1521-0103/353/3/564–572$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.114.215822
J Pharmacol Exp Ther 353:564–572, June 2015
Endothelin Receptor B Protects Granulocyte Macrophage
Colony-Stimulating Factor mRNA from Degradation
David Jungck, Jürgen Knobloch, Sandra Körber, Yingfeng Lin, Jürgen Konradi,
Sarah Yanik, Erich Stoelben, and Andrea Koch
Medical Clinic III for Pneumology, Allergology, Sleep and Respiratory Medicine, Bergmannsheil University Hospital, Bochum,
Germany (D.J., J.Kn., S.K., S.Y., A.K.); Department of Pneumology, Clinic III for Internal Medicine, University of Cologne,
Cologne, Germany (Y.L., J.Ko.); and Department of Thoracic Surgery, Lungenklinik Merheim, Kliniken der Stadt Köln, Cologne,
Germany (E.S.)
Received April 22, 2014; accepted March 30, 2015
Introduction
It is well established that airway smooth muscle cells (ASMCs)
take part in the inflammatory processes of chronic airway
diseases (Clarke et al., 2009; Knobloch et al., 2010, 2013; Barnes,
2011). They are triggered to secrete proinflammatory cytokines
and alter the expression of cell adhesion molecules (Damera
et al., 2009). We have previously shown in human ASMCs
(HASMCs) that tumor necrosis factor a (TNFa) activates an
endothelin-1 (ET-1) autoregulatory positive feedback mechanism that sustains the expression of ET-1 and granulocyte
This work was funded by Actelion Pharmaceuticals, Germany. Actelion
Pharmaceuticals was not involved in study design; in the collection, analysis,
and interpretation of data; in the writing of the manuscript; or in the decision
to submit the paper for publication.
dx.doi.org/10.1124/jpet.114.215822.
whereas there was no significant difference in their effect on GM-CSF
and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA
equally but had no effect on CXCL2. Blocking mitogen-activated
protein kinases revealed that both ETAR and ETBR signal through
p38 mitogen-activated protein kinase, but ETBR also signals
through extracellular signal-regulated kinase (ERK) 1/2 to induce
GM-CSF expression. In the presence of the transcription inhibitor
actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF
but not MMP12 or CXCL3 mRNA. In conclusion, blockade of
each endothelin receptor subtype reduces GM-CSF transcription,
but blocking ETBR additionally protects GM-CSF mRNA from
degradation via ERK-1/2. Accordingly, blocking both ETAR and
ETBR leads to a stronger reduction of TNFa-induced GM-CSF
protein expression. This mechanism might be specific to GMCSF. Our data stress the anti-inflammatory potential of ERA and
warrant further investigation of their utility in chronic inflammatory
airway diseases.
macrophage colony-stimulating factor (GM-CSF) and that is
inhibited by endothelin receptor antagonists (ERAs) (Knobloch
et al., 2009). TNFa induced GM-CSF expression and ET-1
transcription by signaling through p38 mitogen-activated protein kinase (p38MAPK), ET-1 induced its own transcription by
signaling through endothelin receptor subtype A (ETAR) and
p38MAPK, and ET-1 induced GM-CSF transcription through both
endothelin receptor subtypes A and B (ETBR), p38MAPK, and
extracellular signal-regulated kinase (ERK)–1/2. These results
suggested further investigation of the therapeutic potential of
ERA in the early stages of chronic inflammatory airway diseases, although it remained unclear what impact the individual
endothelin receptor subtypes have on GM-CSF expression and
which kinases they signal through.
ET-1 is produced as preproendothelin, released in its precursor
form, big–ET-1, from the synthesizing cells, and is subsequently
ABBREVIATIONS: ASMC, airway smooth muscle cell; BQ123, 2-[(3R,6R,9S,12R,15S)-6-(1H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8,11,14pentaoxo-12-propan-2-yl-1,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid; BQ788, sodium N-{[(2R,6S)-2,6-dimethyl-1-piperidinyl]
carbonyl}-4-methyl-L-leucyl-N-[(1R)-1-carboxylatopentyl]-1-(methoxycarbonyl)-D-tryptophanamide; COPD, chronic obstructive pulmonary disease;
CXCL2, chemokine (C-X-C motif) ligand 2; CXCL3, chemokine (C-X-C motif) ligand 3; ERA, endothelin receptor antagonist; ERK, extracellular
signal-regulated kinase; ET-1, endothelin-1; ETAR, endothelin receptor A; ETBR, endothelin receptor B; GM-CSF, granulocyte macrophage colonystimulating factor; HASMC, human airway smooth muscle cell; PD098059, 2-(29-amino-39-methoxyphenyl)-oxanaphthalen-4-one; PH, pulmonary
hypertension; SB203580, 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine; TNFa, tumor necrosis factor a.
564
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ABSTRACT
Evidence is lacking on the differential effects of the two therapeutic
concepts of endothelin receptor antagonists (ERAs): the blockade
of only the endothelin receptor A (ETAR; selective antagonism)
versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker,
are both available for therapy. We hypothesized that there are
differences in the potential of ERAs to ameliorate inflammatory
processes in human airway smooth muscle cells (HASMCs) and
aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reversetranscription polymerase chain reaction. Tumor necrosis factor a
(TNFa) induced transcription and expression of chemokine (C-X-C
motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3),
granulocyte macrophage colony-stimulating factor (GM-CSF), and
matrix metalloproteinase 12 (MMP12) in HASMCs. In concentrationresponse experiments, bosentan led to a significantly greater
reduction of GM-CSF and MMP12 protein release than ambrisentan,
Endothelin Receptor B Protects GM-CSF mRNA from Degradation
rapidly degraded due to an adenosine-uridine–rich sequence
in its 39 untranslated region (Shaw and Kamen, 1986) but is
stabilized by receptor signaling (Tebo et al., 2003).
There is strong evidence of an involvement of matrix
metalloproteinase 12 (MMP12) in airway inflammation and
remodeling. Genetic polymorphisms of MMP12 have been
associated with the development of COPD and decline in lung
function (Joos et al., 2002; Mukhopadhyay et al., 2010).
The chemokine (C-X-C motif) ligands 2 (CXCL2) and 3
(CXCL3) are activated in bronchoalveolar lavage cells of
asthmatics resistant to corticosteroids (Goleva et al., 2008)
and induce an increased migration of HASMCs from asthmatic patients, suggesting a contribution to airway remodeling (Al-Alwan et al., 2013).
An increased expression of interleukin-32 (IL-32) has been
shown in macrophages, alveolar walls, and bronchiolar epithelium of patients with COPD (Calabrese et al., 2008). Moreover,
IL-32 levels are increased in the serum of asthmatic patients
(Meyer et al., 2012).
The aim of this study was to assess whether differences
between selective versus dual ERA exist in their potential to
ameliorate inflammatory processes in HASMCs, specifically
the expression of GM-CSF, MMP12, CXCL2, CXCL3, and
IL-32 in response to TNFa, and to achieve mechanistic insight
into the associated pathways. To this end, we compared
mRNA and protein expression in response to TNFa stimulation after blocking endothelin receptors with bosentan or
ambrisentan in HASMCs.
Materials and Methods
Acquisition, Isolation, and Cultivation of HASMCs. HASMCs
were dissected from resected lung tissue of patients undergoing
surgery for pulmonary tumors, as previously established (Oltmanns
et al., 2003). Cultivation and characterization were carried out as
stated elsewhere (Raidl et al., 2007). This study was approved by the
ethics committees of the Universities of Cologne (02-004) and Bochum
(4257-12), Germany, and all patients gave written informed consent.
Stimulation of HASMCs. Cells were cultivated until they reached
subconfluence and then deprived of serum for 24 hours before stimulation,
as described elsewhere (Raidl et al., 2007). HASMCs at passages 2–7 were
stimulated with recombinant human TNFa (R&D Systems, Minneapolis,
MN) at 20 ng/ml or with ET-1 (Sigma-Aldrich, Hamburg, Germany)
at 100 nM for the indicated times. Bosentan (Actelion Pharmaceuticals,
Freiburg, Germany), ambrisentan (GlaxoSmithKline, Munich, Germany),
BQ123 (2-[(3R,6R,9S,12R,15S)-6-(1H-indol-3-ylmethyl)-9-(2-methylpropyl)2,5,8,11,14-pentaoxo-12-propan-2-yl-1,4,7,10,13-pentazabicyclo[13.3.0]
octadecan-3-yl]acetic acid; Sigma-Aldrich), and BQ788 (sodium
N-{[(2R,6S)-2,6-dimethyl-1-piperidinyl]carbonyl}-4-methyl-L-leucylN-[(1R)-1-carboxylatopentyl]-1-(methoxycarbonyl)-D-tryptophanamide;
Sigma-Aldrich) were added at 1026 M or at the indicated concentrations
60 minutes before ET-1 or TNFa stimulation. Pretreatment with
SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol5-yl]pyridine; 1 mM; Calbiochem/VWR, Darmstadt, Germany) or PD098059
(2-(29-amino-39-methoxyphenyl)-oxanaphthalen-4-one; 10 mM; Calbiochem/
VWR) was carried out 30 minutes before stimulation. Gene transcription
was inhibited with actinomycin D (Sigma-Aldrich) at 5 mg/ml. Cell
viability was determined by trypan blue staining.
Enzyme-Linked Immunosorbent Assays. GM-CSF enzymelinked immunosorbent assays (R&D Systems) were carried out with
supernatants of subconfluent HASMCs (about 80% density) on 96-well
plates as previously described (Koch et al., 2004). Correspondingly,
intracellular enzyme-linked immunosorbent assays for phosphorylated
p38MAPK (R&D Systems) were performed with subconfluent HASMCs
on 96-well plates as described elsewhere (Knobloch et al., 2009).
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cleaved by endothelin-converting enzyme into the active 21–amino
acid peptide. Discovered as a vasoconstrictor (Yanagisawa et al.,
1988), it is now known to also have an important role in
inflammatory processes. Together with TNFa, ET-1 was described
as a cytokine initiating inflammatory processes in the airways
(Hamilton and Anderson, 2004). It acts mainly locally and signals
through ETA/BR subtypes (Benigni and Remuzzi, 1999), which
have been shown to be present on HASMCs (Flynn et al., 1998).
ERAs were researched extensively after the discovery of
ET-1, and were soon discovered to hold anti-inflammatory
potential (Finsnes et al., 1997). However, many setbacks occurred
during these efforts: tezosentan proved nonbeneficial in acute and
chronic heart failure (McMurray et al., 2007; Kawanabe and
Nauli, 2011), and despite encouraging results from previous
clinical trials, bosentan did not show clinical efficacy in patients
with histologically proven idiopathic pulmonary fibrosis in the
bosentan use in interstitial lung disease-3 [BUILD-3] trial (King
et al., 2011). Nonetheless, clinical efficacy was shown for the
treatment of patients with pulmonary arterial hypertension and
scleroderma-related digital ulcers, and recent results and flaws in
previous trials justify research in abandoned areas (Kohan et al.,
2012).
Bosentan is an endothelin receptor antagonist with almost
equal affinity to both ETAR (Ki = 4.75 6 1.44 nM) and ETBR
(Ki = 40.9 6 12.3 nM; each in human muscle cells) (Yuyama
et al., 2004) and is, therefore, called a dual blocker (Clozel et al.,
1994). A trial published in 2000 involving seven female patients
first showed a positive influence on pulmonary hypertension
(PH) (Williamson et al., 2000). It was subsequently approved for
therapy in the European Union in 2002 as an orphan drug after
two further clinical trials demonstrated efficacy in PH (Channick
et al., 2001; Rubin et al., 2002).
Ambrisentan was approved for therapy by the Food and
Drug Administration in 2007 and the European Medicines
Agency in 2008. Given its much higher affinity for the ETAR
(selective antagonist; ETAR Ki = 0.28 6 0.23 nM, ETBR Ki =
250 6 50 nM in human muscle cells) (Maguire et al., 2012),
the theoretical concept arose that a better clinical outcome
was achieved by conserving the potentially beneficial effects
of stimulation of the ETBR, clearance of ET-1, and vasodilation. Its efficacy in the treatment of pulmonary arterial
hypertension was first shown in a trial with 64 patients (Galié
et al., 2005). However, there is no clinical evidence to support
an advantage of selective over dual ERAs (Opitz et al., 2008).
TNFa is an important cytokine in inflammatory airway
diseases. It is implied in inflammatory and remodeling processes in chronic bronchitis, chronic obstructive pulmonary
disease (COPD), and asthma, and leads to ASMC hyperplasia,
bronchial vasoconstriction, and emphysema- and pulmonary
fibrosis–like changes in animal models (Mukhopadhyay et al.,
2006). Of note, anti-TNFa therapy proved nonbeneficial in
COPD and raised concerns about the possible induction of
malignancies (Bongartz et al., 2006; Rennard et al., 2007).
GM-CSF is involved in inflammatory reactions throughout
the body (Hamilton and Anderson, 2004) with raised mRNA
levels in bronchial bioptic tissue of atopic asthmatics (Bentley
et al., 1993), pulmonary inflammatory processes, and lung
fibrosis (Gajewska et al., 2003). Its expression is induced by
TNFa and prevents eosinophil apoptosis in asthma, leading to
an increased expression of inflammatory mediators (Esnault
and Malter, 2001). GM-CSF levels are raised in induced
sputum of COPD patients (Vlahos et al., 2006). Its mRNA is
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Jungck et al.
Results
Combined Blocking of ETAR and ETBR Leads to
a Greater Reduction of TNFa-Induced GM-CSF and
MMP-12 Protein Release than Blocking Only ETAR. We
have previously shown in time-response experiments that TNFa
induces GM-CSF protein release from cultivated HASMCs
maximally after 72 hours of stimulation (Knobloch et al., 2010,
2013). Using these conditions, GM-CSF protein baseline release
was 22.4 6 5.1 pg/ml (mean 6 S.E.M.), and TNFa clearly
induced GM-CSF release (Fig. 1A). To investigate the effects of
bosentan and ambrisentan in this context, we performed
concentration-response experiments and used the drugs according to their Ki values (see Introduction) and the plasma half-life
of bosentan (Treiber et al., 2007; Croxtall and Keam, 2008) at
a range of 10212 to 1024 M. Both drugs significantly reduced
GM-CSF protein, but bosentan led to a significantly stronger
reduction than ambrisentan (Fig. 1B; Table 1).
TNFa induces GM-CSF mRNA levels in HASMCs at two
maximums: one after 2 hours of stimulation, which is insensitive to bosentan, and a second one after 8 hours, which is
sensitive to bosentan (Knobloch et al., 2009). Both bosentan
and ambrisentan significantly reduced TNFa-induced GMCSF transcription after 8 hours of stimulation—notably,
without significant differences (Fig. 1, C and D; Table 1).
To check if this discrepancy between mRNA and protein
sensitivity to bosentan and ambrisentan is specific for GM-CSF,
we tested four additional cytokines: CXCL2, CXCL3, IL-32, and
MMP12. Previously, mRNA upregulation by TNFa in HASMCs
was shown for all of these cytokines by whole genome microarrays and quantitative reverse-transcription polymerase chain
reaction (Knobloch et al., 2013). TNFa induced the release of
CXCL2, CXCL3, and MMP12 but not IL-32 protein from
HASMCs (Fig. 1, E and H; data not shown). TNFa-induced
CXCL2 release was insensitive to bosentan and ambrisentan
(data not shown). Therefore, IL-32 and CXCL2 were not considered for further analyses. Neither ERA modulated baseline
MMP12 or CXCL3 levels (data not shown). Bosentan clearly
reduced MMP12 protein release in TNFa-exposed cells; however, ambrisentan had no effect, although there was a trend for
the highest concentration (Fig. 1F; Table 1). The difference in
the effects between the two ERAs was statistically significant.
Both drugs reached maximal effects on GM-CSF mRNA at
1 mM (Fig. 1D); therefore, we used this concentration for the
analyses of the effects of the ERAs on MMP12 and CXCL3
mRNA levels. Neither ERA modulated TNFa-induced MMP12
mRNA levels (Fig. 1G). Both bosentan and ambrisentan
partially reduced CXCL3 protein release and mRNA levels in
TNFa-exposed cells but, in both cases, without differences
between the two ERAs (Fig. 1, I and J; Table 1). Notably, the
effect of bosentan on CXCL3 protein had a clear trend but did
not reach statistical significance.
In summary, we found one further gene, MMP12, that showed
a discrepancy in sensitivity to selective and nonselective ERAs
between mRNA transcription and protein expression. These
data are suggestive of a post-transcriptional modification of
GM-CSF and MMP12 mRNA mediated by ETBR, which was
investigated further.
ETAR and ETBR Mediate TNFa-Induced GM-CSF Protein Release via p38MAPK, but Only ETBR Also Signals
through ERK-1/2. ETBR has previously been shown to regulate ET-1 mRNA stability via p38MAPK and ERK in vascular
endothelial cells (Farhat et al., 2008). ERK has also been shown
to mediate GM-CSF mRNA stabilization in peripheral blood
eosinophils after stimulation with TNFa and fibronectin (Esnault
and Malter, 2002). Therefore, we next investigated the role of
mitogen-activated protein kinases in our model. ERAs were used
at a concentration of 1026 M for all further experiments since this
reflects the plasma concentrations reached in patients under
normally dosed therapy (Treiber et al., 2007; Croxtall and Keam,
2008).
Blocking ETAR with either BQ123 or ambrisentan led to
a significant reduction of TNFa-induced GM-CSF release. The
additional blockade of ERK-1/2 activity with PD098059 led to
a further significant reduction in GM-CSF release; this was
also significantly different in comparison with blocking ERK1/2 alone (Fig. 2A). When blocking ETBR with BQ788, there
was also a significant reduction in TNFa-induced GM-CSF
release, but the additional blockade of ERK-1/2 activity with
PD098059 led to no further significant reduction in GM-CSF
levels (Fig. 2A). Hence, ETBR, but not ETAR, mediates TNFainduced GM-CSF release via ERK-1/2. Blocking p38MAPK
activity with SB203580 abolished TNFa-induced GM-CSF
release completely.
To further elucidate the involvement of the individual endothelin receptors in p38MAPK signaling, we conducted activity experiments by measuring the phosphorylation status of p38MAPK.
Our previous experiments showed that TNFa induces p38MAPK
activation time dependently in waves, and that long-term, but not
short-term, stimulation of p38MAPK is a consequence of a reactivation by an ET-1 feedback mechanism (Knobloch et al., 2009).
Accordingly, blocking of ETAR with ambrisentan or ETAR and
ETBR with bosentan had no significant influence on p38MAPK
phosphorylation after stimulation with TNFa for 15 minutes
(Fig. 3). ET-1 induced an increase in p38MAPK phosphorylation,
which was reduced by both ERAs (Fig. 3). Importantly, bosentan,
but not ambrisentan, completely abolished p38MAPK activity, and
the differences in the effects between the two ERAs were
statistically significant (Fig. 3). Summarized, these data show
that both endothelin receptor subtypes signal through p38MAPK,
but only ETBR also signals through ERK-1/2 in HASMCs.
If ETBR signals via both kinases in parallel, TNFa-induced
GM-CSF release should be blocked more efficiently by combined
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Quantitative Reverse-Transcription Polymerase Chain Reaction. Quantitative reverse-transcription polymerase chain reaction was performed on DNA-free total RNA extracted from HASMCs,
as described previously (Knobloch et al., 2009, 2013).
Statistical Analysis. Statistical analyses were performed to
examine the effects of TNFa or ET-1 alone or in combination with
bosentan, ambrisentan, BQ123, BQ788, SB203580, PD098059, and/or
actinomycin D on cytokine release or gene expression from HASMCs.
Data sets were tested and confirmed for a Gaussian distribution by
histogram analysis. The results are indicated as the mean 6 S.E.M.
(Gaussian distribution) or as the median with 25th to 75th interquartile range 6 minimum/maximum (non-Gaussian distribution).
Comparisons in time-response experiments and across more than two
stimulations were performed with one-way repeated-measures analysis of variance (Gaussian distribution) or with the Friedman test (nonGaussian distribution) with 95% confidence intervals. For separate
comparisons of each stimulation, post-hoc Bonferroni Holm or post-hoc
Dunn tests were performed. Comparisons between two parameters
were analyzed by paired, two-tailed Student’s t test. Concentrationresponse curves were calculated by nonlinear (sigmoidal) regression
with variable parameters. All calculations were performed with
GraphPad Prism (GraphPad Software, La Jolla, CA).
Endothelin Receptor B Protects GM-CSF mRNA from Degradation
567
use of ERK-1/2 and p38MAPK inhibitors than by use of the single
inhibitors. To test this, we reduced inhibitor concentrations.
Under these conditions, single use of PD098059 and SB203580
no longer had an effect; however, the combined treatment
showed a reduction (Fig. 2B). ERAs reduced TNFa-induced
GM-CSF release only partially, and from the data obtained so far,
it could not be ruled out that endothelin receptor–independent
GM-CSF expression in response to TNFa is mediated by ERK-1/2,
which could also explain the additive effects of ERK-1/2 and
p38MAPK inhibition on GM-CSF shown in Fig. 2B. However, the
combined treatment with PD098059 and bosentan did not show
additional effects on GM-CSF compared with single treatments
(Fig. 2C), demonstrating that endothelin-independent GM-CSF
regulation by TNFa does not require ERK-1/2 activity.
ETBR Protects GM-CSF but Not MMP12 mRNA from
Degradation. TNFa led to a strong induction of GM-CSF
transcription after 10 hours of stimulation (Fig. 4A). To elucidate
post-transcriptional effects of the endothelin receptor subtypes,
we added actinomycin D after 8 hours of stimulation with TNFa
to stop transcription. Then we added bosentan or ambrisentan
and measured GM-CSF mRNA. Actinomycin D alone did not
significantly reduce GM-CSF mRNA in TNFa-exposed
HASMCs (Fig. 4A). In the presence of actinomycin D, both
drugs significantly reduced GM-CSF mRNA compared with
stimulation with TNFa alone (Fig. 4A), indicating constitutive
mRNA turnover. Importantly, in the presence of actinomycin D,
bosentan, but not ambrisentan, reduced GM-CSF mRNA
compared with incubation with actinomycin D alone (Fig. 4A).
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Fig. 1. Effect of ERAs on cytokine expression in TNFa-exposed HASMCs. HASMCs were stimulated with TNFa at 20 ng/ml for 72 hours. Bosentan
(BOS) or ambrisentan (AMB) was added to the medium at the indicated concentrations 1 hour before stimulation with TNFa. After incubation, RNA was
extracted and subjected to semiquantitative reverse-transcription polymerase chain reaction with subsequent gel electrophoresis, and densitometric
analyses of the polymerase chain reaction signals (C, D, G, and J) or cytokine concentrations in the supernatant were measured by enzyme-linked
immunosorbent assay (A, B, E, F, H, and I). (A, E, and H) Values were normalized to solvent controls. A value of 1 corresponds to an average
concentration of 22.4 6 15.37 (GM-CSF), 135.8 6 8.5 (MMP12), or 480.9 6 199.9 (CXCL3) pg/ml. (C, D, G, and J) Values for target genes were normalized
to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) references and to the unstimulated control, which was set to 1. GM-CSF levels are expressed as
fold induction versus the unstimulated control. Induction data for MMP12 and CXCL3 were already shown in Knobloch et al. (2013); ERA effects shown
in (G) and (J) were investigated in these experimental series. (B, D, F, G, H, and I) Data were calculated as percent reduction (negative values) versus
stimulation with TNFa alone (TNFamax). Data are presented as the mean 6 S.E.M. [n = 9 in (A, B, E, F, and I); n = 11 in (C and D); n = 8 in (G); n = 5 in (J)]
or as the median (bar within box) with 25th to 75th interquartile range (box) 6 minimum/maximum [error bars; n = 9 in (H)]. Curves were created by
nonlinear regression analyses with sigmoidal curve fit using variable slopes. r2 (goodness of fit): bosentan 0.788, ambrisentan 0.8605 (B); bosentan
0.1958, ambrisentan 0.4605 (D); bosentan 0.4932 (F); bosentan 0.4932, ambrisentan 0.4589 (I). (A, C, and E) TNFa effects were analyzed by paired t tests
versus control. In (H), the Wilcoxon signed rank test was used. (B, D, F, G, I, and J) Whole curves or bars were compared using a paired t test and a onesample t test to a hypothetical value of 0 (no reduction). *P , 0.05; **P , 0.01; ***P , 0.001.
568
Jungck et al.
TABLE 1
Effects of bosentan and ambrisentan on TNFa-induced cytokine release or transcription
EC50 and EMAX values were determined from concentration-response curves in Fig. 1.
Cytokine
EMAX 6 S.E.M.
M
% reduction
4.5 1028
1.1 1027
63.71 6 3.026
54.83 6 2.478
60.66 6 14.39
46.66 6 9.635
2.2 1028
4.8 1028
49.04 6 5.432
51.73 6 7.115
52.22 6 19.51
48.70 6 29.13
9.7 1028
—
45.38 6 5.404
—
37.47 6 22.55
9.83 6 25.92
n.d.
n.d.
n.d.
n.d.
22.70 6 14.39
0.25 6 37.83
5.1 1027
2.7 1026
44.48 6 5.862
50.96 6 13.85
29.84 6 18.18
19.36 6 20.92
n.d.
n.d.
n.d.
n.d.
45.77 6 35.87
37.92 6 13.06
—, not calculable as there was no reduction; n.d., not determined.
We conclude that ETBR protects GM-CSF mRNA from
degradation. TNFa also induced MMP12 and CXCL3 mRNA
after 10 hours (Fig. 4, B and C). Actinomycin D reduced
MMP12 mRNA, but there was no additional effect when it was
combined with bosentan or ambrisentan (Fig. 4B). CXCL3
mRNA levels were not modulated by actinomycin D alone or in
combination with ERAs (Fig. 4C). This indicates that ETBR
does not regulate MMP12 and CXCL3 mRNA stability.
TNFa Is Unable to Increase GM-CSF mRNA Levels in
the Presence of ETBR Inhibitors in Long-Term Culture. According to our data, ETBR-mediated GM-CSF mRNA
stability explains the discrepancy between GM-CSF mRNA
and protein sensitivity to bosentan and ambrisentan shown in
Fig. 1, B and D, where GM-CSF mRNA was measured after
8 hours and GM-CSF protein after 72 hours of TNFa
stimulation. However, if ETBR blockade leads to accelerated
GM-CSF mRNA degradation, differences in the effects of
bosentan and ambrisentan on GM-CSF mRNA should be seen
in long-term culture. To test this, we stimulated HASMCs for
24 hours with TNFa. TNFa significantly induced GM-CSF
mRNA levels, and this was not significantly different in the
presence or absence of ambrisentan (Fig. 5). However, in the
presence of bosentan or BQ788, a specific inhibitor for ETBR,
TNFa did not significantly increase GM-CSF mRNA levels.
These data confirm our conclusion stated earlier.
Discussion
We showed that both dual and selective endothelin receptor
antagonism with bosentan or ambrisentan led to a significant
and equal reduction of TNFa-induced GM-CSF and CXCL3,
but not MMP12, transcription in HASMCs after 8 hours of
stimulation. CXCL3 protein was reduced equally by both ERAs,
meaning that there was no difference in sensitivity to selective
and dual ERAs between mRNA and protein. However, the
reduction in release of GM-CSF and MMP12 protein, although
significantly decreased by both ERAs, was significantly greater
after addition of the dual antagonist. This result pointed to an
involvement of ETBR in a post-transcriptional modification of
GM-CSF and MMP12 mRNA or protein. Measuring the amount
of TNFa-induced GM-CSF and MMP12 mRNA after having
stopped transcription and blocked the respective receptors
proved that ETBR has a strong influence on GM-CSF mRNA
stability but no influence on MMP12 mRNA stability. If
a discrepancy in the sensitivity to selective and dual ERAs
between mRNA and protein is explained by ETBR regulation of
mRNA stability, this mechanism should not play a role for
cytokines that do not have this discrepancy in sensitivity. In
accordance with this reasoning, we could not find an indication
that ETBR regulates CXCL3 mRNA stability. In further consideration of the MMP12 results, our data point to a mechanism
that is specific to GM-CSF. This raises the question of a role for
selective ETBR antagonism in certain chronic airway diseases.
TNFa activates an ET-1 autoregulatory positive feedback
mechanism in HASMCs (Knobloch et al., 2009), and we have
shown here that, although both ETAR and ETBR signal through
p38MAPK, only ETBR also signals through ERK-1/2. This suggests
that TNFa-dependent ET-1 signals through ETAR/p38MAPK
and ETBR/p38MAPK pathways to induce GM-CSF transcription, and via the ETBR/ERK pathway to protect GM-CSF mRNA
from degradation (Fig. 6). In support of this, ERK has been
shown to regulate GM-CSF mRNA stability in other cell types
exposed to TNFa (Esnault and Malter, 2002).
Interestingly, whereas ambrisentan has a more favorable
Ki for the ETAR, it had a higher EC50 in comparison with
bosentan (see Introduction). This could mean that, to achieve
the reductive effects of ERA on TNFa-induced cytokine expression, a minimum blockade of ETBR has to be achieved. As
ambrisentan’s Ki for the ETBR is comparatively high, this
would explain the higher concentration needed to achieve the
shown effects.
GM-CSF has been shown to be elevated in chronic inflammatory lung diseases such as COPD and is thought to be an
early driver of lung remodeling. Data from a subchronic smoking
mouse model proved that neutralization of GM-CSF at an early
stage of intense cigarette smoke exposure reduced the number of
macrophages and neutrophils in bronchoalveolar lavage fluid,
suppressed macrophage proliferation, and reduced cytokine,
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
GM-CSF protein
Bosentan
Ambrisentan
GM-CSF mRNA
Bosentan
Ambrisentan
MMP12 protein
Bosentan
Ambrisentan
MMP12 mRNA
Bosentan
Ambrisentan
CXCL3 protein
Bosentan
Ambrisentan
CXCL3 mRNA
Bosentan
Ambrisentan
% Reduction 6 S.E.M. at 1026 M
EC50
Endothelin Receptor B Protects GM-CSF mRNA from Degradation
569
chemokine, and protease mRNA (Vlahos et al., 2010). Hence,
therapeutic strategies to antagonize GM-CSF are worth investigating while reservations exist with regard to possible side
effects such as alveolar proteinosis or an increased susceptibility
to infections upon complete inhibition of GM-CSF (Vlahos et al.,
2006). Since our data show that ERAs decrease TNFa-induced
GM-CSF protein expression from HASMCs, they further
strengthen the argument for ERAs as anti-inflammatory drugs.
Considering that they only reduce but do not abolish GM-CSF
protein expression, and that small amounts of GM-CSF are
sufficient for lung homeostasis (Vlahos et al., 2006), the
induction of side effects becomes less probable.
The ERA-mediated reduction of TNFa-induced CXCL3 and
MMP12 expression further broadens the anti-inflammatory
potential of ERAs and might result in differential effects on
different diseases.
Comparative data on selective and dual ERAs in humans
are scarce. One paper confirmed bosentan’s blockade of ETBR
when compared with sitaxentan in healthy men (MacIntyre
et al., 2010). There are no published clinical trials on the use
of ambrisentan in COPD, but two trials on the efficacy of
bosentan in patients with COPD have been published (Stolz
et al., 2008). The results of the first trial from a Swiss group
were discouraging, as there was no improvement in the primary
endpoint, the 6-minute walking test, in the bosentan group
after 12 weeks of treatment. The chosen population comprised
30 patients (20 of whom received bosentan) with severe or very
severe COPD (Global Initiative for Chronic Obstructive Lung
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 2. Endothelin receptors A and B mediate TNFa-induced GM-CSF expression through p38MAPK, but ERK-1/2 only responds to ETBR. HASMCs were
stimulated with TNFa at 20 ng/ml for 72 hours. SB203580 (SB) at 1 mM and PD098059 (PD) at 10 mM (A and C) or at indicated concentrations (B) were
added 30 minutes before stimulation with TNFa. BQ123, BQ788, ambrisentan (A), and bosentan (C), all at 1026 M, were added 1 hour before stimulation
with TNFa. After incubation, GM-CSF concentrations in the supernatant were measured by enzyme-linked immunosorbent assay, and values were
normalized to solvent controls in (A) and (C). A value of 1 corresponds to an average GM-CSF concentration of 34.71 6 26.34 pg/ml. Data are presented as
the mean 6 S.E.M. of n = 5 individual experiments (A and C) or as the median (bar within box) with 25th to 75th interquartile range (box) 6 minimum/
maximum (errors bars; n = 6) (B). One-way repeated-measures analysis of variance (P , 0.0001) with post-hoc Bonferroni-Holm test (A and C) or paired
t test (B): ##P , 0.01, ###P , 0.001 related to solvent controls; *P , 0.05; **P , 0.01; ***P , 0.001, related to the indicated values or to TNFa stimulation.
570
Jungck et al.
Fig. 3. Bosentan abolishes p38MAPK phosphorylation after stimulation with ET-1. HASMCs were
incubated with ET-1 at 100 nM or TNFa at 20 ng/ml
for 15 minutes. Bosentan at 1026 M or ambrisentan
at 1026 M was added 1 hour before stimulation with
ET-1 or TNFa. After incubation, the activity of
p38MAPK was measured by an intracellular enzymelinked immunosorbent assay for phosphorylated
p38MAPK (p-p38MAPK) with total p38MAPK (t-p38MAPK)
as a reference. Values for p-p38MAPK were normalized to t-p38MAPK and related to solvent controls.
Each graph represents the mean 6 S.E.M. of n = 5
individual experiments. One-way repeated-measures analysis of variance (P , 0.0001) with post-hoc
Bonferroni-Holm tests: P values are indicated in the
graph (related to the indicated values or to solvent
controls if placed on top of a bar); *P , 0.05; **P ,
0.01; ***P , 0.001.
group. However, the trial was not double blinded, and it seems
no primary endpoint had been defined (Valerio et al., 2009).
While these results are interesting, they concern patients
suffering from advanced stages of COPD and PH. In these
patients, extensive lung remodeling with loss of functional
tissue will have already taken place. It is important to stress,
though, that the possible utility of ERAs that results from our
data lies in their potential to ameliorate inflammation and
remodeling in early rather than late stages of chronic
inflammatory airway diseases. Accordingly, the disappointing
results from the BUILD-3 trial also stem from a patient
population with advanced idiopathic pulmonary fibrosis. In
support of this, we have previously shown that ERAs regulate
a broad spectrum of inflammatory and remodeling genes in
HASMCs after stimulation with TNFa. In these experiments,
the addition of bosentan led to the reduction of a larger
Fig. 4. Endothelin receptor subtype B is involved in stabilizing GM-CSF mRNA. HASMCs were stimulated with TNFa at 20 ng/ml for 10 hours. After
8 hours of stimulation with TNFa, 5 mg/ml actinomycin D was added to the medium. After 9 hours of stimulation with TNFa, bosentan or ambrisentan,
each at 1026 M, was added to the medium. After incubation, the RNA was extracted and subjected to semiquantitative reverse-transcription polymerase
chain reaction with subsequent gel electrophoresis and densitometric analyses of the polymerase chain reaction signals. Values for target genes were
normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) references and to the unstimulated control, which was set to 1. GM-CSF, MMP12,
and CXCL3 levels are expressed as fold induction versus the unstimulated control. Each graph represents the mean 6 S.E.M. (A and B) or the median
(bar within box) with 25th to 75th interquartile range (box) 6 minimum/maximum (error bars) (C) of n = 4 individual experiments. One-way repeatedmeasures analysis of variance (P , 0.0001) with post-hoc Bonferroni-Holm tests (A and B) or Friedman test (P = 0.0224) with post-hoc Dunn test (C):
P values are indicated in the graph (related to the indicated values or to solvent controls if placed on top of a bar); **P , 0.01; ***P , 0.001.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Disease stages III and IV), 20 of whom were diagnosed with PH
detected by echocardiography. It should be noted that this is not
the recommended diagnostic tool for PH and may deliver
incorrect results, especially when the systolic pulmonary artery
pressure is below 50 mm Hg (Grünig et al., 2011), as was the
case in this trial. The second trial was carried out by an Italian
group. Forty patients with COPD and PH confirmed by right
heart catheterization were enrolled and randomized to receive
either standard care or standard care plus bosentan for a period
of 18 months. The average forced expiratory volume in 1 second
(FEV1) at baseline was 39% in the standard care group and 37%
in the bosentan group. The authors reported a significant
decrease in pulmonary artery pressure, pulmonary vascular
resistance, and BODE [Body-Mass Index, Airflow Obstruction,
Dyspnea, and Exercise Capacity] index as well as a significant
increase in the 6-minute walking distance for the bosentan
Endothelin Receptor B Protects GM-CSF mRNA from Degradation
number of these genes in comparison with ambrisentan. Also,
bosentan caused a stronger reduction of MMP13 mRNA levels
independent from gene transcription. We concluded that
ETBR was responsible for ameliorating mRNA degradation
(Knobloch et al., 2013).
Since symptoms of chronic inflammatory airway diseases
frequently only occur in advanced stages, the question of how
to identify groups of patients that might profit from the
administration of ERA arises. At least in COPD—where current
therapeutic strategies can at best only slow disease progression
—there is the advantage of knowing that cigarette smoke is the
main risk factor for developing the disease. But merely treating
patients at risk could do more harm than good, especially since
an “at risk” group is no longer part of the guidelines as this
classification proved nonbeneficial. However, increased vigilance will help identify smoking patients who have developed an
early stage of COPD through the use of lung function screening
tests. Moreover, many efforts are under way to identify new
diagnostic methods and subgroups of patients with asthma,
COPD, and idiopathic pulmonary fibrosis expectantly allowing
for an earlier diagnosis and more appropriate therapy (du Bois,
2012; Wenzel, 2012; Vanfleteren et al., 2013).
We have shown here that the anti-inflammatory potential
of dual receptor antagonism with bosentan is greater in HASMCs
than that of selective ETAR antagonism with ambrisentan,
suggesting that there is an advantage of dual over ETAR-selective
Fig. 6. Model to explain the effects of pharmacologic modulation of the
activities of endothelin receptor subtypes on GM-CSF transcription and
expression after stimulation of HASMCs with TNFa. As shown elsewhere
(Knobloch et al., 2009), TNFa stimulation activates an autoregulatory
positive feedback loop of ET-1 expression that leads to transcription and
expression of GM-CSF. In this context, ETAR exclusively signals through
p38MAPK, whereas ETBR also signals through ERK-1/2. Both ETAR and
ETBR signal through p38MAPK to induce GM-CSF transcription. Additionally, GM-CSF mRNA is protected from degradation by ETBR via ERK-1/2.
Consequently, blocking the individual receptor subtypes leads to
differential effects: blocking both ETAR and ETBR with bosentan (BOS)
leads to a greater reduction in GM-CSF expression than blocking ETAR
alone with ambrisentan (AMB) because the protective effect on GM-CSF
mRNA mediated by ETBR is abolished.
antagonism concerning inflammation. We have demonstrated
a mechanism, ETBR-dependent mRNA stability, that explains
the superiority of dual blockers for GM-CSF. However, this
mechanism cannot be generalized for all cytokines with greater
sensitivity to dual than selective blockers as it does not apply to
MMP12. Moreover, we have only examined the effect on four
cytokines in this study and have shown previously that
ambrisentan is more effective in reducing transcription and
release of CXCL10, IL-23A/IL-23, and WISP1 when compared
with bosentan. Given the good safety profile of bosentan and
ambrisentan, their anti-inflammatory properties should be
further elucidated in clinical trials examining early stages of
chronic inflammatory airway diseases.
Acknowledgments
The authors thank Katja Urban for excellent technical assistance
and Arno von dem Bussche-Ippenburg for very valuable help with the
manuscript. Bosentan was provided by Actelion Pharmaceuticals.
Ambrisentan was provided by GlaxoSmithKline.
Authorship Contributions
Participated in research design: Jungck, Knobloch, Koch.
Conducted experiments: Jungck, Knobloch, Körber, Lin, Konradi,
Yanik.
Contributed new reagents or analytic tools: Stoelben.
Performed data analysis: Jungck, Knobloch.
Wrote or contributed to the writing of the manuscript: Jungck,
Knobloch, Koch.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 5. After 24 hours of stimulation with TNFa, a significant difference
in differentially blocking the individual endothelin receptor subtypes
becomes apparent. HASMCs were stimulated with TNFa at 20 ng/ml for
24 hours. One hour before stimulation with TNFa, BQ788, bosentan, or
ambrisentan (at 1026 M each) was added to the medium, respectively.
After incubation, the RNA was extracted and subjected to semiquantitative reverse-transcription polymerase chain reaction with subsequent gel
electrophoresis and densitometric analyses of the polymerase chain
reaction signals. Values for target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) references and to the unstimulated control, which was set to 1. GM-CSF levels are expressed as fold
induction versus the unstimulated control. Each graph represents the
mean 6 S.E.M. of n = 3 individual experiments. One-way repeatedmeasures analysis of variance (P = 0.0054) with post-hoc Bonferroni-Holm
tests: P values are indicated in the graph (related to solvent controls);
**P , 0.01.
571
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Jungck et al.
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