.05, **P#0.01, ***P#0.001 relative to vehicle.
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29756
Dex but not by CpdA or ZK216348. Taken together, these
findings indicate the involvement of the EGF/ERK1/2/MAPK
signalling cascade in the Dex-mediated inhibition of intestinal
epithelial wound closure in vitro. More important, however, is the
fact that neither CpdA nor ZK216348 induce the expression of
MKP-1 and Annexin-1, and thus in their presence the signalling
cascade is still active and wound healing promoted. These data
again match the study in GRdimmice, in which improved wound
healing in comparison to wild type mice suggests that the
inhibitory effect of exogenous GCs is at least to some extent
mediated by the DNA binding activity of the GR (trans-activation
The ability of CpdA and ZK216348 to dissociate between trans-
repression and trans-activation in various inflammatory models
has been shown previously, but for the first time this is here
demonstrated in the context of IBD, where it has successfully been
shown that not only do these SEGRAs have anti-inflammatory
properties, but in addition, they do not negatively interfere with
the process of intestinal epithelial wound healing. However, for
future therapeutic use, a close characterisation of SEGRAs
pharmacology and mode of action is mandatory, e.g. with regard
to substance safety, as the apoptotic and cytotoxic potential of
CpdA in particular results in a narrow therapeutic window .
Nevertheless, CpdA and ZK216348 are suitable model substances
and we suggest that the concept of SEGRAs offer a promising
perspective in the treatment of IBD, where a strong preference is
given to inflammatory gene repression, and where reduction of
GC-linked side effects, e.g. impaired wound repair (mucosal
healing) is of considerable importance.
Materials and Methods
Cell culture and Materials
IEC-6, HeLa, Caco-2, HEK293T and HaCaT cells were
purchased from the Deutsche Sammlung fu ¨r Mikroorganismen
und Zellkulturen (DSMZ, Braunschweig, Germany) and main-
tained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen)
with 10% fetal calf serum (FCS, PAA) 100 U/ml penicillin and
100 mg/ml streptomycin (PAA). For cultivation of Caco-2, this
medium was supplemented with 1% sodium pyruvate (PAA).
HaCaT cells were kept in DMEM high glucose (Invitrogen) with
10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and
2 mM Glutamine (PAA). Stock solutions were prepared by
dissolving Dex (SIGMA-Aldrich) in water, CpdA (Alexis Bio-
chemicals) in PBS (pH 6.0), ZK216348 (kindly provided by Dr.
Scha ¨cke, Bayer Pharma AG, Berlin) in DMSO, all at a
concentration of 10 mM. RU-486 (SIGMA-Aldrich), SB431542
and PD98095 (Calbiochem) were dissolved in DMSO at 10 mM.
For experiments, the cells were seeded on plastic cell culture wells
and allowed to attach for 24–48 h. Prior to Dex or SEGRA
stimulation, cells were cultured in medium containing 1% FCS for
16 h. Dex, SEGRAs and inhibitors, whenever used, were given in
pre-incubations 1 h before addition of other reagents. For
subsequent stimulation, recombinant human TNF-a, IL-1b,
EGF and TGF-b (all PreproTech) were dissolved in water/1%
bovine serum albumin (BSA) and added to the medium to their
final concentrations. Unless otherwise stated, all chemicals were
obtained from Sigma-Aldrich.
Cloning of pGR-FL & pGRE-Luc backbone
An expression plasmid for the human full-length glucocorticoid
receptor (pGR-FL) was generated by cloning GR from HepG2
genomic DNA by PCR using the following primers: GR-FL-for:
59-CGCGGATCCATGGACTCCAAAG-39, GR-FL-rev: 59-CG-
CTCTAGATCACTTTTGATGAAACAG-39 (Eurofins). The re-
sulting PCR fragment was inserted in the pCDNA3 vector
(Invitrogen) after its digestion with BamHI and XbaI enzymes
(Fermentas). The functionality of GR expression was monitored by
Western blot. For the pGRE-Luc backbone construct, the GRE-
enhancer was removed by digestion of pGRE-Luc (reporter gene
construct for GRE-driven luciferase expression, kindly provided by
Prof. Schulzke, Berlin) with NheI and BglII. A custom-made
phosphorylated linker lacking GREs (Eurofins; Forward: 59 –
CTAGCATCGGATCA -39, Reverse: 59- GTAGCCTAGTC-
TAG -39) was inserted before the PTAL-Promoter region.
Transfection and Reporter Gene Assay
Caco-2 cells were transiently transfected with the GR expression
plasmid pGR-FL alone (200 ng, referred to as Caco-2/GR) or like
HEK293T cells, together with the indicated amount of reporter
gene constructs using Lipofectamine 2000 reagent (Invitrogen)
according to manufacturer’s instructions. Briefly, for the reporter
gene assay, cells were seeded in 24-well plates in cell line specific
medium without phenol red. The following plasmid concentra-
tions were used for transfection: 100 ng pGR-FL, 700 ng pGRE-
Luc and 50 ng pSV-40 Renilla as internal standard for Caco-2,
200 ng of the reporter gene construct pSBE4-Luc containing
46SBE (Smad binding elements, generously provided by B.
Vogelstein, Baltimore, USA) and 50 ng pSV-40Renilla for
HEK293T. The medium was changed after 5 h of transfection
and cells were incubated for another 16 h before being treated as
indicated. Reporter gene activity was assayed with the Dual-
Luciferase Reporter Assay System (Promega) and a TECAN
infiniteH M200 Luminometer. Data are shown as relative light
units (RLU) as a percentage of control, normalised to transfection
efficiency (co-transfection of pSV-40-renilla) and normalised to
effects of the respective empty vectors (pCDNA3, pGRE-Luc
backbone construct, pGL3basic or pCGN).
Caco-2 cells plated on sterile chamberslides were treated for
3 h with Dex, SEGRAs or vehicle. The cells were fixed for
20 min with 40% aceton/60% methanol at 220uC and kept in
blocking buffer (3% BSA/PBS/0.1% Tween-20) for 30 min at
room temperature. Subsequent incubation with anti-GR (Santa-
Cruz Biotechnology) diluted in blocking buffer for 30 min at
37uC, and three washing steps with 0.1% Tween-20/PBS/0.1%
BSA, secondary antibody CyTM3-conjugated goat-anti-rabbit
IgG (Zymed) diluted in blocking buffer was applied for 30 min
at room temperature. The slides were counterstained with 49,6-
Diamidino-2-phenylindole (DAPI, Vector Laboratories) mount-
ing medium after another washing and analysed using an
Olympus IX71 microscope at 1006magnification. To quantify
translocation, 100 randomly-chosen cells per treatment were
analysed and processed using GSA Image Analyzer. The
percentage of GR translocation was calculated by dividing the
area of pink (merge of CyTM3 and DAPI) by blue fluorescence
(DAPI) per field.
Electrophoretic mobility shift assay (EMSA)
Consensus oligonucleotide for NF-kB (sc-2505) was obtained
from SantaCruz Biotechnology and endlabeled by polynucleotide
kinase (PNK) using a-ATP (3000 Ci/mM). Binding reactions were
performed for 30 min on ice with 5 mg of IEC-6 nuclear extract
protein as described previously . Polyclonal antibodies used for
supershift experiments were purchased from SantaCruz Biotech-
nology and were added 15 min after addition of the labelled probe
and co-incubated on ice for a further 30 min.
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org11 January 2012 | Volume 7 | Issue 1 | e29756
Cytoplasmic/nuclear extract preparation and NF-kB
Cytoplasmic and nuclear extracts from IEC-6, Caco-2 or HeLa
cells were prepared using the nuclear extraction kit from Active
motif (Active motif) following the manufacturer’s instructions.
Nuclear NF-kB/p65 activity was measured with TransAMH NF-
kB p65 kit (# 40096, Active motif).
Protein extract preparation and Western blot analysis
For the isolation of whole cell extracts, cells were harvested at
4uC in Cell lysis buffer (Cell Signaling) containing multiple
protease inhibitors (Complete MiniH, Roche). Soluble protein
extracts were obtained after sonication of crude lysates and
centrifugation at 10 000 rpm for 10 min at 4uC. Samples were
separated by sodium dodecylsulfate – polyacrylamid gelelectro-
phoresis (SDS-PAGE) and transferred onto nitrocellulose mem-
branes (Hybond C, Amersham) following standard protocols. After
blocking, membranes were incubated overnight at 4uC with the
indicated primary antibodies: anti-GR (sc-8992), anti-p65 (sc-372),
anti-IkB-a (sc-371), anti-EGFR (sc-03) and anti-pEGFR (sc-12351)
(all from SantaCruz Biotechnology) and anti-p42/44 (#4695),
anti-p-p42/44 (#4377S) and anti-Annexin-1 (#3299S) (all from
Cell Signalling) followed by infrared dye-conjugated secondary
antibodies (LI-CORH Biosciences). Bound complexes were
detected with the OdyseeyH infrared imaging system (LI-CORH).
For quantification, the intensities of the total protein were
normalised to b-actin (#A2228, SIGMA-Aldrich) or lamin
(#2032, Cell Signaling) signal.
IEC-6 cells were incubated for 24 h in the presence or absence
of Dex, CpdA or ZK216348 and harvested with 1%-Trypsin-
EDTA (PAA), washed twice with ice-cold PBS, and suspended in
binding buffer as instructed by the manufacturer (Apoptosis
detection kit #559763, BD Biosciences). Aliquots of cells (100 ml)
were incubated with Annexin V/7-AAD and 100 ml counting
beads (CALTAGTMCounting beads; Invitrogen). Data acquisition
(10 000 beads) and analysis were performed in a FACS analyser
(FACSCanto, BD Biosciences).
Caspase-3 activity assay
Caco-2 cells were seeded in 6-well dishes and allowed to reach
confluency. Caspase-3 activity was analysed after 24 h of
incubation with Dex or SEGRAs using a fluorometric immuno-
sorbent enzyme assay (#12 012 952 001, Roche) according to the
manufacturer’s instructions with the following modification: All
cells per treatment were harvested and cell lysate used for Caspase-
3 activity assay. After fluorometrical determination of free
fluorescent AFC, total protein concentration of the samples was
measured and adapted to the activity to correct for variation in
Cytotoxicity and Cell proliferation assay
Cells were cultured at a density of 0.016106cells in 96-well
dishes, allowed to attach over night and treated for 24 h with
different concentrations of Dex or SEGRAs as indicated.
Cytotoxicity was determined by measuring lactate dehydrogenase
(LDH) release using a commercially available kit (Cytotoxicity
detection kit (LDH), Roche) following the manufacturers instruc-
tions. Proliferation of IEC-6 cells was determined by analysing 59-
Bromo-29-Deoxy-uridine (BrdU) incorporation into newly synthe-
sised DNA using a cell proliferation enzyme linked immunosor-
bent assay (ELISA) (Cell proliferation ELISA (BrdU), Roche).
Migration (Restitution) Assay
An epithelial cell wound healing model was performed using a
modified version of the previously described techniques [18,48].
Confluent monolayers of IEC-6 or HaCaT cells were wounded in
a standardised procedure. Three independent wounds (,20–
25 mm, horizontal) per dish were established with a sterile pipette
tip and areas marked with a sterile razor blade (vertical). After
scraping, cells were washed with fresh medium and wounds
photographed (Sony, DSC-S75) at 100-fold magnification (Ax-
iovert 135, Carl Zeiss) using an ocular reticle, which allowed clear
recovery of the photographed area. Cells were cultured for a
further 24 h in fresh, serum-deprived medium in the presence or
absence of Dex or SEGRAs, individually or in combination with
EGF, TGF-b, SB431542 or PD98059. Hereafter, wound areas
were photographed again and migration of cells was assessed by
comparing the calculated (WEGA-Image Viewer, M.O.S.S.)
wound area before and after 24 h. At least 8 wounded areas per
dish were analysed and three independent experiments performed.
IL-8 and TGF-b ELISA
Cells were seeded in 6-well dishes and treated as described. The
concentration of Interleukin 8 (IL-8) in the supernatants was
measured by commercially available QuantikineH ELISA kit
(R&D Systems). In IEC-6 supernatants for the determination of
total (latent plus bioactive) TGF-b, cell culture supernatants were
first activated by acidification with 1 N HCl for 10 min, followed
by neutralisation with 1.2 N NaOH/0.5 M HEPES at room
temperature. TGF-b concentration was determined using the rat
TGF-b QuantikineH ELISA (R&D Systems). The ELISAs were
performed according to the manufacturer’s instructions.
Isolation of Total RNA and qPCR
For isolation of total RNA, cells were purified with TRIzolH
(Invitrogen) according to the manufacturer’s instructions. 2 mg of
total RNA underwent DNAse I digestion (Fermentas) and
subsequently were subsequently reverse transcribed using the
iScript cDNA Synthesis kit (Bio-Rad). Real-time PCR for gene
expression of MKP-1, Annexin-1 or rTGF-b was performed in
cDNA (diluted 1 : 5) using the Power SYBR Green PCR master
mix with the AB StepOnePlus (Applied Biosystems) Sequence
Detector system under the standard protocol. Human or rat b-
Actin was used to normalise the results. The relative mRNA
expression of each studied gene was calculated with the
comparative DCt method using the formula 22DDCt.
The data are expressed as means 6 S.E.M. Analysis of variance
(ANOVA) was performed when more than two groups were
compared, and when significant (P,0.05), multiple comparisons
were performed with the Tukey test. A P value#0.05 was
considered to be significant.
repression effects of SEGRAs in different concentra-
tions. (A) Relative Luciferase Activity of Caco-2/GR cells
transfected with the glucocorticoid response element (GRE)-driven
luciferase construct (pGRE-luc) and pSV-40 Renilla after 24 h of
treatment with or without Dex [1–50 mM], CpdA or ZK216348
[1–50 mM]. (B) Caco-2 cells were pre-treated with Dex [0.1–
10 mM], CpdA or ZK216348 [1–50 mM] for 1 h before 15 min
cultivation in co-presence or absence of TNF-a [0.5 nM] and
harvesting for nuclear protein extract preparations. NF-kB activity
Comparison of trans-activation and trans-
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org12January 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.
1. Summers RW, Switz DM, Sessions JT, Jr., Becktel JM, Best WR, et al. (1979)
National Cooperative Crohn’s Disease Study: results of drug treatment.
Gastroenterology 77: 847–869.
2. Kozuch PL, Hanauer SB (2008) Treatment of inflammatory bowel disease: a
review of medical therapy. World J Gastroenterol 14: 354–377.
3. Rogler G (2010) Gastrointestinal and liver adverse effects of drugs used for
treating IBD. Best Pract Res Clin Gastroenterol 24: 157–165.
4. Stanbury RM, Graham EM (1998) Systemic corticosteroid therapy–side effects
and their management. Br J Ophthalmol 82: 704–708.
5. Stahn C, Lowenberg M, Hommes DW, Buttgereit F (2007) Molecular
mechanisms of glucocorticoid action and selective glucocorticoid receptor
agonists. Mol Cell Endocrinol 275: 71–78.
6. Heitzer MD, Wolf IM, Sanchez ER, Witchel SF, DeFranco DB (2007)
Glucocorticoid receptor physiology. Rev Endocr Metab Disord 8: 321–330.
7. Barnes PJ, Adcock I (1993) Anti-inflammatory actions of steroids: molecular
mechanisms. Trends Pharmacol Sci 14: 436–441.
8. Resche-Rigon M, Gronemeyer H (1998) Therapeutic potential of selective
modulators of nuclear receptor action. Curr Opin Chem Biol 2: 501–507.
9. Schacke H, Berger M, Rehwinkel H, Asadullah K (2007) Selective glucocor-
ticoid receptor agonists (SEGRAs): novel ligands with an improved therapeutic
index. Mol Cell Endocrinol 275: 109–117.
10. Kleiman A, Tuckermann JP (2007) Glucocorticoid receptor action in beneficial
and side effects of steroid therapy: lessons from conditional knockout mice. Mol
Cell Endocrinol 275: 98–108.
11. Rosen J, Miner JN (2005) The search for safer glucocorticoid receptor ligands.
Endocr Rev 26: 452–464.
12. De Bosscher K, Haegeman G (2009) Minireview: latest perspectives on
antiinflammatory actions of glucocorticoids. Mol Endocrinol 23: 281–291.
13. De Bosscher K, Vanden Berghe W, Beck IM, Van Molle W, Hennuyer N, et al.
(2005) A fully dissociated compound of plant origin for inflammatory gene
repression. Proc Natl Acad Sci U S A 102: 15827–15832.
14. Schacke H, Schottelius A, Docke WD, Strehlke P, Jaroch S, et al. (2004)
Dissociation of transactivation from transrepression by a selective glucocorticoid
receptor agonist leads to separation of therapeutic effects from side effects. Proc
Natl Acad Sci U S A 101: 227–232.
15. Dewint P, Gossye V, De Bosscher K, Vanden Berghe W, Van Beneden K, et al.
(2008) A plant-derived ligand favoring monomeric glucocorticoid receptor
conformation with impaired transactivation potential attenuates collagen-
induced arthritis. J Immunol 180: 2608–2615.
16. van Loo G, Sze M, Bougarne N, Praet J, Mc Guire C, et al. (2010)
Antiinflammatory properties of a plant-derived nonsteroidal, dissociated
glucocorticoid receptor modulator in experimental autoimmune encephalomy-
elitis. Mol Endocrinol 24: 310–322.
17. Yemelyanov A, Czwornog J, Gera L, Joshi S, Chatterton RT, Jr., et al. (2008)
Novel steroid receptor phyto-modulator compound a inhibits growth and
survival of prostate cancer cells. Cancer Res 68: 4763–4773.
18. Jung S, Fehr S, Harder-d’Heureuse J, Wiedenmann B, Dignass AU (2001)
Corticosteroids impair intestinal epithelial wound repair mechanisms in vitro.
Scand J Gastroenterol 36: 963–970.
19. Goke MN, Schneider M, Beil W, Manns MP (2002) Differential glucocorticoid
effects on repair mechanisms and NF-kappaB activity in the intestinal
epithelium. Regul Pept 105: 203–214.
20. Anstead GM (1998) Steroids, retinoids, and wound healing. Adv Wound Care
21. Rieder F, Brenmoehl J, Leeb S, Scholmerich J, Rogler G (2007) Wound healing
and fibrosis in intestinal disease. Gut 56: 130–139.
22. Sturm A, Dignass AU (2008) Epithelial restitution and wound healing in
inflammatory bowel disease. World J Gastroenterol 14: 348–353.
23. Park JE, Barbul A (2004) Understanding the role of immune regulation in
wound healing. Am J Surg 187: 11S–16S.
24. Wilson AJ, Gibson PR (1997) Epithelial migration in the colon: filling in the
gaps. Clin Sci (Lond) 93: 97–108.
25. Okamoto R, Watanabe M (2005) Cellular and molecular mechanisms of the
epithelial repair in IBD. Dig Dis Sci 50 Suppl 1: S34–38.
26. Podolsky DK (1997) Healing the epithelium: solving the problem from two sides.
J Gastroenterol 32: 122–126.
27. Dignass AU, Podolsky DK (1993) Cytokine modulation of intestinal epithelial
cell restitution: central role of transforming growth factor beta. Gastroenterology
28. Tetreault MP, Chailler P, Rivard N, Menard D (2005) Differential growth factor
induction and modulation of human gastric epithelial regeneration. Exp Cell
Res 306: 285–297.
29. Beer HD, Fassler R, Werner S (2000) Glucocorticoid-regulated gene expression
during cutaneous wound repair. Vitam Horm 59: 217–239.
30. Herr I, Gassler N, Friess H, Buchler MW (2007) Regulation of differential pro-
and anti-apoptotic signaling by glucocorticoids. Apoptosis 12: 271–291.
31. Wust S, Tischner D, John M, Tuckermann JP, Menzfeld C, et al. (2009)
Therapeutic and adverse effects of a non-steroidal glucocorticoid receptor ligand
in a mouse model of multiple sclerosis. PLoS One 4: e8202.
32. Pineton de Chambrun G, Peyrin-Biroulet L, Lemann M, Colombel JF (2010)
Clinical implications of mucosal healing for the management of IBD. Nat Rev
Gastroenterol Hepatol 7: 15–29.
33. Durer U, Hartig R, Bang S, Thim L, Hoffmann W (2007) TFF3 and EGF
induce different migration patterns of intestinal epithelial cells in vitro and
trigger increased internalization of E-cadherin. Cell Physiol Biochem 20:
34. Znalesniak EB, Hoffmann W (2010) Modulation of cell-cell contacts during
intestinal restitution in vitro and effects of epidermal growth factor (EGF). Cell
Physiol Biochem 25: 533–542.
35. Engelbrecht Y, de Wet H, Horsch K, Langeveldt CR, Hough FS, et al. (2003)
Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase
phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase
and impair proliferation in human and mouse osteoblast cell lines. Endocrinol-
ogy 144: 412–422.
36. Bladh LG, Johansson-Haque K, Rafter I, Nilsson S, Okret S (2009) Inhibition of
extracellular signal-regulated kinase (ERK) signaling participates in repression of
nuclear factor (NF)-kappaB activity by glucocorticoids. Biochim Biophys Acta
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org13 January 2012 | Volume 7 | Issue 1 | e29756
37. Bonner GF (1996) Current medical therapy for inflammatory bowel disease.
South Med J 89: 556–566.
38. Barnes PJ, Karin M (1997) Nuclear factor-kappaB: a pivotal transcription factor
in chronic inflammatory diseases. N Engl J Med 336: 1066–1071.
39. Gossye V, Elewaut D, Van Beneden K, Dewint P, Haegeman G, et al. (2010) A
plant-derived glucocorticoid receptor modulator attenuates inflammation
without provoking ligand-induced resistance. Ann Rheum Dis 69: 291–296.
40. Zhang Z, Zhang ZY, Schluesener HJ (2009) Compound A, a plant origin ligand
of glucocorticoid receptors, increases regulatory T cells and M2 macrophages to
attenuate experimental autoimmune neuritis with reduced side effects.
J Immunol 183: 3081–3091.
41. Wicke C, Halliday B, Allen D, Roche NS, Scheuenstuhl H, et al. (2000) Effects
of steroids and retinoids on wound healing. Arch Surg 135: 1265–1270.
42. Reed BR, Clark RA (1985) Cutaneous tissue repair: practical implications of
current knowledge. II. J Am Acad Dermatol 13: 919–941.
43. Grose R, Werner S, Kessler D, Tuckermann J, Huggel K, et al. (2002) A role for
endogenous glucocorticoids in wound repair. EMBO Rep 3: 575–582.
44. Yan F, Hui YN, Li YJ, Guo CM, Meng H (2007) Epidermal growth factor
receptor in cultured human retinal pigment epithelial cells. Ophthalmologica
45. Piette C, Deprez M, Roger T, Noel A, Foidart JM, et al. (2009) The
dexamethasone-induced inhibition of proliferation, migration, and invasion in
glioma cell lines is antagonized by macrophage migration inhibitory factor (MIF)
and can be enhanced by specific MIF inhibitors. J Biol Chem 284:
46. Suer S, Ampasala D, Walsh MF, Basson MD (2009) Role of ERK/mTOR
signaling in TGFbeta-modulated focal adhesion kinase mRNA stability and
protein synthesis in cultured rat IEC-6 intestinal epithelial cells. Cell Tissue Res
47. Eberhardt W, Pluss C, Hummel R, Pfeilschifter J (1998) Molecular mechanisms
of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat
mesangial cells. J Immunol 160: 4961–4969.
48. Sato Y, Rifkin DB (1988) Autocrine activities of basic fibroblast growth factor:
regulation of endothelial cell movement, plasminogen activator synthesis, and
DNA synthesis. J Cell Biol 107: 1199–1205.
SEGRAs and Intestinal Epithelial Repair
PLoS ONE | www.plosone.org 14 January 2012 | Volume 7 | Issue 1 | e29756