downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated...

Downregulation of protease-activated receptor-1 inhuman lung fibroblasts is specifically mediated by theprostaglandin E2 receptor EP2 through cAMP elevationand protein kinase AElena Sokolova1, Roland Hartig2 and Georg Reiser1

1 Institut fur Neurobiochemie, Medizinische Fakultat, Otto-von-Guericke-Universitat Magdeburg, Germany

2 Institut fur Immunologie, Medizinische Fakultat, Otto-von-Guericke-Universitat 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)-b1 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 E2; protease-

activated receptor-1

Correspondence

G. Reiser, Institut fur Neurobiochemie,

Medizinische Fakultat, Otto-von-Guericke-

Universitaet Magdeburg, Leipziger Strasse

44, D-39120 Magdeburg, Germany

Fax: +49 391 6713097

Tel: +49 391 6713088

E-mail: [email protected]

(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)E2, 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 PGE2. Activation of other receptors

coupled to cAMP elevation, such as b-adrenergic and adenosine receptors,

does not reproduce the effects of PGE2. PGE2-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.

PGE2-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 PGE2 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)E2. PGE2 is a

metabolite of arachidonic acid derived via the cyclo-

oxygenase pathway. PGE2 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], PGE2 can mediate its

antifibrotic effects via downregulation of the PAR-1

expression level on lung fibroblasts [3].

In the present work, we show that PGE2 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 PGE2, but not by other receptors coupled to

cAMP elevation, such as b-adrenergic receptor (AR)

and adenosine receptor A2B. PGE2-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 PGE2 in fibrotic lung

diseases.

Results

Downregulation of PAR-1 expression in human

lung fibroblasts (hLFs) is mediated via the PGE2

receptor EP2, but not by other receptors coupled

to cAMP elevation

In human lung fibroblasts, PGE2 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 PGE2 on PAR-1

expression is concentration-dependent, with an EC50

value of approximately 5 nm. The maximal effect

was reached at 100–200 nm PGE2 (data not shown).

We observed that concentrations of PGE2 higher

than 500 nm induced changes in fibroblast morp-

hology. We next examined whether activation of other

Gs-coupled receptors that are expressed on hLFs, such

as b-AR and adenosine receptor A2B, can induce down-

regulation of the PAR-1 level. We treated fibroblasts

with the b2-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

PGE2 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 PGE2, forskolin, butaprost, ISO, and

NECA.

Surprisingly, ISO and NECA appeared to be less

potent than PGE2 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. PGE2, forskolin and butaprost reduced the

PAR-1 density on the plasma membrane by 31%, 27%

and 30%, respectively (P < 0.001 for PGE2, 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.

PGE2 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 PGE2 resulted in reduction of func-

tional responses caused by the PAR receptor. For

this purpose we performed free intracellular

Ca2+concentration ([Ca2+]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-NH2). The cells

that were preincubated with PGE2 for 18 h before

the experiment exhibited a significantly lower rise of

[Ca2+]i in response to TRag (15 lm) than the

control cells (Fig. 2A). The Ca2+response of PGE2-

pretreated cells was reduced by 22% (Fig. 2D).

Pretreatment of the cells with forskolin resulted in

similar reduction of the Ca2+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 Ca2+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 PGE2-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 PGE2-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 PGE2, 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

A D

B E

C F

Fig. 1. Comparative effects of PGE2, 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 nM PGE2, 10 lM FSK, 5 lM But,

1 lM ISO, or 10 lM NECA. (A) PAR-1 mRNA levels after treatment with PGE2 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 PGE2 (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


Epac with a subsequent increase in GTP-bound Rap1

(Fig. 3A, lower panel).

Addition of H-89 (1 lm) abolished the PGE2-

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 Ca2+

responses to PAR-1 agonist TRag of hLFs treated

with PGE2 overnight in the absence and presence of

H-89, respectively. In preliminary experiments, we

showed that H-89 alone did not alter cellular

Ca2+responsiveness as compared to untreated cells. As

shown in Fig. 3B by the Ca2+response traces and the

statistical evaluation, H-89 reversed the reduction of

the Ca2+response induced by PGE2. Therefore, the

effect of PGE2 is fully PKA-dependent.

Effect of PGE2 on PAR-1 mRNA stability and

involvement of protein synthesis in the PGE2-

induced downregulation of PAR-1 expression

We evaluated whether the reduction of the steady-

state level of PAR-1 transcript after PGE2 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 PGE2

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 PGE2 is not due to decreased stability of the

mRNA.

As the effect of PGE2 becomes detectable with a

stimulus lasting for at least 3 h, we also checked

whether PGE2 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 PGE2 application and

assessed the PAR-1 transcript level after 3 or 6 h.

Pretreatment with actinomycin D did not abrogate

PGE2-mediated downregulation of the PAR-1

mRNA level. In parallel experiments, we evaluated

A B

C D

Fig. 2. Effect of PGE2, forskolin (FSK) and NECA pretreatment on PAR-1-mediated Ca2+mobilization. hLFs were pretreated with 50 nM PGE2

(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 lM TRag for 60 s. The changes of [Ca2+]i indicated by the changes in the fluorescence ratio

(F340 nm ⁄ F380 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.



the influence of inhibition of protein synthesis on

PGE2-induced downregulation of PAR-1 level. Cells

were preincubated with cycloheximide (CHX)

(10 lgÆmL)1) for 30 min before PGE2 application,

and then mRNA and protein levels of PAR-1 were

determined. We found that CHX did not influence

PGE2-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 PGE2 and

CHX further decreased the PAR-1 protein level as

compared to treatments with PGE2 alone or CHX

alone. Therefore, protein synthesis is involved neither

in control of PAR-1 gene expression under resting

conditions nor in the PGE2-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. PGE2 can induce activation

A B

Fig. 3. Involvement of PKA and Epac in PGE2-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 lM), PGE2 (50 nM), PGE2 in

the presence of the PKA inhibitor H-89 (1 lM), or H-89 alone. H-89 was added 30 min before PGE2. 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 PGE2 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 lM) 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 nM PGE2, 1 lM H-89 or both PGE2 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 lM 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. Ca2+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 PGE2 in the

presence of H-89.

CHX control

100

Cou

nts

200

160

120

8040

0

101 102

FL1-H103 104

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.



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 PGE2 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 PGE2, 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 PGE2 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 PGE2. 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 Gs-coupled recep-

tor for PGE2. Our finding that EP2 has a major role

in mediating the inhibitory effect of PGE2 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 nM 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 nM). 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 nM PGE2

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.



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-b1-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 PGE2-

mediated inhibition of collagen synthesis in lung fibro-

blasts were documented [16]. Suppression of fibroblast

chemotaxis and TGF-b1-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, PGE2-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 PGE2, we suggest that PGE2 regulates the activities

of transcription factors responsible for regulation of

PAR-1 gene expression. We observed attenuation of

the effect of PGE2 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 PGE2 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 PGE2 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 A2B and b-AR, reproduced the

effect of PGE2 on PAR-1 mRNA level with kinetics

identical to that of PGE2, but PAR-1 protein level and

receptor responsiveness remained unchanged. This dis-

crepancy in the action of PGE2 and ligands of receptor

A2B and b2-AR (NECA and ISO) might result from

different effects of those compounds on PAR-1 mRNA

stability. However, PGE2 did not influence the rate of

PAR-1 mRNA degradation. Another explanation for

the fact that only PGE2 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 b2-AR signaling was docu-

mented [36,37]. The receptor can couple to both Gs

and Gi proteins. Moreover, b2-AR coupling can be

switched from Gs to Gi protein after PKA activation

[38,39]. This duality is likely to underlie differences in

the effects of activation of EP2, A2B and b2-AR in

lung fibroblasts observed in the present work.

Additionally, a cell-specific action of PGE2 to modu-

late PAR-1 level was observed. Apparently, the expres-

sion profile of receptors for PGE2, i.e. the

predominance of either Gs or Gi 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 PGE2 at

the same concentrations was almost ineffective. This

may result from simultaneous activation of Gi-coupled

EP3 receptor by PGE2 [40]. Human airway epithelial

cells were insensitive to PGE2 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, PGE2 acts as a specific factor



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 PGE2 treatment. This broadens the spectrum of

antifibrotic effects of PGE2 and highlights the great

therapeutic potential of PGE2 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). PGE2, 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, Gottingen, Germany). DMEM, fetal

bovine serum and antibiotics (penicillin and streptomycin)

were from Biochrom KG (Berlin, Germany), Accutase�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% CO2. 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) CO2.

Cytosolic Ca2+ measurement

The [Ca2+]i was measured, as previously described [41],

using the Ca2+-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 MgCl2,

1.8 mm CaCl2, 25 mm 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 [Ca2+]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 (F340 nm ⁄F380 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¢-CCAGCAGGTCGGTGAACTCTT-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. Theamplification 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 PGE2 for 16 h. After completion of the

incubation period, cells were washed twice with NaCl ⁄Pi

and detached from flasks by treatment with nonenzymatic

Cell Dissociation Solution (Sigma) on a rocking platform



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 ⁄Pi and centri-

fuged at 300 g for 4 min. The cells were incubated with

antibodies against PAR-1 (5.0 lgÆmL)1 in NaCl ⁄Pi contain-

ing 1.0% BSA) for 1 h at 4 �C, rinsed in NaCl ⁄Pi, and

incubated with secondary antibodies conjugated to

Alexa 488 (10 lgÆmL)1) at 4 �C for 1 h. Then, cells were

rinsed with NaCl ⁄Pi 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

PGE2 (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 PGE2 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, Gottingen, 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 ⁄Pi 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

Na3VO4, 1 mm 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 fur Bildung und Forschung (BMBF, grant

01ZZ0407).

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downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated...

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