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Am J Physiol Gastrointest Liver Physiol 293: G128-G136, 2007. First published March 22, 2007; doi:10.1152/ajpgi.00031.2007
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LIVER AND BILIARY TRACT

Thrombin inhibits migration of human hepatic myofibroblasts

Jennifer Gillibert-Duplantier,1,2 Véronique Neaud,1,2 Jean-Frédéric Blanc,1,3 Paulette Bioulac-Sage,1,2 and Jean Rosenbaum1,2

1Institut National de la Santé et de la Recherche Médicale, U-889, 2Institut Fédératif de Recherche 66, Université Victor Segalen Bordeaux 2, and 3Centre Hospitalier Universitaire de Bordeaux, Groupement des Spécialités Digestives, Bordeaux, France

Submitted 15 January 2007 ; accepted in final form 21 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E2 and its EP2 receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.

liver fibrosis; hepatic stellate cell; cyclooxygenase; protease-activated receptor; phosphatidylinositol 3-kinase

FIBROSIS IS THE COMMON RESPONSE of the liver to many chronic liver injuries such as chronic viral hepatitis, alcoholic liver disease, and others. Liver fibrosis is characterized by an excessive deposition of extracellular matrix (ECM) components, consecutive to both an increased synthesis and a relative failure of degradation. The synthesis of ECM components is mainly due to liver myofibroblasts (25). Myofibroblasts are absent from the normal liver and arise from the differentiation of precursor cells, both hepatic stellate cells and portal fibroblasts (3, 20, 47). The phenotype of activated myofibroblasts includes the ability to synthesize high levels of ECM components and inhibitors of matrix degradation (i.e., tissue inhibitor of matrix metalloproteinases-1), a high proliferation rate, a high-level expression of {alpha}-smooth muscle actin (a cytoskeletal protein involved in contraction and migration), and an increased ability to migrate. The control of myofibroblast migration is likely to play a significant role in liver fibrogenesis. Indeed, in the early phases of liver injury, migration enables the rapid recruitment of fibrogenic cells to the lesion site before their proliferation. On the other hand, in later stages, inhibition of migration may be an efficient mechanism to stabilize activated fibrogenic cells on the site of the lesion.

The mechanisms of liver fibrogenic cell migration have already been addressed, and many studies have identified active mediators. Besides cytokines such as monocyte chemotactic protein 1 (30), RANTES (regulated on activation normal T-expressed and presumably secreted) (40), or endothelin-1 (45), growth factors like transforming growth factor-beta1 or epidermal growth factor (EGF) (49), and ECM components (49), these studies have highlighted the role of platelet-derived growth factor-BB (PDGF-BB) as a major chemotactic agent for these cells (19, 28). Because PDGF-BB and its receptor are overexpressed during liver fibrosis (37, 38), it is likely that PDGF-BB is involved in the control of myofibroblast migration in vivo. However, fibrogenesis is a complex process in which many different active molecules are generated at the same time and at the same location. Thus the resulting chemotactic effect of PDGF-BB may be influenced by environmental molecules.

It has been shown that the blood-coagulation process is activated during liver fibrogenesis, as evidenced by the deposition of fibrin (34). This implies that thrombin generation occurs in the liver. Thrombin converts soluble fibrinogen into insoluble fibrin. Besides this major hemostatic function, thrombin can also activate intracellular signal transduction via the activation of specific receptors, the protease-activated receptors (PARs). This family includes four receptors (reviewed in Ref. 6) belonging to the larger family of seven-transmembrane-domain receptors, and they are coupled to heterotrimeric G proteins. The mechanism of activation of these receptors is quite unusual. Indeed, thrombin cleaves the NH2-terminal part of the receptor, revealing a new NH2-terminal extremity that acts as a ligand for the receptor. Thrombin mainly signals by using the PAR-1 receptor but is also able to activate PAR-3 and PAR-4 (6). It has been shown that PAR-1 expression is upregulated in acute and chronic liver diseases (27, 39). There is now a strong case for a role of thrombin and PAR-1 in liver fibrosis, because 1) in vitro, thrombin is mitogenic for rat and human liver fibrogenic cells (12, 26, 29), and 2) we have shown that inhibition of thrombin protects rats against experimental liver fibrosis induced by carbon tetrachloride intoxication (14), and others have shown beneficial effects of the blockade of PAR-1 in the bile duct ligation model (11).

The effects of thrombin on cell migration are highly dependent on the cell type. For instance, thrombin inhibits human iliac endothelial cell (9) and MDAMB231 breast cancer cell migration (18), whereas it induces migration of microvascular endothelial cells (41), vascular smooth muscle cells (35), and fibroblasts (8).

Thus the aim of this study was to investigate the effect of thrombin on human liver myofibroblast migration, with an emphasis on the interactions of thrombin with PDGF-BB.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. PDGF-BB was from Eurobio (les Ulis, France). EGF was from Peprotech (Tebu, Le Perray en Yvelines, France). Human {alpha}-thrombin, hirudin, prostaglandin E2 (PGE2), 8-bromo-cAMP, DAPI, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NS-398, nimesulide, and sodium orthovanadate were from Sigma (Saint Quentin Fallavier, France). The SFLLRNPNDKYEPF peptide was from Neosystem (Strasbourg, France). Butaprost and AH-6809 were from Cayman Chemical (Interchim, Asnières, France). Phospho-Thr308 Akt-1 and Akt-1 antibodies and phosphotyrosine and phospho-PDGF-beta receptor (Tyr751) antibodies were from Cell Signaling Technology (Ozyme, Saint Quentin Yvelines, France). Thrombin receptor H-111 antibody, PDGF-beta receptor, and PDGF-beta receptor agarose-conjugated antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Superscript II was from Invitrogen (Cergy-Pontoise, France), Taq polymerase was from Promega (Charbonnières-les-Bains, France), and RNeasy extraction kit was from Qiagen (Courtaboeuf, France). Fetal calf serum (FCS) was from Dutscher (Brumath, France), and human serum was from Etablissement Français du Sang (Bordeaux, France).

Cell isolation and culture. Human hepatic myofibroblasts were obtained from explants of nontumoral liver resected during partial hepatectomy and characterized as described previously (48). Specifically, the procedure, based on the selective growth advantage of myofibroblasts in the culture conditions used, allowed for a 100% pure myofibroblast population, as shown by positive staining for {alpha}-smooth muscle actin and vimentin and negative staining for CD68 (a Kupffer cell marker), von Willebrand factor (an endothelial cell marker), and cytokeratin (an epithelial cell marker). This procedure is in accordance with Institut National de la Santé et de la Recherche Médicale ethical regulation imposed by French legislation. Myofibroblasts were grown in DMEM containing 5% FCS, 5% pooled human serum, and 5 ng/ml EGF. EGF was removed from the medium at least 3 days before experiments.

Cell-migration assay in Boyden chamber. Cell-migration assays were performed by using a Boyden-chamber method as described (32). Briefly, an 8-µm polycarbonate filter was inserted in 24-well plates. Twenty-thousand cells were suspended in 200 µl DMEM containing 0.05% FCS and were added to the upper chamber, while 800 µl of DMEM-0.05% FCS containing agonists or vehicle were added to the lower chamber. After 6 h at 37°C in a 5% CO2 incubator, the cells on top of the membrane were removed with a cotton swab. The filters were fixed for 30 min with methanol, and nuclear DNA was stained with DAPI. Cells that had migrated to the lower surface of the filter were counted by using a Zeiss Axioplan fluorescence microscope on a 10x central field (which represents the fifth of the whole membrane surface).

Cell-migration assay in monolayer wounding-repair model. This was done as described (15). Briefly, confluent monolayers were serum starved for 48 h in Waymouth medium. Then a wound was made in the monolayer by using a pipette tip. Cells were then washed three times with Waymouth medium, and medium containing thrombin (18 nM) and/or hirudin (2 U/ml) was added for 24 h. Cells were then fixed in chilled methanol, washed, and stained in hemalun solution.

Cell migration was evaluated under a microscope with a 10x objective by counting the number of cells that migrated to repair the wound in a 25-mm2 square (which represents 12 consecutive fields). Each experiment was performed in triplicate.

Viability assay. Myofibroblasts (2 x 104 per 200 µl) were seeded in 96-well plates in DMEM-0.05% FCS and were treated with agonists or vehicle. After 6 h at 37°C in a 5% CO2 incubator, the medium was replaced by 1 mg/ml MTT for 1.5 h. The tetrazolium crystals were then solubilized with DMSO, and the optical density was read at 540 nm.

RNA isolation and RT-PCR analysis for EP receptors. Total RNA was prepared by using the Qiagen RNeasy kit. Total RNA (1 µg) were reverse transcribed with Superscript II. An aliquot was then amplified with Promega Taq polymerase by using the following PGE receptor primers, which were previously described (24): EP1, sense primer 5'-ACG CGG CCG CTG CTC CAC GCC-3' and antisense primer 5'-CAG TTG GCG CAG CAC GGC CTG-3'; EP2, sense primer 5'-CGG ACC GCT TAC CTG CAG CTG-3' and antisense primer 5'-TAA TGA AAT CCG ACA ACA GAG-3'; EP3, sense primer 5'-ACG AGC GTT GGG AGC ACA TCG-3' and antisense primer 5'-GCA GT CTC AAC TGA TT CTG-3'; and EP4, sense primer 5'-ATC GAC TGG ACC ACC AAC GTG ACG-3' and antisense primer 5'-TCT ATT GCT TTA CTG AGC ACT GTC-3'. PCR conditions were as follows: denaturation for 2 min at 94°C, followed by 40 cycles at 94°C for 30 s; 61°C (EP1), 56°C (EP2), or 58°C (EP3 and EP4) for 30 s; and 72°C for 45 s. Negative control was performed for each reaction, including the omission of the RT step or the omission of the cDNA in the PCR mix.

Western blot analysis of Akt-1 phosphorylation and PDGF-beta receptor phosphorylation. Hepatic myofibroblasts were seeded at a density of 10,000 cells/cm2 in 35-mm-diameter dishes, grown to confluence, and made quiescent in serum-free Waymouth medium over 24 h. Cells were then incubated with thrombin or other agents. At the end of the incubation, dishes were put on ice and washed with phosphate-buffered saline, and the cells were solubilized for 30 min under shaking at 4°C in lysis buffer containing 50 mM Tris·HCl, pH 7.5, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM beta-glycerophosphate, 1 mM activated sodium orthovanadate, 0.1% beta-mercaptoethanol, and 1 µM microcystin. Protein concentrations were measured with a reagent from Bio-Rad. Fifty micrograms of total protein were loaded on either 7% (PDGF-beta receptor) or 10% (Akt-1) SDS-PAGE and were transferred on a polyvinylidene fluoride membrane. The membranes were blocked with 2.5% bovine serum albumin in 1x TBS-0.1% Tween and were incubated with a rabbit polyclonal anti-phospho-Akt-1 antibody diluted 1/1,000 when investigating Akt-1 phosphorylation or with a mouse monoclonal anti-phosphotyrosine antibody diluted 1/2,000 when investigating PDGF receptor phosphorylation. In some experiments, we used a rabbit polyclonal phospho-PDGF-beta receptor antibody. The blots were washed with 1x TBS-0.1% Tween. The appropriate peroxidase-conjugated secondary antibody was applied in the same buffer containing 5% skimmed dry milk for 1 h, and the immunodetected proteins were visualized by using an enhanced chemiluminescence assay (Amersham Biopharmacia, Orsay, France). Membranes were stripped and reblotted by using a rabbit polyclonal Akt-1 antibody or a rabbit polyclonal PDGF-beta receptor antibody, respectively. Signals were acquired on a Macintosh computer by using a Kodak Digital Science DC 120 digital camera and were quantified by using NIH Image software.

Immunoprecipitation of PDGF-beta receptor. Quiescent cells were incubated for 10 min with PDGF-BB and/or thrombin, dishes were put on ice and washed with phosphate-buffered saline, and the cells were solubilized in lysis buffer containing 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM beta-glycerophosphate, 10 µl/ml Sigma inhibitor cocktail, 1 mM activated sodium orthovanadate, and 10 mM sodium fluoride. One milligram of total protein was immunoprecipitated with PDGF-beta receptor agarose-conjugated antibody overnight at 4°C. Immunoprecipitates were washed and analyzed by Western blot as previously described for PDGF-beta receptor phosphorylation.

Statistical analysis. All data are expressed as means ± SD. Differences between means were determined by the Kruskall-Wallis nonparametric test by using the Statcrunch package (htpp://www.statcrunch.com). Values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Thrombin inhibits human hepatic myofibroblast migration. It has been shown that thrombin could promote or inhibit cell migration depending on the cell type. According to data shown in Fig. 1A, in a Boyden-chamber assay, thrombin at 18 nM (1 U/ml) inhibited human hepatic myofibroblast migration by 49 ± 3% (Fig. 1A) compared with control. This inhibition was dose dependent (Fig. 1B) and was not consecutive to a deleterious effect of thrombin on cell adhesion or survival (MTT assay; data not shown). The inhibitory effect was confirmed in the monolayer wound-repair model, where thrombin at 18 nM inhibited migration by 19 ± 4% (Fig. 1C; n = 5; P < 0.005). Subsequent experiments were performed by using the Boyden-chamber assay.


Figure 1
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  		Fig. 1. Thrombin inhibits human hepatic myofibroblast migration. A: Boyden chamber assay. Myofibroblasts were seeded in the upper compartment and were exposed for 6 h to either control medium or medium containing 18 nM thrombin. Cells that had migrated to the bottom side of the filter were counted. Graph shows quantification of 30 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. B: same experimental design as in A. Cells were exposed to indicated concentrations of thrombin. Graph shows quantification of 3 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. C: wound-repair assay. Quiescent confluent monolayers were wounded with a pipette tip, then cultivated for 24 h with or without thrombin (18 nM). Cells that migrated to repopulate the wound were counted. Graph shows quantification of 5 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. D: same experimental design as in A. Platelet-derived growth factor (PDGF)-BB was used at 20 ng/ml and thrombin at 18 nM. Graph shows quantification of 16 independent experiments performed in triplicate. Results are presented as fold variation over control values (without PDGF-BB) and are shown as means ± SD.

 
PDGF-BB is a potent chemotactic agent for human hepatic myofibroblasts. We thus examined the effect of thrombin on PDGF-BB-induced migration. Figure 1D shows that PDGF-BB stimulated by about threefold the migration of human liver myofibroblasts; this was greatly reduced by thrombin because the effect of PDGF-BB was not different from baseline in the presence of thrombin.

Thrombin effects on migration require PAR activation. Most thrombin effects are consecutive to activation of PARs in a catalytic activity-dependent manner (6). The role of PARs in the effects of thrombin on migration was evaluated with several experiments. We first used the specific inhibitor of thrombin catalytic activity, hirudin. Whereas hirudin alone had no effect on cell migration, it totally reversed thrombin-induced inhibition of migration in both basal (Fig. 2A) and PDGF-BB-stimulated conditions (Fig. 2B). These data show that inhibition of migration requires the catalytic activity of thrombin, thus strongly suggesting that thrombin's effect is mediated by one of the PARs. Because thrombin has a high affinity for PAR-1, we investigated whether PAR-1 activation was responsible for the effect of thrombin on migration. PAR-1 activation can be induced by using synthetic peptides that mimic the NH2 terminus generated after thrombin cleavage. Similar to thrombin, the SFLLRNPNDKYEPF peptide induced a dose-dependent inhibition of migration, although with a lower potency (compare Fig. 2C with Fig. 1B). Finally, the inhibitory effect of thrombin on migration was completely abolished in the presence of a blocking antibody to PAR-1 that recognizes the specific thrombin-cleavage sequence on PAR-1 (Fig. 2D). Together, these data show a central role for PAR-1 in the mediation of thrombin's effect on migration.


Figure 2
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  		Fig. 2. Thrombin catalytic activity and protease-activated receptor (PAR)-1 activation are required for inhibition of migration. A: inhibition of basal migration. Same experimental design as in Fig. 1A. Thrombin (18 nM) was preincubated with 2 U/ml hirudin or control medium for 2 h. Quantification of 3 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. *P = 0.01 compared with control; #P = 0.02 compared with thrombin alone. B: inhibition of PDGF-BB-induced migration. Same experimental design as in Fig. 1A. PDGF-BB was used at 20 ng/ml. Thrombin (18 nM) was preincubated with 2 U/ml hirudin or control medium for 2 h. Results are means of 3 independent experiments performed in triplicate and are presented as fold variation over control values. *P ≤ 0.05 compared with PDGF alone. NS, not significant. C: effect of the thrombin mimetic peptide SFLLRNPNDKYEPF. Myofibroblasts seeded in the upper compartment of the migration chamber were exposed for 6 h to SFLLRNPNDKYEPF at the indicated concentrations. Graph shows the mean of 2 experiments performed in triplicate. Results are presented as fold variation over control values. At the highest concentration tested (150 µM), a scrambled peptide (SLNNPLEPKRYFDF) had no significant effect on migration (101.8 ± 1.9% of control; n = 3). D: a PAR-1 blocking antibody reverses thrombin's effect. Myofibroblasts were exposed for 1 h to blocking PAR-1 antibody or rabbit immunoglobulin at 20 µg/ml before thrombin treatment for 6 h. Results are means of 3 independent experiments performed in triplicate and are presented as fold variation over control values. Closed bars, control; open bars, thrombin. *P ≤ 0.05 compared with control values. #P ≤ 0.05 compared with control rabbit IgG values.

 
Cyclooxygenase-2 activation is responsible for the generation of negative migratory signals. We (33) and others (26) have previously shown that thrombin stimulates cyclooxygenase (COX)-2 expression and activity in human liver myofibroblasts and that COX-2 activity in these cells can be efficiently blocked by NS-398 (7). Furthermore, COX-2 products can regulate cell migration (21, 22, 41). We thus investigated whether COX-2 was involved in the regulation of cell migration in response to thrombin. Indeed, the specific COX-2 inhibitor NS-398 reversed thrombin's effect on basal migration (Fig. 3A). This effect was dose dependent (data not shown). Similar results were obtained with another COX-2 antagonist, nimesulide, that dose-dependently reverted the effects of thrombin (Fig. 3B). On the other hand, NS-398 failed to reverse thrombin's effect on PDGF-BB-induced migration (Fig. 3C).


Figure 3
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  		Fig. 3. Cyclooxygenase (COX)-2 is involved in thrombin inhibition of basal migration but not in inhibition of PDGF-BB-induced migration. A: NS-398 inhibits thrombin's effect on basal migration. Myofibroblasts were exposed for 1 h to 10 µM NS-398 or its vehicle before to thrombin treatment for 6 h. Quantification of 9 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. *P ≤ 0.0001 compared with control; #P ≤ 0.0003 compared with thrombin alone. B: nimesulide (Nim) inhibits thrombin's (Thr) effect on basal migration. Myofibroblasts were exposed for 1 h to the indicated concentrations of nimesulide before thrombin treatment for 6 h. Quantification of 2 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. C: NS-398 does not inhibit thrombin's effect on PDGF-BB-induced migration. Myofibroblasts were exposed for 1 h to 10 µM NS-398 or its vehicle before treatment with 20 ng/ml PDGF-BB with or without 18 nM thrombin for 6 h. Quantification of 5 independent experiments performed in triplicate. Results are presented as fold variation over control values (without PDGF-BB) and are shown as means ± SD. *P ≤ 0.03 compared with PDGF-BB alone.

 
PGE2 is one of the major prostaglandins produced in response to COX-2 activation. Specifically, PGE2 is produced in response to thrombin in human liver myofibroblasts (26). PGE2 can act through several PGE receptors labeled EP1–EP4. We first examined whether myofibroblasts expressed EP receptors. By RT-PCR, we showed that human hepatic myofibroblasts express EP1, EP2, and EP3 but not EP4 transcripts (Fig. 4A). PGE2 used instead of thrombin significantly decreased myofibroblast migration (Fig. 4B). In human hepatic myofibroblasts, thrombin is known to increase cAMP levels via its effect on prostaglandin synthesis (26). We found that the effect of PGE2 could also be reproduced by 8-bromo-cAMP, a cell-permeable cAMP analog (Fig. 4C). The latter finding suggested that PGE2's effect could be mediated by EP2 or EP4 receptors, which are positively coupled to adenylyl cyclase. Because EP4 was not expressed in myofibroblasts, we turned our attention to EP2. As shown in Fig. 4D, the specific EP2 agonist butaprost inhibited migration to the same extent as thrombin. Moreover, the specific antagonist of the EP2 receptor, AH-6809, almost completely abolished the inhibitory effect of thrombin on migration of myofibroblasts (Fig. 4E). Together, these data show that thrombin inhibition of basal migration is due to activation of COX-2 and to the production of PGE2 acting via EP2 receptors.


Figure 4
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  		Fig. 4. Prostaglandin E2 (PGE2) and its EP2 receptor are involved in inhibition of myofibroblast migration. A: expression of EP receptors was examined by RT-PCR with specific primers. PCR product sizes for EP1, EP2, EP3, and EP4 are 280, 432, 597, and 513 bp, respectively. For all receptors, lanes 1–4 show PCR products from 4 different preparations of myofibroblasts and lane 5 shows positive control (for EP1, K562; for EP2 and EP3, bone marrow mononuclear cells; for EP4, bone marrow stromal cells). Control with omission of the cDNA in PCR mix was always negative (lane 6; not shown). B: effect of PGE2 on migration. Myofibroblasts were exposed for 6 h to control medium in the presence or absence of 300 nM PGE2 or of 18 nM thrombin. Quantification of 4 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. *P ≤ 0.02 compared with control. C: effect of 8-bromo-cAMP on migration. Myofibroblasts were exposed for 6 h to control medium in the presence or absence of 1 mM 8-bromo-cAMP (8brcAMP) or 18 nM thrombin. Quantification of 3 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. *P ≤ 0.04 compared with control. D: effect of an EP2 agonist on thrombin-induced migration. Myofibroblasts were exposed to 18 nM thrombin or 10 µM butaprost for 6 h. Quantification of 3 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. *P ≤ 0.04 compared with control. E: effect of an EP2 antagonist on thrombin-induced migration. Myofibroblasts were exposed for 1 h to 10 or 20 µM AH-6809 or its vehicle before treatment with 18 nM thrombin for 6 h. Quantification of 3 independent experiments performed in triplicate. Results are presented as fold variation over control values and are shown as means ± SD. *P ≤ 0.04 compared with control; #P ≤ 0.05 compared with thrombin alone.

 
Thrombin inhibits PDGF-BB-induced migration via an inhibition of the phosphatidylinositol 3-kinase pathway. Activation of phosphatidylinositol 3-kinase (PI3K) has been shown to be the major pathway responsible for PDGF-BB-induced migration in human hepatic myofibroblasts (28). We thus asked whether thrombin interfered with this pathway. As shown in Fig. 5A, PDGF-BB induced the expected phosphorylation of the PI3K downstream effector Akt-1. Thrombin alone did not induce Akt-1 phosphorylation. On the contrary, thrombin inhibited PDGF-BB-induced Akt-1 phosphorylation by 50% (Fig. 5). Because specific inhibition of PI3K by LY-294002 or Wortmannin is enough to inhibit the chemotactic effect of PDGF-BB on human hepatic myofibroblasts (28), our results strongly suggest that thrombin inhibits PDGF-BB-stimulated migration by interfering with PI3K activation.


Figure 5
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  		Fig. 5. Thrombin inhibits PDGF-BB-induced activation of the phosphatidylinositol 3-kinase (PI3K) pathway. A: myofibroblasts were treated for 10 min with 20 ng/ml PDGF-BB, 18 nM thrombin, or both. Cell lysates were analyzed by Western blot with a phospho-Akt-1 antibody (top). Blot shows duplicates for each condition. It was stripped and rehybridized with an Akt-1 antibody (bottom). B: quantification of 19 independent experiments measuring thrombin's effect on Akt-1 phosphorylation. Results are presented as fold variation over PDGF-BB-induced Akt-1 phosphorylation and are shown as means ± SD. *P ≤ 0.0001 compared with PDGF-BB alone.

 
Because PDGF-beta receptor autophosphorylation is essential for activation of the PI3K pathway in response to PDGF-BB, we investigated whether it was affected by thrombin. Indeed, thrombin significantly reduced (51.8 ± 7.8%) PDGF-BB-induced phosphorylation of a 190-kDa protein (Fig. 6, A and C) identified as the PDGF-beta receptor following immunoprecipitation with a PDGF-beta receptor antibody (Fig. 6B).


Figure 6
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  		Fig. 6. Thrombin inhibits PDGF-BB-induced PDGF-beta receptor phosphorylation. A: myofibroblasts were treated for 10 min with 20 ng/ml PDGF-BB, 18 nM thrombin, or both. Cell lysates were analyzed by Western blot with a phosphotyrosine antibody (top). Blot was stripped and then rehybridized with a PDGF-beta receptor antibody (bottom). B: myofibroblasts were treated for 10 min with 20 ng/ml PDGF-BB, 18 nM thrombin, or both. Cell lysates were immunoprecipitated with a PDGF-beta receptor antibody. Immunoprecipitates were analyzed by Western blot with a phosphotyrosine antibody (top). Blot was stripped and then rehybridized with a PDGF-beta receptor antibody (bottom). C: quantification of 6 independent experiments measuring thrombin's effect on PDGF-beta receptor phosphorylation, analyzed by anti-phosphotyrosine Western blot. Results are presented as fold variation over PDGF-BB-induced PDGF-beta receptor phosphorylation and are shown as means ± SD. *P ≤ 0.003 compared with PDGF-BB alone.

 
The inhibitory effect of thrombin on PDGF-beta receptor phosphorylation (Fig. 7, A and B) and Akt-1 phosphorylation (Fig. 7, C and D) was abolished when cells were treated with the broad-spectrum tyrosine phosphatase inhibitor sodium orthovanadate. In addition, in cells treated with sodium orthovanadate in the absence of PDGF-BB, thrombin reproducibly elicited a slight phosphorylation of Akt-1.


Figure 7
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  		Fig. 7. Thrombin inhibits PDGF-beta receptor activation and Akt-1 phosphorylation through a tyrosine phosphatase-dependent mechanism. A: thrombin inhibition of PDGF-beta receptor phosphorylation is dependent on the activation of a tyrosine phosphatase. Myofibroblasts were incubated for 1 h with 1 mM sodium orthovanadate before treatment for 10 min with 20 ng/ml PDGF-BB, 18 nM thrombin, or both. Cell lysates were analyzed by Western blot with a phospho-PDGF-beta receptor antibody (top). Blot was stripped and then reblotted with a PDGF-beta receptor antibody (bottom). B: quantification of 3 independent experiments with sodium orthovanadate. Results are presented as fold variation of PDGF-BB-induced PDGF-beta receptor phosphorylation and are shown as means ± SD. *P ≤ 0.05 compared with PDGF-BB alone. C: thrombin inhibition of Akt-1 phosphorylation is dependent on the activation of a tyrosine phosphatase. Myofibroblasts were incubated for 1 h with 1 mM sodium orthovanadate before incubation for 10 min with 20 ng/ml PDGF-BB, 18 nM thrombin, or both. Cell lysates were analyzed by Western blot with a phospho-Akt-1 antibody (top). Blot was stripped and then reblotted with Akt-1 antibody (bottom). D: quantification of 3 independent experiments with sodium orthovanadate. Results are presented as fold variation of PDGF-BB-induced Akt-1 phosphorylation and are shown as means ± SD. *P ≤ 0.05 compared with PDGF-BB alone.

 
Together, these results strongly suggest that thrombin inhibits PDGF-BB-induced migration through increased PDGF-beta receptor dephosphorylation and subsequent inhibition of the PI3K pathway.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Liver fibrogenesis is a complex process that implies the coordinate involvement of many signaling molecules. Examples of biologically active molecules for which expression and/or activity is increased in this setting are transforming growth factor-beta1, endothelin-1, and monocyte chemotactic protein-1, as well as PDGF-BB and thrombin. The role of these factors has been studied in much detail, although how they interact remains in most cases not explored. Here, with the use of a Boyden-chamber assay, we demonstrate that thrombin inhibits human hepatic myofibroblast migration when stimulated by PDGF-BB, a well-known chemotactic agent for these cells (28). Other experiments showed that thrombin also inhibits spontaneous myofibroblast migration in the absence of PDGF-BB. Although the effects were less conspicuous, thrombin definitely also inhibited myofibroblast migration in an independent wounding-repair assay. As already stated, the effects of thrombin on cell migration are highly variable. Thus we decided to obtain data on the mechanism used by thrombin to inhibit human liver myofibroblast migration.

Most of thrombin's cellular effects are consecutive to signalization via PAR receptors, especially PAR-1, although a nonproteolytic pathway has also been described (44). We have shown that human liver myofibroblasts express PAR-1, PAR-3, and PAR-4 (33), the three PARs mediating thrombin's effects. In our experiments, the involvement of thrombin catalytic activity and the role of PAR-1 receptor were demonstrated by three lines of evidence: 1) the specific inhibitor of thrombin catalytic activity, hirudin, abolished thrombin's effect on both basal and PDGF-BB-induced migration; 2) the PAR-1 agonist peptide SFLLRNPNDKYEPF reproduced the inhibitory effect of thrombin; and 3) blocking PAR-1 antibody abolished the effect of thrombin.

Thrombin can induce the expression and the activation of COX-2 in human liver (26, 33) and colonic myofibroblasts (42), as well as the corresponding production of PGE2. As for thrombin, the effects of prostaglandins on cell migration are quite different among cell types (17, 21, 36, 43). We provide evidence that COX-2 and PGE2 are responsible for the inhibitory effect of thrombin on spontaneous migration of human liver myofibroblasts. We show that the inhibitory effect of thrombin on spontaneous cell migration is abolished by two selective COX-2 inhibitors, NS-398 and nimesulide. In addition, exogenous PGE2 used alone was able to reproduce the effects of thrombin. PGE2 interacts with four subtypes of prostanoid receptors called EP1–EP4. We have now shown that human hepatic myofibroblasts express EP1, EP2, and EP3 but not EP4. Butaprost, a specific EP2 agonist, reproduced the effects of thrombin, and AH-6809, a specific antagonist of the EP2 receptor, abolished almost completely thrombin's effects on migration. This demonstrates that most if not all of thrombin's effects are relayed through PGE2 binding to the EP2 receptor.

Whereas COX-2 is involved in the inhibitory effect of thrombin on spontaneous migration, our data show that it is not responsible for the inhibitory effect of thrombin on PDGF-BB-stimulated chemotaxis. Indeed, addition of NS-398 did not reverse the effect of thrombin on PDGF-BB-stimulated chemotaxis. Since COX-2 activation did not explain the effect of thrombin on PDGF-BB-stimulated migration, we examined another possibility. Previous studies have shown the dependence of PDGF-BB-induced migration on the activity of PI3K in human hepatic myofibroblasts (13, 28) and other cell types (5, 46). We thus hypothesized that thrombin could act by antagonizing the PI3K pathway. This was investigated in our experiments by monitoring the phosphorylation of the PI3K downstream effector Akt-1. Indeed, thrombin strongly decreased Akt phosphorylation evoked by PDGF-BB. In additional experiments, we found that thrombin also inhibited Akt phosphorylation following stimulation by EGF, and this effect was correlated to an inhibition of EGF-induced migration (data not shown). Marra et al. (28) have shown that specific inhibition of PI3K by Wortmannin or LY-294002 is enough to inhibit PDGF-BB-induced chemotaxis in human hepatic myofibroblasts. Thus our data strongly suggest that thrombin inhibits PDGF-BB-induced chemotaxis through decreased activation of the PI3K pathway.

We further show that inhibition of Akt-1 activation is a downstream effect of thrombin-induced inhibition of PDGF-beta receptor autophosphorylation. This implies that besides migration, thrombin may also affect other PDGF-mediated effects. Receptor tyrosine kinase activation is shut down through dephosphorylation by tyrosine phosphatases. We show that thrombin inhibition of PDGF-beta receptor phosphorylation, and subsequent Akt activation, is abolished when cells are pretreated with the tyrosine phosphatase inhibitor sodium orthovanadate, indicating that thrombin trans-inactivates PDGF-beta receptor through a tyrosine phosphatase. Such a mechanism has already been put forward to explain the effects of other G protein-coupled receptor ligands such as bradykinin (1, 4, 16) and somatostatin (2, 10). It should also be noted that whereas Akt-1 phosphorylation was not detectable with thrombin alone (see Fig. 7C, lanes 1 and 2), it became apparent when cells were also treated with sodium orthovanadate (Fig. 7C, lanes 5 and 6). Thus the apparent lack of PI3K activation by thrombin in myofibroblasts, in contrast to platelets (31) or other cells (23), is due to the simultaneous activation of a tyrosine phosphatase.

In conclusion, we have shown that thrombin inhibits human liver myofibroblast migration. Spontaneous migration is inhibited through a COX-2-PGE2-EP2 mechanism, whereas PDGF-BB-induced migration is inhibited via dephosphorylation of PDGF-beta receptor and subsequent inhibition of signaling. Given that PDGF-BB and its receptors are highly expressed in the course of liver fibrosis, it is likely that thrombin may help fine-tune the chemotactic effects of PDGF-BB on fibrogenic cell migration. Our results may at first glance appear counterintuitive, because in vivo inhibition of thrombin or its receptor resulted in decreased experimental liver fibrogenesis (11, 14). However, whereas there is good evidence that thrombin antagonism in vivo is beneficial in the prevention of fibrosis, there are no comparable in vivo data at the stage of established fibrosis in the presence of fully activated myofibroblasts, such as the ones used in this study. In addition, the antagonistic effects of thrombin on migration may actually participate to the fibrogenic process via prevention of migration of the myofibroblasts from the sites of injury.


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This work was funded in part by grants from Comité de la Dordogne from the Ligue Nationale Contre le Cancer and Conseil Régional d'Aquitaine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Rosenbaum, INSERM U889, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France (e-mail: jean.rosenbaum{at}gref.u-bordeaux2.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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