Báo cáo khoa học: Downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated by the prostaglandin E2 receptor EP2 through cAMP elevation and protein kinase A pot

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

activation of protease-activated receptors (PARs) [1,2].
Human lung fibroblasts express three PAR subtypes,
PAR-1 to PAR-3. PAR-1 has been shown by us to be
the most abundant and the main functional receptor
among the PARs in primary human lung fibroblasts
[3].
PAR-1 activation has a strong impact on the devel-
opment of fibrosis and accompanied inflammation.
Studies with fibroblast cell lines revealed that activa-
tion of PAR-1 mediates many profibrotic effects, such
as cell proliferation, collagen synthesis, release of the
chemokines interleukin-8, monocyte chemotactic pro-
tein-1, and interleukin-6, and the profibrotic growth
factors connective tissue growth factor (CTGF) and
platelet-derived growth factor [4–7]. In PAR-1-deficient
mice, inflammatory cell recruitment, pulmonary edema,
collagen accumulation and expression of CTGF and
transforming growth factor (TGF)-b
1
was reduced in
response to bleomycin-induced fibrosis [8,9]. Moreover,
the PAR-1 protein level is increased in lung tissues of
patients with pulmonary fibrosis [8] and in early stages
Keywords
cAMP; E prostanoid receptor; lung
fibroblasts; prostaglandin E
2
; protease-
activated receptor-1
Correspondence

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

2
. PGE
2
-mediated downregulation of
PAR-1 depends mainly on protein kinase A activity, but does not depend
on another cAMP effector, the exchange protein activated by cAMP.
PGE
2
-induced reduction of PAR-1 level is not due to a decrease of PAR-1
mRNA stability, but rather to transcriptional regulation. The present
results provide further insights into the therapeutic potential of PGE
2
to
specifically control fibroblast function in fibrotic diseases.
Abbreviations
AR, adrenergic receptor; CHX, cycloheximide; CTGF, connective tissue growth factor; Epac, exchange protein activated by cAMP; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; hLF, human lung fibroblast; ISO, isoproterenol; NECA, adenosine-5¢-N-ethylcarboxamide; PAR,
protease-activated receptor; PG, prostaglandin; PKA, protein kinase A; siRNA, small interfering RNA; TGF, transforming growth factor; TRag,
thrombin receptor agonist.
FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3669
of pulmonary fibrosis associated with scleroderma (sys-
temic sclerosis) [10]. Thus, PAR-1 activation in fibro-
blasts seems to play an important role during the
development of fibrotic diseases.
One of the factors that can suppress functions of
lung fibroblasts is prostaglandin (PG)E
2
. PGE
2
is a

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

-induced reduction
of the PAR-1 level is likely to be due to a decrease in
gene transcription but not increased mRNA degra-
dation. These findings extend our knowledge of the
control of fibroblast functions and shed further light
on the therapeutic potential of PGE
2
in fibrotic lung
diseases.
Results
Downregulation of PAR-1 expression in human
lung fibroblasts (hLFs) is mediated via the PGE
2
receptor EP2, but not by other receptors coupled
to cAMP elevation
In human lung fibroblasts, PGE
2
causes downregula-
tion of PAR-1 gene expression in a time-dependent
manner via the EP2 receptor [3]. In the present
work, we found that the effect of PGE
2
on PAR-1
expression is concentration-dependent, with an EC
50
value of approximately 5 nm. The maximal effect
was reached at 100–200 nm PGE
2
(data not shown).
We observed that concentrations of PGE

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

NECA.
Surprisingly, ISO and NECA appeared to be less
potent than PGE
2
and the other cAMP-inducing
agents in terms of reduction of PAR-1 protein on the
cell surface, as assessed by flow cytometry analysis
(Fig. 1B–E). The statistical evaluation is given in
Fig. 1F. PGE
2
, forskolin and butaprost reduced the
PAR-1 density on the plasma membrane by 31%, 27%
and 30%, respectively (P < 0.001 for PGE
2
, P < 0.01
for butaprost and forskolin, n = 5), whereas the
reduction by ISO and NECA amounted to only 5–7%.
Therefore, we conclude that the regulation of PAR-1 is
a specific process triggered by activation of a specific
receptor, namely EP2.
PGE
2
and forskolin but not ISO and NECA reduce
cell responsiveness to the PAR-1-specific agonist
thrombin receptor agonist (TRag)
We checked whether the reduction of PAR-1 protein
on the plasma membrane of hLFs after treatment of
the cells with PGE
2
resulted in reduction of func-

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

similar reduction of the Ca
2+
response (by 20%)
(Fig. 2B,D). The degree of decrease is comparable to
the degree of reduction of PAR-1 protein on the cell
surface. Consistent with the flow cytometry data, no
changes in the Ca
2+
response were observed after
pretreatment of the cells with NECA (Fig. 2C,D)
and ISO (data not shown).
PAR-1 downregulation by EP2 E. Sokolova et al.
3670 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS
Involvement of alternative cAMP-induced
signaling pathways in the PGE
2
-induced
downregulation of PAR-1
At present, two distinct cAMP-dependent signaling
pathways are known. The first one includes the activa-
tion of PKA by cAMP, followed by phosphorylation
of the transcription factor cAMP response element-
binding protein. Activated cAMP response
element-binding protein then binds to cAMP response
elements on the DNA and thereby regulates the tran-
scription of genes, either positively or negatively.
Another pathway includes the direct activation of
Epac (exchange protein directly activated by cAMP)
by cAMP. Epac works as cAMP-sensitive guanine
nucleotide exchange factor (cAMP-GEF) for the Ras-

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

and used for real-time PCR. Modulation of mRNA expression was calculated using the GAPDH gene as a reference gene. Data are mean-
s ± SE of three independent experiments. (B–F) Flow cytometry analysis of PAR-1 surface expression. Cells were incubated with cAMP-ele-
vating agents for 16 h, collected using nonenzymatic cell dissociation solution, stained with antibodies against PAR-1, and analyzed by flow
cytometry. (B–E) Representative histograms obtained by flow cytometry analysis in unstimulated hLFs and cells treated with PGE
2
(B), But
(C), ISO and NECA (D), and FSK (E). (F) Quantification of the data, expressed as percentage change of mean fluorescence intensity, gives
the reduction of PAR-1 expression on hLFs. Each value represents the mean ± SE of at least three independent experiments. *P < 0.05,
**P < 0.01, ***P < 0.001; significant difference as compared with unstimulated conditions.
E. Sokolova et al. PAR-1 downregulation by EP2
FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3671
Epac with a subsequent increase in GTP-bound Rap1
(Fig. 3A, lower panel).
Addition of H-89 (1 lm) abolished the PGE
2
-
induced downregulation of PAR-1 mRNA by 78%
(P < 0.01, n = 4), whereas this PKA inhibitor alone
did not influence the PAR-1 level (Fig. 3A). Similar
results were obtained with another PKA inhibitor,
KT-5720 (1.5 lm; data not shown). To confirm the
data obtained on mRNA level, we compared Ca
2+
responses to PAR-1 agonist TRag of hLFs treated
with PGE
2
overnight in the absence and presence of
H-89, respectively. In preliminary experiments, we
showed that H-89 alone did not alter cellular
Ca

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

)1
) did not appreciably decrease the basal
expression of PAR-1 over a period of 6 h. More-
over, there was no alteration in the degradation
rate of PAR-1 mRNA in cells stimulated with PGE
2
as compared to unstimulated cells. In additional
control experiments, we showed the ability of actino-
mycin D to inhibit transcription of collagen a1 type I
gene (COL1A1) in hLFs (data not shown). There-
fore, the reduced expression of PAR-1 after exposure
to PGE
2
is not due to decreased stability of the
mRNA.
As the effect of PGE
2
becomes detectable with a
stimulus lasting for at least 3 h, we also checked
whether PGE
2
induces the transcription and protein
synthesis of factors that participate in further steps
leading to decreased PAR-1 expression. We added
actinomycin D 30 min before PGE
2
application and
assessed the PAR-1 transcript level after 3 or 6 h.
Pretreatment with actinomycin D did not abrogate
PGE

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

2
-induced downregulation of PAR-1 level. Cells
were preincubated with cycloheximide (CHX)
(10 lgÆmL
)1
) for 30 min before PGE
2
application,
and then mRNA and protein levels of PAR-1 were
determined. We found that CHX did not influence
PGE
2
-induced downregulation of the PAR-1 mRNA
level, as detected by real-time PCR analysis (data
not shown). Incubation of the cells with CHX alone
resulted in a decreased amount of PAR-1 on the
plasma membrane, with a reduction by  28% as
compared to untreated cells (Fig. 4). Furthermore,
simultaneous treatment of the cells with PGE
2
and
CHX further decreased the PAR-1 protein level as
compared to treatments with PGE
2
alone or CHX
alone. Therefore, protein synthesis is involved neither
in control of PAR-1 gene expression under resting
conditions nor in the PGE
2
-induced downregulation

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

detected by western blot analysis. (B) The cells were pretreated with 50 n
M PGE
2
,1lM H-89 or both PGE
2
and H-89 for 16 h 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, as described in Fig. 2.
The traces are the mean value of at least 50 single cells measured in one experiment and are representative of three different experiments.
In the histogram, each value represents the mean ± SE of three independent experiments. Ca
2+
responses of the cells treated with H-89
were undistinguishable from those of untreated cells and were taken as a control. *P < 0.05 as compared to stimulation with PGE
2
in the
presence of H-89.
CHX
control
10
0
Counts
20016012080400
10
1
10
2
FL1-H
10
3
10

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

strongly decreased the COL1A1 mRNA level by
80–96% in hLFs, as determined by real-time PCR.
However, there was only a negligible effect of mithra-
mycin A on the PAR-1 mRNA level in hLFs (Fig. 5A).
For comparison, we tested whether mithramycin A
has the ability to influence the basal gene expression of
PAR-1 in other cell lines expressing this receptor. We
used the human astrocytoma cell line 1321N1 and the
human alveolar epithelial cell line A549. In 1321N1
cells, mithramycin A reduced the PAR-1 mRNA level
by 60–75%, whereas in A549 cells, this drug did not
exert any noticeable effect (Fig. 5A). Therefore, we can
conclude that in different cells the PAR-1 gene is regu-
lated differentially by Sp1.
The involvement of the second transcription factor,
AP-2, in PAR-1 expression was tested by small inter-
fering RNA (siRNA) methodology. When we knocked
down the endogenous AP-2 by transfection of fibro-
blasts with specific AP-2 siRNA (100 nm), the expres-
sion of AP-2 was reduced by 75% after 24–36 h of
transfection, and by 60% after 48 h of transfection, as
determined by real-time PCR. Scrambled siRNA did
not affect the AP-2 expression, confirming the speci-
ficity of AP-2 siRNA. Reduction of AP-2 was also
confirmed by western blot analysis (Fig. 5B, inset).
Silencing of AP-2 itself did not affect the PAR-1
expression level. After treatment with PGE
2
, fibro-
blasts with knocked down AP-2 expressed higher levels

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

. Our finding that EP2 has a major role
in mediating the inhibitory effect of PGE
2
on human
A
B
Fig. 5. Involvement of transcription factors Sp1 and AP-2 in the
regulation of PAR-1 expression. (A) hLFs, A549 cells and 1321N1
cells were incubated with 50 n
M mithramycin A for 24 h. Then,
total RNA was isolated and used for real-time PCR for detection of
PAR-1 expression level (gray bars). The level of collagen (COL1A1)
in hLFs (dashed bar) was determined as a positive control. (B) hLFs
were transfected with AP-2 siRNA (100 n
M). AP-2 knockdown was
determined by western blot analysis 36 h after transfection.
b-Tubulin served as a loading control. For experiments, after 24 h
of incubation with AP-2 siRNA, cells were treated with 50 n
M PGE
2
for an additional 7 h. Total RNA was isolated and used for real-time
PCR. Control cells were transfected with scrambled siRNA. Data
are means ± SE of four independent experiments. *P < 0.05; sig-
nificant difference as compared with scrambled siRNA-transfected
cells.
PAR-1 downregulation by EP2 E. Sokolova et al.
3674 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS
lung fibroblasts, as observed in the present work, is in
good agreement with studies of other research groups.
EP2 activation resulted in inhibition of collagen

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.

Downregulation of PAR-1 is likely to be regulated
at the transcriptional level rather than by an altered
mRNA degradation rate. As we showed that de novo
protein synthesis is not required to mediate the effects
of PGE
2
, we suggest that PGE
2
regulates the activities
of transcription factors responsible for regulation of
PAR-1 gene expression. We observed attenuation of
the effect of PGE
2
by silencing of the transcription fac-
tor AP-2. The role of AP-2 as a repressor of PAR-1
gene expression was proposed for human melanoma
cells [19,28]. Moreover, AP-2 can be activated by
signals leading to cAMP elevation [29].
However, AP-2 silencing resulted in partial reduc-
tion of the PGE
2
effect in hLFs. Moreover, we did not
observe an effect of inhibition of Sp1, which is a posi-
tive transcriptional regulator of the PAR-1 gene and a
competitor of AP-2 for binding to the regulatory
region of the PAR-1 gene [19,29]. Interestingly, in the
human alveolar epithelial A549 cell line, Sp1 inhibi-
tion, as in human lung fibroblasts, did not influence
the PAR-1 basal transcription, whereas in the human
astrocytoma cell line 1321N1, the inhibition of Sp1

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

2B
and b
2
-AR (NECA and ISO) might result from
different effects of those compounds on PAR-1 mRNA
stability. However, PGE
2
did not influence the rate of
PAR-1 mRNA degradation. Another explanation for
the fact that only PGE
2
stimulation results in the
reduction of PAR-1 protein on the cell surface could
be differential modulation of the translation rate or
the rate of internalization and degradation of PAR-1
protein.
As we and others [15–17,26,33] have shown the role
of cAMP in the suppression of fibroblast function and
promotion of the antifibrotic phenotype, we assume
that non-cAMP-dependent mechanisms may account
for the lack of the effects of ISO and NECA. Indeed,
in different cell types, NECA and ISO were shown to
exert their effects via cAMP-independent mechanisms
[34,35]. The duality of b
2
-AR signaling was docu-
mented [36,37]. The receptor can couple to both G
s
and G
i

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

smooth muscle cells an efficient negative regulator of
PAR-1 expression was prostacyclin, whereas PGE
2
at
the same concentrations was almost ineffective. This
may result from simultaneous activation of G
i
-coupled
EP3 receptor by PGE
2
[40]. Human airway epithelial
cells were insensitive to PGE
2
in terms of PAR regula-
tion, as was found by us (Sokolova and Reiser, unpub-
lished results). Thus, given the important role of lung
fibroblasts and their PAR-1 in the development of
pulmonary fibrosis, PGE
2
acts as a specific factor
E. Sokolova et al. PAR-1 downregulation by EP2
FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3675
that downregulates PAR-1 in this cell type to provide
the antifibrotic phenotype.
In summary, we revealed that PAR-1 level and
PAR-1 responsiveness can be decreased selectively
after PGE
2
treatment. This broadens the spectrum of
antifibrotic effects of PGE

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

with 10% fetal bovine serum and 100 lgÆmL
)1
penicillin
and streptomycin at 37 °C in a humidified atmosphere of
10% CO
2
. Confluent cells were enzymatically passaged with
a split ratio of 1 : 3 to 1 : 4, using Accutase to minimize
the proteolytic activation of PARs. A549 cells from ATCC
and 1321N1 cells were cultured in DMEM supplemented
with 10% fetal bovine serum and 100 lgÆmL
)1
penicillin
and streptomycin and kept at 37 °C in a humidified atmo-
sphere of 5% (A549 cells) and 10% (1321N1 cells) CO
2
.
Cytosolic Ca
2+
measurement
The [Ca
2+
]
i
was measured, as previously described [41],
using the Ca
2+
-sensitive fluorescent dye fura-2 ⁄ AM. For
dye loading, the cells grown on a coverslip were placed in
1 mL of Hepes-buffered saline (NaHBS, containing 20 mm

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

Total RNA was isolated from the cells with the RNeasy
Kit (Qiagen, Hilden, Germany). The isolation included
DNase treatment. Reverse transcription was carried out
with 1 lg of each RNA with an iScript cDNA synthesis kit
(Bio-Rad, Munich, Germany) in a final volume of 20 lL,
according to the manufacturer’s protocol. Real-time PCR
was performed on the iCycler (Bio-Rad) in a 25 lL reaction
volume using SYBR green PCR Master Mix (Bio-Rad), as
described by the manufacturer. The primers used were as fol-
lows: PAR-1, forward 5¢-CCTGCTTCAGTCTGTGC-3 ¢,
reverse 5¢-CCAGGTGCAGCATGTACA-3¢; COL1A1,
forward 5¢-CAAGACGAAGACATCCCACCA-3¢, reverse
5¢-CAGATCACGTCATCGCACAACA-3¢; AP-2, forward
5¢-ATGCCGTCTCCGCCATCCCTAT-3¢, reverse 5¢-CCA
GCAGGTCGGTGAACTCTT-3¢; and glyceraldehyde-
3-phosphate dehydrogenase (GAPDH), forward 5¢-CAAAA
TCAAGTGGGGCGATGCT-3¢, reverse 5¢-ACCACCTGG
TGCTCAGTGTAGC-3¢. The use of intron-flanking prim-
ers, in addition to DNase treatment during RNA isolation,
excludes the possibility of genomic DNA amplification. The
thermal cycling conditions included a denaturation step at
95 °C for 3 min, followed by 30 cycles at 94 °C for 30 s,
58 °C (PAR-1, AP-2, and GAPDH) or 55 °C (COL1A1) for
90 s, and 72 °C for 1 min, and the final melting curve pro-
gram with a ramping rate of 0.5 ° CÆs
)1
from 55 to 95 °C. The
amplification specificity of PCR products was confirmed by
melting curve analysis and agarose gel electrophoresis. All
mRNA measurements were normalized to the GAPDH

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

Alexa 488 (10 lgÆ mL
)1
)at4°C for 1 h. Then, cells were
rinsed with NaCl ⁄ P
i
and stored in 1.0% paraformaldehyde
at 4 °C until they were measured by flow cytometry. An
unstained sample and a sample stained only with the sec-
ondary antibody were analyzed in each experiment. Cell
surface-bound fluorescence was analyzed by flow cytometry
(LSR I; BD Biosciences, San Jose, CA, USA) and quanti-
fied using cell quest software (BD Biosciences).
mRNA stability experiments
Cells were serum-starved and then incubated with either
PGE
2
(50 nm) or with vehicle control for 4 or 6 h. Then,
actinomycin D (7 lgÆmL
)1
) was added to stop gene tran-
scription. Total RNA was isolated at 0, 1, 3 and 6 h after
addition of actinomycin D. In another set of experiments,
total RNA from the cells exposed to PGE
2
without actino-
mycin D was isolated at the same time points. The PAR-1
expression level was quantified by real-time PCR analysis
and normalized to the GAPDH level.
GTP-Rap1 affinity purification
Rap1 activity was measured using the EZ-Detect RAP1

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

i
and lysed in modified
RIPA buffer (50 nm Tris ⁄ HCl, pH 7.4, 150 nm NaCl, 1%
Igepal, 0.25% sodium deoxycholate, 1 mm EDTA, 1 mm
Na
3
VO
4
,1mm NaF, protease inhibitor cocktail). Cell sus-
pensions were rotated for 15 min at 4 °C and centrifuged at
14 000 g for 15 min at 4 °C. The protein concentration was
determined by the Bradford method (Bio-Rad Protein
Assay; Bio-Rad), using BSA as standard. Samples contain-
ing equal amounts of protein (30 lg) were separated by
12.5% SDS ⁄ PAGE, transferred to nitrocellulose mem-
branes (Hybond C; Amersham Biosciences), and blocked
with 3% BSA. The blots were developed by incubation
with antibodies against AP-2a (1 : 200) overnight at 4 °C,
followed by incubation with horseradish peroxidase-conju-
gated anti-mouse IgG (1 : 20 000) for 1 h at room tempera-
ture. Bands were visualized by enhanced chemiluminescence
(Super-Signal West Pico; Pierce) and Hyperfilm ECL
(Amersham Biosciences). After stripping, the membranes
were reprobed with antibodies against b-tubulin
(1 : 40 000). Quantification of the band densities was
carried out using a GS-800 calibrated densitometer and
quantity one software (Bio-Rad).
Statistical analysis
Statistical evaluation was carried out by t-test and multiple
comparisons by one-way ANOVA with Dunnett’s correc-

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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