Downregulation of protease-activated receptor-1 in
human lung fibroblasts is specifically mediated by the
prostaglandin E
2
receptor EP2 through cAMP elevation
and protein kinase A
Elena Sokolova
1
, Roland Hartig
2
and Georg Reiser
1
1 Institut fu
¨
r Neurobiochemie, Medizinische Fakulta
¨
t, Otto-von-Guericke-Universita
¨
t Magdeburg, Germany
2 Institut fu
¨
r Immunologie, Medizinische Fakulta
¨
t, Otto-von-Guericke-Universita
¨
t Magdeburg, Germany
Lung fibroblasts actively participate in wound healing
after tissue injury and in inflammatory responses by
production of a vast variety of proinflammatory medi-
ators, growth factors, and extracellular matrix compo-
nents. Many of those mediators are released upon

G. Reiser, Institut fu
¨
r Neurobiochemie,
Medizinische Fakulta
¨
t, Otto-von-Guericke-
Universitaet Magdeburg, Leipziger Strasse
44, D-39120 Magdeburg, Germany
Fax: +49 391 6713097
Tel: +49 391 6713088
E-mail:
(Received 17 October 2007, revised 3 April
2008, accepted 19 May 2008)
doi:10.1111/j.1742-4658.2008.06511.x
Many cellular functions of lung fibroblasts are controlled by protease-acti-
vated receptors (PARs). In fibrotic diseases, PAR-1 plays a major role in
controlling fibroproliferative and inflammatory responses. Therefore, in
these diseases, regulation of PAR-1 expression plays an important role.
Using the selective prostaglandin EP2 receptor agonist butaprost and
cAMP-elevating agents, we show here that prostaglandin (PG)E
2
, via the
prostanoid receptor EP2 and subsequent cAMP elevation, downregulates
mRNA and protein levels of PAR-1 in human lung fibroblasts. Under
these conditions, the functional response of PAR-1 in fibroblasts is
reduced. These effects are specific for PGE
2
. Activation of other receptors
coupled to cAMP elevation, such as b-adrenergic and adenosine receptors,
does not reproduce the effects of PGE

metabolite of arachidonic acid derived via the cyclo-
oxygenase pathway. PGE
2
is the major prostanoid syn-
thesized by lung fibroblasts [11]. It can also act on
fibroblasts in a paracrine fashion after release from the
adjacent epithelial layer [12]. In addition to antifibrotic
properties, such as inhibition of fibroblast prolifera-
tion, differentiation, chemotaxis, and synthesis of col-
lagen by the cells [13–17], PGE
2
can mediate its
antifibrotic effects via downregulation of the PAR-1
expression level on lung fibroblasts [3].
In the present work, we show that PGE
2
decreases
the abundance of PAR-1 on the cell surface and the
receptor responsiveness to PAR-1 activators. The regu-
lation occurs in a cAMP- and protein kinase A
(PKA)-dependent manner. PAR-1 downregulation is
mediated exclusively by the EP2 receptor, a receptor
for PGE
2
, but not by other receptors coupled to
cAMP elevation, such as b-adrenergic receptor (AR)
and adenosine receptor A
2B
. PGE
2

2
higher
than 500 nm induced changes in fibroblast morp-
hology. We next examined whether activation of other
G
s
-coupled receptors that are expressed on hLFs, such
as b-AR and adenosine receptor A
2B
, can induce down-
regulation of the PAR-1 level. We treated fibroblasts
with the b
2
-AR agonist isoproterenol (ISO) and with
the adenosine receptor agonist adenosine-5¢-N-ethyl-
carboxamide (NECA) for 3, 6 and 24 h. ISO (1 lm)
and NECA (10 lm) downregulated the PAR-1 mRNA
level with a time dependence similar to that of
PGE
2
and the other cAMP-inducing agents (the specific
EP2 agonist butaprost, and the activator of adenylyl
cyclase forskolin). A plateau was observed during the
first 3 h of treatment, followed by a rapid decrease of
the PAR-1 mRNA level (by  70%). The effect per-
sisted for up to 24 h. Figure 1A shows the steady-state
expression level of PAR-1 after 7 h of treatment of
hLFs with PGE
2
, forskolin, butaprost, ISO, and

tional responses caused by the PAR receptor. For
this purpose we performed free intracellular
Ca
2+
concentration ([Ca
2+
]
i
) measurements in Fura-2-
AM-loaded fibroblasts and stimulated the cells with
the synthetic PAR-1-activating peptide TRag (Ala-
pFluoro-Phe-Arg-Cha-homoArg-Tyr-NH
2
). The cells
that were preincubated with PGE
2
for 18 h before
the experiment exhibited a significantly lower rise of
[Ca
2+
]
i
in response to TRag (15 lm) than the
control cells (Fig. 2A). The Ca
2+
response of PGE
2
-
pretreated cells was reduced by 22% (Fig. 2D).
Pretreatment of the cells with forskolin resulted in

like small GTPases Rap1 and Rap2.
In our work, we tested the involvement of PKA and
Epac in the PGE
2
-induced regulation of PAR-1 using
the specific PKA inhibitor H-89 and the Epac activator
8-CPT-2¢-O-Me-cAMP. PAR-1 levels were detected by
real-time PCR and by flow cytometry analysis. Appli-
cation of the Epac activator (50–400 lm) did not
reproduce the inhibitory effects of PGE
2
, butaprost
and forskolin on PAR-1 mRNA levels (Fig. 3A). Com-
parable data were obtained for PAR-1 protein levels
(data not shown). By pull-down experiments, we con-
firmed the ability of 8-CPT-2¢-O-Me-cAMP to activate
AD
BE
CF
Fig. 1. Comparative effects of PGE
2
, forskolin (FSK), butaprost (But), ISO, and NECA on PAR-1 mRNA level and receptor surface expres-
sion. hLFs were serum-starved overnight in medium containing 0.1% BSA, and then incubated with 50 n
M PGE
2
,10lM FSK, 5 lM But,
1 l
M ISO, or 10 lM NECA. (A) PAR-1 mRNA levels after treatment with PGE
2
and cAMP-elevating agents for 7 h. Total RNA was isolated

2+
responsiveness as compared to untreated cells. As
shown in Fig. 3B by the Ca
2+
response traces and the
statistical evaluation, H-89 reversed the reduction of
the Ca
2+
response induced by PGE
2
. Therefore, the
effect of PGE
2
is fully PKA-dependent.
Effect of PGE
2
on PAR-1 mRNA stability and
involvement of protein synthesis in the PGE
2
-
induced downregulation of PAR-1 expression
We evaluated whether the reduction of the steady-
state level of PAR-1 transcript after PGE
2
treatment
was due to an increase in mRNA degradation. For
this purpose, we determined the half-life of PAR-1
mRNA in the presence of the transcriptional inhibitor
actinomycin D. The treatment with actinomycin D
(7 lgÆmL

2
-mediated downregulation of the PAR-1
mRNA level. In parallel experiments, we evaluated
AB
CD
Fig. 2. Effect of PGE
2
, forskolin (FSK) and NECA pretreatment on PAR-1-mediated Ca
2+
mobilization. hLFs were pretreated with 50 nM PGE
2
(A), 10 lM FSK (B), or 10 lM NECA (C) for 16 h prior to experiments in the medium containing 2.5% fetal bovine serum. Then, cells were
loaded with fura-2 ⁄ AM and exposed to 15 l
M TRag for 60 s. The changes of [Ca
2+
]
i
indicated by the changes in the fluorescence ratio
(F
340 nm
⁄ F
380 nm
) were measured. The solid trace is the mean response of control untreated cells; the dashed trace is the mean response of
pretreated cells. Traces obtained from at least 50 single cells measured in one experiment were averaged. (D) Individual traces were ana-
lyzed and quantified. Each value represents the mean ± SE of three independent experiments. *P < 0.05; significant difference as compared
with control cells.
PAR-1 downregulation by EP2 E. Sokolova et al.
3672 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS
the influence of inhibition of protein synthesis on
PGE

of PAR-1 gene expression. However, ongoing protein
synthesis is required for maintaining the level of
PAR-1 on the cell surface.
Transcription factors potentially involved in the
regulation of PAR-1 expression
Downregulation of the PAR-1 level could be also due
to decreased transcription. PGE
2
can induce activation
AB
Fig. 3. Involvement of PKA and Epac in PGE
2
-induced downregulation of PAR-1 level. (A) Upper panel: hLFs were serum-starved overnight
in medium containing 0.1% BSA and then incubated for 7 h with the Epac agonist 8-CPT-2¢-O-Me-cAMP (200 l
M), PGE
2
(50 nM), PGE
2
in
the presence of the PKA inhibitor H-89 (1 l
M), or H-89 alone. H-89 was added 30 min before PGE
2
. Control cells were incubated with med-
ium. Total RNA was isolated and used for real-time PCR. Modulation of mRNA expression was calculated using the GAPDH gene as a refer-
ence gene. Data are means ± SE of three independent experiments. **P < 0.01; significant difference between cells treated with PGE
2
in
the presence and absence of H-89. Lower panel: hLFs were serum-starved overnight in medium containing 0.1% BSA and then treated with
the Epac agonist 8-CPT-2¢-O-Me-cAMP (200 l
M) for 15 min. GTP-Rap1 was isolated by affinity purification. Total and active Rap1 were

4
Fig. 4. Influence of inhibition of protein synthesis on PAR-1 expres-
sion level on hLFs. Flow cytometry analysis of PAR-1 surface
expression. Cells were incubated with CHX (10 lgÆmL
)1
) for 16 h,
collected using nonenzymatic cell dissociation solution, and stained
with antibodies against PAR-1.
E. Sokolova et al. PAR-1 downregulation by EP2
FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3673
of negative regulators or suppress the activity of posi-
tive regulators of the PAR-1 gene. There is evidence in
the literature that PAR-1 gene expression is under the
control of two transcription factors, i.e. Sp1 and AP-2.
Sp1 acts as a positive regulator, and AP-2 as a nega-
tive regulator [18,19]. Moreover, in cancer cell lines
and cells isolated from malignant tissues, the inverse
correlation of expression levels of AP-2 and PAR-1
was shown [19]. We tested the involvement of Sp1 and
AP-2 transcription factors in PAR-1 expression and
the possible influence of PGE
2
on their activity.
For the analysis of Sp1 involvement, we used its
specific inhibitor mithramycin A. This drug interferes
with Sp1 binding to GC-rich elements in promoter
regions. Mithramycin A activity was controlled by detec-
tion of the expression of COL1A1, which is well known
to be under the strong positive regulation of Sp1 in
human fibroblasts [20]. Indeed, 50 nm mithramycin A

of PAR-1 mRNA than untransfected cells. Silencing of
AP-2 partially reversed the effect of PGE
2
by 34%
(P < 0.05, n = 4) (Fig. 5B).
Discussion
It is now well established that PAR-1 plays a harmful
role in the development of lung fibrosis [2]. PAR-1
activation results in proliferation of lung fibroblasts,
production of extracellular matrix, and secretion of
profibrotic growth factors and cytokines [4,6,9,21,22].
In addition, PAR-1 activation in human lung fibro-
blasts protects the cells from apoptosis induced by sev-
eral apoptotic stimuli [10] and induces transformation
of fibroblasts into the myofibroblast phenotype [23].
Therefore, blocking of PAR-1 activity represents a
promising target for interfering with this lesion.
As we show here, one of the factors capable of
controlling PAR-1 on lung fibroblasts is PGE
2
. The
prostanoid suppresses PAR-1 gene expression, protein
presentation on the cell surface, and responsiveness of
PAR-1 to its specific agonist. The downregulation of
PAR-1 is a cAMP ⁄ PKA-dependent process, which is
modulated by activation of EP2, the G
s
-coupled recep-
tor for PGE
2

synthesis [16], fibroblast proliferation [16,24], differen-
tiation [15], cell migration [17], apoptosis [25], and
TGF-b
1
-induced production of profibrotic CTGF [26].
Two downstream effectors of cAMP, PKA and
Epac, a guanine nucleotide exchange factor, can be
activated in lung fibroblasts [16]. Using a specific acti-
vator of Epac, 8-CPT-2¢-O-Me-cAMP, we have shown
that Epac is not involved in downregulation of mRNA
and protein levels of PAR-1. On the other hand, inhi-
bition of PKA by its inhibitor H-89 prevented PAR-1
downregulation. Similarly, the involvement of the
PKA pathway and the lack of a role of Epac in PGE
2
-
mediated inhibition of collagen synthesis in lung fibro-
blasts were documented [16]. Suppression of fibroblast
chemotaxis and TGF-b
1
-induced synthesis of CTGF
was shown to be fully PKA-dependent [14,26]. Inter-
estingly, prostacyclin, another arachidonic acid-derived
mediator, exerted its inhibitory effect on lung fibro-
blasts via a cAMP ⁄ PKA- but not Epac-dependent
pathway [27]. Therefore, PGE
2
-induced antifibrotic
effects in lung fibroblasts are likely to be mediated
mainly by PKA.

dramatically reduced PAR-1 transcription. This implies
cell type-specific transcriptional regulation of the PAR-
1 gene. Thus, other transcription factors are responsi-
ble for basal transcription of PAR-1 and may account
for PGE
2
effects in lung fibroblasts. Recently, it was
shown that the transcription factor early growth
response-1 partially controls PAR-1 expression in
malignant cancer cells [30]. Early growth response-1
has been proposed to play an important role in the
pathogenesis of fibrosis [31,32], and therefore might be
a positive regulator of PAR-1 gene expression in lung
fibroblasts.
It is of interest to note that activation of other
receptors coupled to cAMP elevation, such as the
adenosine receptor A
2B
and b-AR, reproduced the
effect of PGE
2
on PAR-1 mRNA level with kinetics
identical to that of PGE
2
, but PAR-1 protein level and
receptor responsiveness remained unchanged. This dis-
crepancy in the action of PGE
2
and ligands of receptor
A

proteins. Moreover, b
2
-AR coupling can be
switched from G
s
to G
i
protein after PKA activation
[38,39]. This duality is likely to underlie differences in
the effects of activation of EP2, A
2B
and b
2
-AR in
lung fibroblasts observed in the present work.
Additionally, a cell-specific action of PGE
2
to modu-
late PAR-1 level was observed. Apparently, the expres-
sion profile of receptors for PGE
2
, i.e. the
predominance of either G
s
or G
i
protein-coupled
receptors (EP2 and EP3, respectively), is responsible
for its selective action on different cell types. For
example, in contrast to lung fibroblasts, in vascular

2
and highlights the great
therapeutic potential of PGE
2
or related drugs for the
treatment of fibrotic diseases.
Experimental procedures
Materials
The synthetic thrombin receptor agonist peptide TRag was
from NeoMPS SA (Strasbourg, France). PGE
2
, H-89, ISO
and NECA were purchased from Sigma (Schnelldorf,
Germany). 8-CPT-2 ¢-O-Me-cAMP, cycloheximide, actino-
mycin D and mithramycin A were from Calbiochem (La
Jolla, CA, USA). Butaprost was from Cayman Chemical
(Ann Arbor, MI, USA). Antibodies against PAR-1
(WEDE15) were from Immunotech, antibodies against AP-2
were from Abcam (Biozol, Eching, Germany), and anti-
bodies against b-tubulin were from Sigma. Alexa Fluor 488
goat anti-(mouse IgG) and fura-2 ⁄ AM were from Molecular
Probes (MoBiTec, Go
¨
ttingen, Germany). DMEM, fetal
bovine serum and antibiotics (penicillin and streptomycin)
were from Biochrom KG (Berlin, Germany), AccutaseÔ
was from PAA Laboratories (Coelbe, Germany).
Cell cultures
Primary human lung fibroblasts (CCD-25Lu) (ATCC,
Wesel, Germany) were cultured in DMEM supplemented

Hepes, pH 7.4, 145 mm NaCl, 5.4 mm KCl, 1 mm MgCl
2
,
1.8 mm CaCl
2
,25mm glucose) supplemented with 2 lm
fura-2 ⁄ AM for 30 min at 37 °C. Loaded cells were trans-
ferred into a perfusion chamber with a bath volume
of about 0.2 mL and mounted on an inverted microscope
(Axiovert 135; Zeiss, Jena, Germany). During the experi-
ments, the cells were continuously superfused with NaHBS
heated to 37 °C.
Single cell fluorescence measurements of [Ca
2+
]
i
were
performed using an imaging system from TILL Photonics
GmbH (Munich, Germany). Cells were excited alternately
at 340 nm and 380 nm for 25–75 ms at each wavelength
with a rate of 0.33 Hz, and the resultant emission was col-
lected above 510 nm. Images were stored on a personal
computer, and subsequently the changes in fluorescence
ratio (F
340 nm
⁄ F
380 nm
) were determined from selected
regions of interest covering a single cell.
Real-time RT-PCR analysis

mRNA level, which was unchanged in control and treated
cells.
Flow cytometry analysis
Lung fibroblast monolayers in 12-well tissue culture dishes
were serum-starved in DMEM containing 0.1% BSA and
treated with 50 nm PGE
2
for 16 h. After completion of the
incubation period, cells were washed twice with NaCl ⁄ P
i
and detached from flasks by treatment with nonenzymatic
Cell Dissociation Solution (Sigma) on a rocking platform
PAR-1 downregulation by EP2 E. Sokolova et al.
3676 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS
for 20 min at 37 °C. The cells were then fixed briefly at
4 °C with an equal volume of 0.2% paraformaldehyde to
preserve cell integrity during subsequent centrifugation
steps. The fixed cells were rinsed in NaCl ⁄ P
i
and centri-
fuged at 300 g for 4 min. The cells were incubated with
antibodies against PAR-1 (5.0 lgÆmL
)1
in NaCl ⁄ P
i
contain-
ing 1.0% BSA) for 1 h at 4 °C, rinsed in NaCl ⁄ P
i
, and
incubated with secondary antibodies conjugated to

activation kit (Pierce, Rockford, IL, USA) according to
the manufacturer’s protocol. Briefly, lung fibroblasts in
100 mm plates were serum-starved in DMEM containing
0.1% BSA overnight and then treated with the Epac acti-
vator 8-CPT-2¢-O-Me-cAMP or forskolin for 15 min.
Cells were washed in NaCl ⁄ Tris and lysed using the pro-
vided lysis ⁄ wash buffer containing a protease inhibitor
cocktail (Roche Molecular Biochemicals, Mannheim,
Germany). Cell lysates were incubated with Rap-binding
domain RalGDS-RBD fused to a glutathione S-transfer-
ase carrier disk. After repeated washing steps, bound
GTP-Rap1 was removed from the disk by boiling in SDS
sample buffer and analyzed by western blotting using
Rap1 antibody.
siRNA
siRNA against AP-2 and nonsilencing siRNA labeled with
Alexa Fluor 488 as a scrambled siRNA control were from
Qiagen (Heidelberg, Germany). hLFs were transfected at
70–80% density with AP-2 siRNA using MATra-A (mag-
net-assisted transfection for adherent cells) reagent (IBA
GmbH, Go
¨
ttingen, Germany), according to the manu-
facturer’s protocol. AP-2 knockdown was assessed by
real-time RT-PCR and western blotting at 24, 36 and 48 h
after transfection.
Western blot analysis
Fibroblasts were transfected with AP-2 siRNA and incu-
bated in full medium for 36 and 48 h. Then, cells were
washed twice with ice-cold NaCl⁄ P

tion, with P < 0.05 considered as significant.
Acknowledgements
This work was supported by grants from the Bundes-
ministerium fu
¨
r Bildung und Forschung (BMBF, grant
01ZZ0407).
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