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Am J Physiol Heart Circ Physiol 285: H745-H754, 2003. First published May 1, 2003; doi:10.1152/ajpheart.01042.2002
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Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation

Régis Bobe, Xing Yin, Marie-Cécile Roussanne, Olivier Stepien, Evelyne Polidano, Claude Faverdin, and Pierre Marche

Unité Mixte de Recherche 7131, Centre Nationale de la Recherche Scientifique, Hôpital Broussais, 75014 Paris, France

Submitted 16 December 2002 ; accepted in final form 25 April 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.

calcium; thrombin signaling; vascular smooth muscle cells; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor

ABNORMAL PROLIFERATION of vascular smooth muscle cells (VSMCs) is a key event in various vascular diseases, including arteriosclerosis, hypertension, or postangioplasty restenosis, and thrombin is one of the various growth factors involved in the control of VSMC growth (20, 30). Thrombin stimulation leads to protein phosphorylation and gene expression through activation of its receptor, described as a member of the family of G protein-coupled receptors. Thrombin binding to its receptor stimulates phospholipase C and results in intracellular Ca2+ elevation and PKC activation. These events participate in the stimulation of a MAPK cascade that mediates cell growth and differentiation (8, 10, 20, 25). Among the members of the MAPK family, the 42- and 44-kDa isoforms of MAPK (p42/p44 MAPK or ERK1/2) have been shown to participate in the mitogenic mechanism triggered by thrombin in VSMCs (21, 23). Transactivation of the epidermal growth factor receptor (EGFR) appears to be involved in the response to angiotensin II or thrombin stimulation of VSMCs (4, 15, 24), whereas in astrocytes EGFR transactivation is not necessary for thrombin to induce ERK phosphorylation (38). EGFR transactivation has been shown to be Ca2+ and PKC dependent in some cases (7, 9, 44), and it has been suggested that kinase(s) of the Src family play an essential role upstream of the EGFR (2, 31, 37, 43). Furthermore, a controversial role is given to Ca2+-dependent proline-rich tyrosine kinase (Pyk2), which is described to activate Src as well as be activated by Src (1, 6). Nonetheless, mechanisms whereby thrombin transactivates the EGFR and induces ERK1/2 activation are still poorly understood.

To clarify the signaling pathways involved in thrombin-triggered ERK1/2 activation, we investigated the role of activation of Src family kinase, Pyk2, and the EGFR through phosphorylation of specific Tyr residues. The roles played by intracellular Ca2+ concentration ([Ca2+]i) and PKC activity in these processes were also examined using the cell-permeable Ca2+ chelator BAPTA-AM and the PKC specific inhibitor GF-109203X, respectively. In this respect, the effects of thrombin were compared with those of thapsigargin because in VSMCs both agents have been shown to increase intracellular Ca2+ and to induce signaling events sharing some similarities (32).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. Cell culture materials and media were obtained from Costar and Life Technologies, respectively. Fetal calf serum (FCS) was from Roche Diagnostic (Gagny, France). Fura 2-AM and thapsigargin were purchased from Molecular Probes (Eugene, OR) and Biomedical Research Chemicals (Euromedex; Souffelweyersheim, France), respectively. EGFR kinase inhibitors (tyrphostin AG-1478 and PD-153035), BAPTA-AM, the Src family kinase inhibitor PP2, MEK-specific inhibitors (U-0126 and PD-98059), the phophatidylinositol 3-kinase (PI3K) inhibitor LY-294002, and the PKC inhibitor bisindolylmaleimide (GF-19203X) were from Calbiochem (Merck Eurolab; Fontenay-sous-Bois, France). Thrombin and all other chemicals were from Sigma Aldrich (St. Quentin Fallavier, France).

Antibodies. p44/42 MAPK antibody, phospho-p44/42 MAPK (Thr202/Tyr204) E10 monoclonal antibody, phospho-Src family (Tyr416) antibody, phospho-EGFR (Tyr1068) antibody, and anti-mouse or anti-rabbit IgG horseradish peroxidase-linked antibodies were purchased from Cell Signaling Technology (Beverly, MA), and anti-Pyk2 (pTyr402), phospho-specific antibody was purchased from BioSource International (Camarillo, CA). Briefly, the antibody used against phospho-Src family kinase detects endogenous levels of activated Src kinases through phosphorylation of Tyr416, which is reported to be the major activation site (22). The antibody used against phospho-Pyk2 detects the enzyme when activated via its autophosphorylation of Tyr402, which is independent on Src kinase activity (6). The antibody used against the phospho-EGFR detects the receptor when phosphorylated on Tyr1068, which is one of the major autophosphorylation sites of the receptor, described to be involved in the association with the complex Grb2/Sos-1 leading to Ras kinase activation (27). The antibody used against phospho-p44/42 MAPK detects the enzyme when dually phosphorylated on Thr202 and Tyr204, which is the activated form of the enzyme.

Cell cultures. Experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. VSMCs were isolated from the thoracic aortas of 10-wk-old Wistar rats by the explant technique of Ross (29), as previously detailed (12). Cells were grown in DMEM supplemented with 8 mM HEPES buffer, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (vol/vol) FCS and were subcultured weekly. In this study, subconfluent VSMCs between passages 4 and 12 were used. For each experiment, VSMCs were cultured for 3 days in 10% FCS and then made quiescent by serum deprivation for 2 days. Cell viability, as assessed by lactate dehydrogenase activity measurement, was not affected by the various experimental conditions.

[Ca2+]i measurement. [Ca2+]i was determined by using the fluorescent indicator fura 2 as already detailed (32).

Western immunoblotting analysis. Quiescent VSMCs were pretreated and stimulated as indicated in the figures. After protein extraction, SDS-PAGE was carried out essentially as described (40). For immunoblotting, the level of protein was controlled using antibodies directed against both phosphorylated and nonphosphorylated forms of ERK1/2 and processed as recommended by the manufacturer. Horseradish peroxidase-linked anti-rabbit IgG was used as a secondary antibody, and immunoreactive bands were detected by using an ECL kit from Amersham Pharmacia Biotech (Orsay, France). The same membranes were stripped at 65°C for 45 min in stripping buffer (pH 6.8) containing 0.7% {beta}-mercaptoethanol, SDS 2%, and 62.5 mM Tris and treated for immunoblotting with specific anti-phospho-antibodies as described above. Densitometry analysis of immunoblots was carried out using NIH Image software.

Statistical analysis. Values are given as means ± SD of n experiments. Each experiment involved one distinct culture. Multiple comparisons and time-dependence effects were examined by one-way ANOVA. P values of <0.05 were considered to represent significant differences.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Kinetics of phosphorylation induced by thrombin and thapsigargin. In the first series of experiments, the kinetics of phosphorylation by thrombin (1 U/ml) of Tyr416 Src, Tyr402 Pyk2, Tyr1068 EGFR, and Thr202/Tyr204 ERK1/2 were examined. Figure 1A shows that maximums of Src, Pyk2, and EGFR phosphorylations were obtained for 1–3 min of thrombin stimulation. The time course of activation of ERK1/2 by thrombin showed that the maximum of activation was reached after 5 min, as previously reported for human VSMCs (33).



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  		Fig. 1. Kinetics of phosphorylation induced by thrombin, thapsigargin, and di-(tert-butyl)-1,4-hydroquinone (tBHQ). A: quiescent vascular smooth muscle cells (VSMCs) were stimulated with 1 U/ml thrombin for various times as indicated before lysis. Equivalent amounts of proteins were loaded, and the level of phosphorylation for each cell lysate was analyzed by immunoblotting with antibodies specific for the phosphorylated (pTyr416) form of Src (P-Src), the phosphorylated (pTyr402) form of Ca2+-dependent proline-rich tyrosine kinase (P-Pyk2), the phosphorylated (pTyr1068) form of the epidermal growth factor receptor (P-EGFR), and the phosphorylated form of ERK1/2 (P-ERK). B: cells were stimulated with 1 µM thapsigargin or 20 µM tBHQ for various times as indicated, and cell lysates were immunoblotted for P-ERK. Each blot is representative of 3 or 4 distinct experiments.

 

Thrombin-triggered Ca2+ movements have been recently shown to consist in a rapid mobilization of Ca2+ followed by Ca2+ influx (32). To evaluate the influence of the increase in [Ca2+]i on ERK1/2 activation, VSMCs were stimulated by thapsigargin (1 µM) and di-(tert-butyl)-1,4-hydroquinone (tBHQ; 20 µM), two compounds well known for their ability to inhibit sarco(endo)plasmic Ca2+-ATPases and to induce [Ca2+]i increase by mobilizing Ca2+ through depletion of intracellular Ca2+ pools (17, 34). When cells were stimulated with thapsigargin and tBHQ, the kinetics of phosphorylation of Src, Pyk2, and EGFR resembled those observed with thrombin (data not shown). However, thapsigargin and tBHQ induced ERK1/2 phosphorylation in a time-dependent manner that was slightly delayed compared with thrombin (Fig. 1B). Indeed, ERK1/2 activation was maximal for 5–15 min of thapsigargin stimulation or for 30 min of tBHQ stimulation and returned to basal levels by 2 h.

For the rest of the study, the phosphorylation levels of Src, Pyk2, and EGFR were observed for 2 min of thrombin and thapsigargin stimulation, whereas ERK1/2 stimulation was observed after a stimulation of 5 and 10 min of thrombin and thapsigargin, respectively.

Sequential inhibition of ERK1/2 signaling pathways. To clearly identify the primary events that lead to activation of ERK1/2, VSMCs were pretreated with inhibitors of Src family or EGFR kinases, and the levels of phosphorylation of Src, Pyk2, EGFR, and ERK1/2 were observed after stimulation by thrombin or thapsigargin. Dose-response assays of the Src family kinase inhibitor PP2 on the thrombin signaling pathway showed that 1 µM PP2 significantly decreased both EGFR and ERK1/2 phosphorylation and that the inhibition was maximal for 10 µM PP2 (data not shown). Figure 2 shows that PP2 (10 µM) decreased by 50–60% both the basal and thrombin-stimulated levels of Src phosphorylation. Figure 2 also shows that pretreatment of VSMCs by PP2 resulted in an almost complete inhibition of thrombin-induced phosphorylation of Pyk2, EGFR, and ERK1/2. On the other hand, the pretreatment of VSMCs by the EGFR kinase inhibitor AG-1478 (10 µM) resulted in the complete inhibition of thrombin-induced phosphorylation of the EGFR (Fig. 2). Such a pretreatment considerably inhibited thrombin-induced ERK1/2 phosphorylation but had no significant effect on Src and Pyk2 phosphorylation levels. Similar results were obtained when PD-153035, another EGFR kinase inhibitor, was used (data not shown). Additionally, thapsigargin-induced ERK1/2 phosphorylation also appeared to be dependent on EGFR activity because after AG-1478 (10 µM) pretreatment, ERK1/2 phosphorylation decreased by {approx}75% and was not significantly different from the basal level (data not shown).



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  		Fig. 2. Effect of Src and EGFR kinase inhibitors on thrombin-induced phosphorylation of Src, Pyk2, EGFR, and ERK1/2. Quiescent VSMCs were pretreated for 20 min with 10 µM of the Src family kinase inhibitor PP2 or 10 µM of the EGFR kinase inhibitor AG-1478 (AG) before stimulation with 1 U/ml thrombin (Thr) (or solvent for control) for 2 min to study Src, Pyk2, and EGFR phosphorylation and for 5 min to study ERK1/2 phosphorylation. These signaling proteins were then analyzed using specific anti-phospho antibodies as described in EXPERIMENTAL PROCEDURES. For each experiment, thrombin-induced phosphorylation was quantified by densitometry. Each tracing is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thrombin alone, taken as 100%.

 

Effects of [Ca2+]i on thrombin- and thapsigargin-induced ERK1/2 signaling pathways. To examine the effect of [Ca2+]i elevation on the thrombin-induced ERK1/2 activation signaling pathway, we studied the effects of an intracellular Ca2+ chelator, BAPTA, on Src, Pyk2, EGFR, and ERK1/2 phosphorylation levels induced by thrombin. To do so, VSMCs were pretreated with 10 µM BAPTA-AM for 30 min before stimulation with 1 U/ml thrombin. As shown in Fig. 3A, BAPTA-AM treatment did not affect the resting [Ca2+]i level but did cause a complete inhibition of the thrombin-induced [Ca2+]i increase. Similarly, such a treatment completely inhibited the thapsigargin- and tBHQ-induced [Ca2+]i increase (data not shown). Nevertheless, BAPTA-AM pretreatment did have a small but significant effect on Src and Pyk2 phosphorylation induced by thrombin and resulted in a more than 60% inhibition of EGFR phosphorylation (Fig. 3B). However, ERK1/2 phosphorylation was not significantly affected by BAPTA-AM pretreatment (Fig. 3B).



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  		Fig. 3. Effect of BAPTA pretreatment on thrombin-induced phosphorylation of Src, Pyk2, EGFR, and ERK1/2. A: fura 2-labeled, quiescent VSMCs were either untreated or pretreated with 10 µM BAPTA-AM for 30 min before stimulation with 1 U/ml thrombin (arrowhead) in Ca2+-free medium. B: as indicated, cells were pretreated for 30 min with 10 µM BAPTA-AM and then stimulated with 1 U/ml thrombin for 2 min for Src, Pyk2, and EGFR phosphorylation and for 5 min for ERK1/2 phosphorylation. These proteins were then analyzed using specific anti-phospho antibodies as described in EXPERIMENTAL PROCEDURES. For each experiment, thrombin-induced phosphorylation was quantified by densitometry. Each tracing is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thrombin alone, taken as 100%.

 

To confirm the role of [Ca2+]i on the ERK1/2 activation pathway, VSMCs were pretreated with 10 µM BAPTA-AM for 30 min before stimulation with 1 µM thapsigargin. The results showed that phosphorylation of Src, Pyk2, and EGFR was also dependent on the increase of [Ca2+]i because, in presence of BAPTA, their levels of phosphorylation were markedly decreased if not abolished and were not significantly different from basal levels (Fig. 4). However, it was noticeable that BAPTA-AM pretreatment did not affect the level of ERK1/2 phosphorylation induced by thapsigargin (Fig. 4). These results strongly suggested that ERK1/2 phosphorylation can be, in particular conditions, independent of EGFR transactivation.



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  		Fig. 4. Effect of BAPTA pretreatment on thapsigargin (Tg)-induced phosphorylation of Src, Pyk2, EGFR, and ERK1/2. Quiescent VSMCs were pretreated (or not) with 10 µM BAPTA-AM for 30 min before stimulation with 1 µM thapsigargin. Src, Pyk2, EGFR, and ERK1/2 phosphorylation was determined using specific anti-phospho antibodies as described in EXPERIMENTAL PROCEDURES. For each experiment, thapsigargin-induced phosphorylation was quantified by scanning densitometry. Each blot and plot is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thapsigargin alone, taken as 100%.

 

Effect of BAPTA-AM pretreatment on thrombin-induced phosphorylation of ERK1/2 in the presence of EGFR kinase inhibitors. To further ascertain that ERK1/2 phosphorylation could be independent of EGFR transactivation, the effect of BAPTA on thrombin stimulation was analyzed in the presence of EGFR kinase inhibitors. In this series of experiments, 10 µM BAPTA-AM (or the diluent) was added to VSMCs for 10 min before the addition of AG-1478 or PD-153035 (10 µM each) for an additional 20 min before stimulation by thrombin (1 U/ml). Pretreatment with the EGFR kinase inhibitor AG-1478 resulted in a complete inhibition of thrombin-induced EGFR phosphorylation, and cotreatment with BAPTA did not recover EGFR phosphorylation (Fig. 5A). This indicates that, in the presence of BAPTA, AG-1478 still exhibits its EGFR kinase inhibitor property. As already observed from the results shown in Figs. 3B and 4, BAPTA did not significantly affect the thrombin-induced phosphorylation of ERK1/2, whereas pretreatment with the EGFR kinase inhibitors AG-1478 or PD-153035 almost completely inhibited the ERK1/2 phosphorylation (Fig. 5, A and B). However, ERK1/2 activation by thrombin appeared to be partially and significantly preserved against AG-1478 or PD-153035 inhibition in VSMCs pretreated with BAPTA-AM (Fig. 5, A and B).



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  		Fig. 5. Effect of the combination of BAPTA and EGFR kinase inhibitors on thrombin-induced phosphorylation of EGFR and ERK1/2. A: quiescent VSMCs were in the presence or absence of 10 µM BAPTA-AM for 10 min before the addition of the EGFR inhibitors AG-1478 (10 µM) or PD-153035 (PD; 10 µM) for an additional 20 min. Cells were then stimulated with 1 U/ml thrombin for 2 and 5 min for the analysis of EGFR and ERK1/2 phosphorylation, respectively. B: for each experiment, thrombin-induced phosphorylation of ERK1/2 was quantified by densitometry. Each blot and plot is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thrombin alone, taken as 100%; $P < 0.05 compared with VSMCs pretreated with AG-1478 and stimulated with thrombin; §P < 0.05 compared with VSMCs pretreated with PD-153035 and stimulated with thrombin.

 

Effect of the Src family kinase inhibitor PP2 on EGFR-independent thrombin-induced ERK1/2 phosphorylation. To evaluate the importance of Src kinase activity in EGFR-independent activation of ERK1/2 by thrombin, the effects of the Src kinase inhibitor PP2 were examined. In these experiments, 10 µM PP2 (or the diluent) was added to VSMCs pretreated with a combination of BAPTA and AG-1478 before stimulation. BAPTA and AG-1478 pretreatment did not significantly modify the level of phosphorylation of Src, Pyk2, and ERK1/2 induced by thrombin, whereas the phosphorylation of the EGFR was completely abolished (Fig. 6). The additional inhibition of Src family kinase activity by PP2 resulted in the total inhibition of the phosphorylation of all the signaling proteins under-studied (Fig. 6). On the other hand, in VSMCs that were only BAPTA pretreated, inhibition of Src with PP2 lead to similar results. Under such conditions, thrombin-induced phosphorylation of Src, Pyk2, EGFR, and ERK1/2 was significantly inhibited by 64%, 49%, 82%, and 65%, respectively (n = 5; data not shown). These results suggest that Src kinase activity should be involved in both EGFR-dependent and -independent phosphorylation of ERK1/2 by thrombin.



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  		Fig. 6. Role of Src on thrombin-induced activation of ERK1/2 in BAPTA-AM- and AG-1478-pretreated VSMCs. Quiescent VSMCs were pretreated or not with 10 µM BAPTA-AM and 10 µM AG-1478 for 15 and 5 min, respectively, before the addition of 10 µM PP2 for 15 min before thrombin stimulation. Phosphorylations of Src, Pyk2, EGFR, and ERK1/2 were analyzed using specific anti-phospho antibodies as described. For each experiment, thrombin-induced phosphorylation was quantified by densitometry. Each blot and plot is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thrombin alone, taken as 100%. $P < 0.05 compared with thrombin in the presence of BAPTA and AG-1478.

 

Role of MEK and PI3K in EGFR-dependent and -independent thrombin-induced ERK1/2 phosphorylation. To examine the importance of MEK activity in the EGFR-independent activation of ERK1/2 by thrombin, the effects of the MEK inhibitor U-0126 were examined. In these experiments, VSMCs were pretreated (or not) with BAPTA and AG-1478 and then 10 µM U-0126 (or its diluent) was added before thrombin stimulation. As shown in Fig. 7, the MEK inhibitor did not affect the thrombin-induced EGFR phosphorylation, whereas the drug inhibited ERK1/2 phosphorylation. Under these experimental conditions, similar results were observed when U-0126 was replaced with PD-98059, another MEK inhibitor (data not shown). These results clearly indicate that MEK activity is involved in both the thrombin-induced EGFR-dependent and -independent phosphorylation of ERK1/2.



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  		Fig. 7. Role of MEK on thrombin-induced EGFR-dependent and -independent ERK1/2 activation in VSMCs. Quiescent cells were pretreated or not with 10 µM BAPTA-AM and 10 µM AG-1478 for 15 and 5 min, respectively, before the addition of U-0126 (10 µM) for 15 min before thrombin stimulation. EGFR and ERK1/2 phosphorylations were analyzed as described. For each experiment, thrombin-induced phosphorylation was quantified by densitometry. Each blot and plot is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thrombin alone, taken as 100%.

 

The inhibition of PI3K by LY-294002 has been shown to affect thrombin-induced ERK1/2 phosphorylation (25). The possible influence of the PI3K/Akt pathway on ERK1/2 phosphorylation was therefore explored in VSMCs pretreated with BAPTA-AM. To do so, VCMCs were pretreated (or not) with BAPTA-AM as described above before the addition of 20 µM LY-294002 (or its diluent) 15 min before thrombin stimulation. In BAPTA-treated VSMCs, LY-294002 significantly reduced the thrombin-induced ERK1/2 phosphorylation by 38 ± 14%, whereas in BAPTA-untreated VSMCs this reduction was 70 ± 8% (n = 3, P < 0.05 vs. BAPTA-treated VSMCs).

Role of PKC in thrombin-induced EGFR-independent and -dependent phosphorylation of ERK1/2. In addition to an increase of [Ca2+]i, thrombin also triggers PKC activation. To assess whether PKC plays a role in thrombin-induced ERK1/2 signaling pathways under the conditions described above, the specific PKC inhibitor GF-109203X (35) was added for 20 min before thrombin stimulation to VSMCs that were or were not pretreated with BAPTA-AM. As shown in Fig. 8, GF-109203X (1–10 µM) had no significant effect on thrombin-induced Src phosphorylation but inhibited, in a dose-dependent manner, EGFR and ERK1/2 phosphorylation. Inhibition of 60% and 70% of thrombin-induced EGFR and ERK phosphorylation, respectively, was obtained when cells were pretreated with 10 µM GF-109203X. However, with the use of the same experimental protocol, no significant effect of GF-109203X (1–10 µM) on ERK1/2 phosphorylation could be observed when VSMCs were stimulated by 1 µM thapsigargin (data not shown). Additionally, pretreatment with both BAPTA-AM and GF-109203X (10 µM) resulted in an additive inhibition of thrombin-induced EGFR autophosphorylation, whereas thrombin-induced ERK1/2 activity was protected (Fig. 8). This suggests that the EGFR-independent thrombin-induced ERK1/2 phosphorylation observed in the presence of BAPTA was not due to the PKC activity that is actually involved in the EGFR-dependent ERK1/2 phosphorylation pathway.



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  		Fig. 8. Role of PKC on thrombin-induced phosphorylation of Src, EGFR, and ERK1/2: influence of BAPTA pretreatment. A: quiescent VSMCs preincubated or not with 10 µM BAPTA-AM for 10 min were additionally pretreated for 20 min with 1 or 10 µM of the PKC inhibitor GF-109203X (GF) before thrombin stimulation. Src, EGFR, and ERK1/2 phosphorylation was analyzed using specific anti-phospho-antibodies as described. B: for each experiment, thrombin-induced phosphorylation was quantified by densitometry. Each blot is representative of at least 3 distinct experiments. Values are means ± SE; n = 3. *P < 0.05 compared with thrombin alone, taken as 100%; $P < 0.05 compared with VSMCs pretreated with 10 µM GF-109203X and stimulated with thrombin.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
In an attempt to unravel the signaling pathways whereby thrombin exerts its growth effect in VSMCs through ERK1/2 activation, we investigated the phosphorylation of signaling tyrosine kinases, namely, Src, Pyk2, and the EGFR, that are known to participate in thrombin-triggered ERK1/2 activation of VSMCs. We used phosphorylation state-specific antibodies directed against phospho-Tyr416 of the Src family, phospho-Tyr402 of Pyk2, phospho-Tyr1068 of the EGFR, and phospho-Thr202/Tyr204 of ERK1/2 because these phosphorylation sites have been described as representative of the activated state of each enzyme (6, 22, 27, 43). First, we determined the appropriate conditions to observe the phosphorylation of the proteins of interest. The results confirmed that in VMSCs, in contrast to astrocytes (38), thrombin induced the phosphorylation of the EGFR (15, 16) as well as that of Src, Pyk2, and ERK1/2. The results shown in Fig. 2 allowed us to draw an organization of the thrombin-induced signaling pathway wherein Src kinase activation occurs upstream of Pyk2, which is itself upstream of EGFR transactivation, that promotes ERK1/2 activation. This is consistent with the roles played by Src in agonist-stimulated vascular contraction, ERK1/2 activation, and cytoskeletal reorganization (13, 14, 36). Our results also indicate that the nonreceptor tyrosine kinase Pyk2 likely participates in the cascade of events upstream of EGFR, as suggested by its lack of inhibition by two EGFR kinase activity inhibitors, namely, tyrphostins AG-1478 and PD-153035. Such a role has been already ascribed to Pyk2 in angiotensin II-stimulated VSMCs (26). In addition, the almost complete inhibition of thrombin-induced ERK1/2 phosphorylation by both AG-1478 and PD-153035 suggests that ERK1/2 activation is dependent on EGFR transactivation, in agreement with the central role described for the EGFR in G protein-coupled receptor signaling (43). Moreover, thrombin-induced ERK1/2 phosphorylation was not affected by pretreatment of VSMCs with 10 µM AG-1295, another tyrphostin that is a selective inhibitor of PDGF receptor kinase (results not shown), indicating that thrombin signaling does not involve PDGF receptor transactivation. This also suggests that the effects observed with both tyrphostins AG-1478 and PD-153035 resulted from their EGFR kinase activity inhibitor property rather than from some nonspecific effects.

Our observation that thapsigargin-induced stimulation of the ERK1/2 pathway is also inhibited by AG-1478 (data not shown), together with recent reports on the involvement of Ca2+ in EGFR transactivation (7, 9, 44), strongly suggest a role for [Ca2+]i in the ERK1/2 activation signaling pathway. To further investigate the role of [Ca2+]i on the phosphorylation levels of these signaling proteins, VSMCs were pretreated with the Ca2+ chelator BAPTA-AM, which completely inhibits thrombin- and thapsigargin-induced [Ca2+]i elevation (Fig. 3A and data not shown). This pretreatment resulted in a complete loss of phosphorylation of the EGFR induced by both thrombin and thapsigargin, but only thapsigargin-induced Src and Pyk2 phosphorylation was noticeably inhibited (Figs. 3 and 4). These results suggest that increased [Ca2+]i is acting upstream of Src/Pyk2 in thapsigargin stimulation, as described for ionomycin, ATP, or PDGF (9). However, thrombin stimulation appears to be more complex. BAPTA-AM pretreatment promoted a small inhibition of the phosphorylation of Src/Pyk2, suggesting other pathway(s) for Src and Pyk2 activation. Surprisingly, BAPTA-AM pretreatment failed to inhibit the ERK1/2 phosphorylation induced by thrombin or thapsigargin (Figs. 3 and 4), suggesting a possible dissociation between the activations of ERK1/2 and the EGFR. This observation questioned the state of activation of EGFR. As only the phosphorylation of Tyr1068, one of the major EGFR autophosphorylation sites, was observed in our study, the EGFR may well have been activated through other phosphorylation sites (39). Indeed, other tyrosine autophosphorylation sites of the EGFR such as Tyr1086, Tyr1148, and Tyr1173 appeared to be also involved in its receptor kinase activity (3, 11). However, it has been reported that BAPTA-AM pretreatment of VSMCs results in the inhibition of all phosphorylation sites of the EGFR (16). To strengthen the existence of an EGFR-independent pathway leading to ERK1/2 phosphorylation, VSMCs were pretreated with a combination of BAPTA-AM and the EGFR kinase inhibitor AG-1478. Under these conditions, thrombin stimulation resulted in a complete absence of EGFR phosphorylation (Fig. 5A), whereas ERK1/2 activation was still partially preserved (Fig. 5). Under these conditions, the role for Src kinase activity in an EGFR-independent ERK1/2 activation pathway was also analyzed using the Src kinase inhibitor PP2. The results shown in Figs. 2 and 6 clearly show that both EGFR-dependent and -independent ERK1/2 phosphorylation pathways are dependent on Src kinase activity. However, it is noticeable that in presence of BAPTA, the level of phosphorylation of Src induced by thrombin and thapsigargin was not higher that the basal level, whereas no inhibition was observed for ERK1/2 phosphorylation (Figs. 3 and 4). Therefore, it is possible that the minimum of Src activity observed in the basal condition is enough but necessary to lead to the activation of ERK1/2.

To determine whether EGFR-independent ERK1/2 phosphorylation resulted from activation of the Ras/Raf/MEK pathway, the effect of potent and specific MEK inhibitors, U-0126 and PD-98059, was investigated. The results show that these drugs strongly reduced the phosphorylation of ERK1/2 without affecting EGFR phosphorylation. Similar inhibition of the thrombin-induced phosphorylation of ERK1/2 was observed when cells were pretreated with a combination of BAPTA-AM and AG-1478 (Fig. 7). These results indicate that both EGFR-dependent and -independent pathways phosphorylate ERK1/2 via MEK. Our observation that LY-294002 partially but significantly inhibited the thrombin-induced ERK1/2 phosphorylation in both BAPTA-treated and -untreated VSMCs (25) suggests that thrombin-induced ERK1/2 activation should be partially mediated by PI3K/Akt signaling. It has been demonstrated that Akt negatively regulates the Ras/Raf/MEK/ERK1/2 pathway via the inactivation of Akt (28, 42). Whether such an interaction occurs in BAPTA-treated VSMCs remains to be established.

Because PKC signaling participates in EGFR activation (16, 43) and can induce ERK1/2 activation directly through Raf/MEK (5), we investigated whether PKC activity could be involved in EGFR-independent ERK1/2 activation. The results clearly show that the inhibition of PKC activity by GF-109203X, while expectedly reducing thrombin-induced phosphorylation of EGFR irrespective of the [Ca2+]i level, does no longer exert its inhibitory effect on thrombin-induced ERK1/2 activation when [Ca2+]i elevation has been prevented by BAPTA-AM pretreatment (Fig. 8). This further confirms that ERK1/2 can be activated through an EGFR-independent pathway.

Taken together, our results indicate that under regular/physiological conditions, thrombin-induced ERK1/2 activation of VSMCs occurs through events that are dependent on PKC, Src, MEK, and EGFR activation, as already reported (8, 10, 16). Our results also indicate that under some particular conditions resulting in [Ca2+]i depletion, thrombin-induced ERK1/2 activation may occur through pathways that are dependent on Src and MEK activity but independent of both EGFR kinase and PKC activity (Fig. 9). This is the first report on the involvement of such pathways in thrombin signaling in VSMCs.



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  		Fig. 9. Schematic diagram of thrombin signaling in VSMCs resulting in ERK1/2 phosphorylation: effect of BAPTA pretreatment. A: in quiescent VSMCs, thrombin stimulation results in the activation of Src family kinases, Pyk2, and PKC and mobilization of calcium. These induce the activation of ERK1/2 through EGFR transactivation. B: in VSMCs pretreated with BAPTA, thrombin-induced ERK1/2 phosphorylation is preserved, independent of EGFR and PKC activity but still dependent on Src kinase activity. GPCR, G protein-coupled receptor; ERK1/2-P, phosphorylated ERK1/2.

 

Phosphatases are known to participate in MAP kinase signaling (18, 41). Thus it is possible that BAPTA-AM pretreatment results in inhibition of calcium-dependent phosphatases that control phosphorylation level of ERK1/2 in the nucleus. This may result in a lower rate of dephosphorylation of ERK1/2, unmasking a new EGFR-independent signaling pathway in thrombin- and thapsigargin-stimulated VSMCs. Moreover, the fact that EGFR-independent ERK1/2 phosphorylation in presence of BAPTA is also observed under thapsigargin stimulation, i.e., under conditions that block intracellular calcium pumps, suggests a role for either the calcium pumps or those intracellular pools that are depleted in calcium. Indeed, the calcium-emptied reticulum has recently been reported to be associated with a prolonged phosphorylation of ERK1/2 through inhibition of MKP1 expression (19). Such an event, which can be associated with the activation of ERK1/2, remains under investigation.

This work provides evidence for a new pathway of ERK1/2 activation and potentially of VSMC proliferation/differentiation. This pathway seems to be independent of EGFR transactivation or PKC activation but dependent on Src family kinase activation and is triggered in absence of an elevation of [Ca2+]i during VSMC stimulation. The importance of such pathway is relevant as many compounds used in therapy modify Ca2+ homeostasis. It has been, for example, recently reported that in cultured VSMCs, the antihypertensive drug amlodipine might interact with the intracellular Ca2+ pump and might empty the Ca2+ pool (32). Because amlodipine pretreatment appears to affect calcium mobilization without interfering with the level of phosphorylation of ERK1/2 (33), potential effects of the drug on the presently reported pathway are currently under investigation.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was partially supported by Pfizer International.


   ACKNOWLEDGMENTS
 
We are indebted to Francine Amicel-Lourtil for valuable assistance. We thank Drs. C. Bernaud (Paris, France) and Jan Buch (New York, NY) for steady encouragement throughout the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Marche, CNRS UMR 7131, Hôpital Broussais, 102 rue Didot, 75014 Paris, France (E-mail: pierre.marche{at}brs.ap-hop-paris.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.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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