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 Elena Sokolova1, Roland Hartig2 and Georg Reiser1 Institut fur Neurobiochemie, Medizinische Fakultat, Otto-von-Guericke-Universitat Magdeburg, Germany ¨ ¨ ¨ Institut fur Immunologie, Medizinische Fakultat, Otto-von-Guericke-Universitat Magdeburg, Germany ă ă ă Keywords cAMP; E prostanoid receptor; lung fibroblasts; prostaglandin E2; proteaseactivated receptor-1 Correspondence G Reiser, Institut fur Neurobiochemie, ă Medizinische Fakultat, Otto-von-Guerickeă Universitaet Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany Fax: +49 391 6713097 Tel: +49 391 6713088 E-mail: georg.reiser@med.ovgu.de (Received 17 October 2007, revised 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-activated 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 Lung fibroblasts actively participate in wound healing after tissue injury and in inflammatory responses by production of a vast variety of proinflammatory mediators, growth factors, and extracellular matrix components 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 development of fibrosis and accompanied inflammation Studies with fibroblast cell lines revealed that activation of PAR-1 mediates many profibrotic effects, such as cell proliferation, collagen synthesis, release of the chemokines interleukin-8, monocyte chemotactic protein-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 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 PAR-1 downregulation by EP2 E Sokolova et al of pulmonary fibrosis associated with scleroderma (systemic sclerosis) [10] Thus, PAR-1 activation in fibroblasts 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 cyclooxygenase pathway PGE2 is the major prostanoid synthesized 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 proliferation, differentiation, chemotaxis, and synthesis of collagen 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 regulation 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 degradation 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 downregulation 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 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 morphology 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 downregulation of the PAR-1 level We treated fibroblasts with the b2-AR agonist isoproterenol (ISO) and with 3670 the adenosine receptor agonist adenosine-5¢-N-ethylcarboxamide (NECA) for 3, 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 h of treatment, followed by a rapid decrease of the PAR-1 mRNA level (by  70%) The effect persisted for up to 24 h Figure 1A shows the steady-state expression level of PAR-1 after 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 functional responses caused by the PAR receptor For this purpose we performed free intracellular Ca2+concentration ([Ca2+]i) measurements in Fura-2AM-loaded fibroblasts and stimulated the cells with the synthetic PAR-1-activating peptide TRag (AlapFluoro-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 PGE2pretreated 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) FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS E Sokolova et al PAR-1 downregulation by EP2 A D B E C F Fig Comparative effects of PGE2, forskolin (FSK), butaprost (But), ISO, and NECA on PAR-1 mRNA level and receptor surface expression hLFs were serum-starved overnight in medium containing 0.1% BSA, and then incubated with 50 nM PGE2, 10 lM FSK, lM But, lM ISO, or 10 lM NECA (A) PAR-1 mRNA levels after treatment with PGE2 and cAMP-elevating agents for 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 means ± SE of three independent experiments (B–F) Flow cytometry analysis of PAR-1 surface expression Cells were incubated with cAMP-elevating 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 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 activation of PKA by cAMP, followed by phosphorylation of the transcription factor cAMP response elementbinding protein Activated cAMP response element-binding protein then binds to cAMP response elements on the DNA and thereby regulates the transcription 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 Raslike 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 Application 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) Comparable data were obtained for PAR-1 protein levels (data not shown) By pull-down experiments, we confirmed the ability of 8-CPT-2¢-O-Me-cAMP to activate FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3671 PAR-1 downregulation by EP2 E Sokolova et al A B C D Fig 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 analyzed and quantified Each value represents the mean ± SE of three independent experiments *P < 0.05; significant difference as compared with control cells Epac with a subsequent increase in GTP-bound Rap1 (Fig 3A, lower panel) Addition of H-89 (1 lm) abolished the PGE2induced 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 PGE2induced downregulation of PAR-1 expression We evaluated whether the reduction of the steadystate level of PAR-1 transcript after PGE2 treatment 3672 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 lgỈmL)1) did not appreciably decrease the basal expression of PAR-1 over a period of h Moreover, 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 actinomycin D to inhibit transcription of collagen a1 type I gene (COL1A1) in hLFs (data not shown) Therefore, 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 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 before PGE2 application and assessed the PAR-1 transcript level after or h Pretreatment with actinomycin D did not abrogate PGE2-mediated downregulation of the PAR-1 mRNA level In parallel experiments, we evaluated FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS E Sokolova et al PAR-1 downregulation by EP2 A B control 40 80 120 160 200 CHX the influence of inhibition of protein synthesis on PGE2-induced downregulation of PAR-1 level Cells were preincubated with cycloheximide (CHX) (10 lgỈmL)1) for 30 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 Counts Fig 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 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 before PGE2 Control cells were incubated with medium 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 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 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, 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 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-89 100 101 102 103 104 FL1-H Fig Influence of inhibition of protein synthesis on PAR-1 expression level on hLFs Flow cytometry analysis of PAR-1 surface expression Cells were incubated with CHX (10 lgỈmL)1) for 16 h, collected using nonenzymatic cell dissociation solution, and stained with antibodies against PAR-1 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 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3673 PAR-1 downregulation by EP2 E Sokolova et al of negative regulators or suppress the activity of positive 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 negative 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 detection 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 A B 80–96% in hLFs, as determined by real-time PCR However, there was only a negligible effect of mithramycin 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 regulated differentially by Sp1 The involvement of the second transcription factor, AP-2, in PAR-1 expression was tested by small interfering RNA (siRNA) methodology When we knocked down the endogenous AP-2 by transfection of fibroblasts with specific AP-2 siRNA (100 nm), the expression 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 specificity 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, fibroblasts 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 Fig 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 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; significant difference as compared with scrambled siRNA-transfected cells 3674 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 fibroblasts protects the cells from apoptosis induced by several 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 receptor for PGE2 Our finding that EP2 has a major role in mediating the inhibitory effect of PGE2 on human FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS E Sokolova et al 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], differentiation [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 activator 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, inhibition 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 PGE2mediated inhibition of collagen synthesis in lung fibroblasts were documented [16] Suppression of fibroblast chemotaxis and TGF-b1-induced synthesis of CTGF was shown to be fully PKA-dependent [14,26] Interestingly, prostacyclin, another arachidonic acid-derived mediator, exerted its inhibitory effect on lung fibroblasts 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 factor 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 reduction of the PGE2 effect in hLFs Moreover, we did not observe an effect of inhibition of Sp1, which is a positive 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 inhibition, 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 PAR1 gene Thus, other transcription factors are responsible 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 PAR-1 downregulation by EP2 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 discrepancy 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 documented [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 modulate PAR-1 level was observed Apparently, the expression 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 regulation, as was found by us (Sokolova and Reiser, unpublished 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 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3675 PAR-1 downregulation by EP2 E Sokolova et al 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 (Axiovert 135; Zeiss, Jena, Germany) During the experiments, 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 collected 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 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, actinomycin 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 antibodies 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 ă 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 lgỈmL)1 penicillin and streptomycin at 37 °C in a humidified atmosphere of 10% CO2 Confluent cells were enzymatically passaged with a split ratio of : to : 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 lgỈmL)1 penicillin and streptomycin and kept at 37 °C in a humidified atmosphere 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 mL of Hepes-buffered saline (NaHBS, containing 20 mm Hepes, pH 7.4, 145 mm NaCl, 5.4 mm KCl, mm MgCl2, 1.8 mm CaCl2, 25 mm glucose) supplemented with lm fura-2 ⁄ AM for 30 at 37 °C Loaded cells were transferred into a perfusion chamber with a bath volume of about 0.2 mL and mounted on an inverted microscope 3676 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 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 follows: 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 glyceraldehyde3-phosphate dehydrogenase (GAPDH), forward 5¢-CAAAA TCAAGTGGGGCGATGCT-3¢, reverse 5¢-ACCACCTGG TGCTCAGTGTAGC-3¢ The use of intron-flanking primers, 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 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 min, and the final melting curve program with a ramping rate of 0.5 °CỈs)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 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 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS E Sokolova et al for 20 at 37 °C The cells were then fixed briefly at °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 centrifuged at 300 g for The cells were incubated with antibodies against PAR-1 (5.0 lgỈmL)1 in NaCl ⁄ Pi containing 1.0% BSA) for h at °C, rinsed in NaCl ⁄ Pi, and incubated with secondary antibodies conjugated to Alexa 488 (10 lgỈmL)1) at °C for h Then, cells were rinsed with NaCl ⁄ Pi and stored in 1.0% paraformaldehyde at °C until they were measured by flow cytometry An unstained sample and a sample stained only with the secondary antibody were analyzed in each experiment Cell surface-bound fluorescence was analyzed by flow cytometry (LSR I; BD Biosciences, San Jose, CA, USA) and quantified 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 or h Then, actinomycin D (7 lgỈmL)1) was added to stop gene transcription Total RNA was isolated at 0, 1, and h after addition of actinomycin D In another set of experiments, total RNA from the cells exposed to PGE2 without actinomycin 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 activator 8-CPT-2¢-O-Me-cAMP or forskolin for 15 Cells were washed in NaCl ⁄ Tris and lysed using the provided 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-transferase 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 (magnet-assisted transfection for adherent cells) reagent (IBA PAR-1 downregulation by EP2 GmbH, Gottingen, Germany), according to the manuă facturers 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 incubated 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, mm EDTA, mm Na3VO4, mm NaF, protease inhibitor cocktail) Cell suspensions were rotated for 15 at °C and centrifuged at 14 000 g for 15 at °C The protein concentration was determined by the Bradford method (Bio-Rad Protein Assay; Bio-Rad), using BSA as standard Samples containing equal amounts of protein (30 lg) were separated by 12.5% SDS ⁄ PAGE, transferred to nitrocellulose membranes (Hybond C; Amersham Biosciences), and blocked with 3% BSA The blots were developed by incubation with antibodies against AP-2a (1 : 200) overnight at °C, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (1 : 20 000) for h at room temperature Bands were visualized by enhanced chemiluminescence (Super-Signal West Pico; Pierce) and Hyperfilm ECL (Amersham Biosciences) 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A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi Demonstration in a reconstituted system J Biol Chem 277, 31249–31256 40 Pape R, Rauch BH, Rosenkranz AC, Kaber G & Schror K (2008) Transcriptional inhibition of proteaseactivated receptor-1 expression by prostacyclin in human vascular smooth muscle cells Arterioscler Thromb Vasc Biol 28, 534–540 41 Kahlert S & Reiser G (2004) Glial perspectives of metabolic states during cerebral hypoxia – calcium regulation and metabolic energy Cell Calcium 36, 295–302 FEBS Journal 275 (2008) 3669–3679 ª 2008 The Authors Journal compilation ª 2008 FEBS 3679 ... cyclooxygenase pathway PGE2 is the major prostanoid synthesized by lung fibroblasts [11] It can also act on fibroblasts in a paracrine fashion after release from the adjacent epithelial layer [12] In addition... 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... fibrotic gingiva: interactions between cAMP and MAPK signaling pathways, and prostaglandin E2- EP3 receptor mediated activation of the c-JUN N-terminal kinase J Biol Chem 282, 15416–15429 27 Kamio