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(Hypertension. 2001;37:148.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Salicylate Inhibits Phosphorylation of the Nonreceptor Tyrosine Kinases, Proline-Rich Tyrosine Kinase 2 and c-Src

Zhongyan Wang; Peter Brecher

From the Whitaker Cardiovascular Institute and Department of Biochemistry, Boston University School of Medicine, Boston, Mass.

Correspondence to Peter Brecher, PhD, Boston University School of Medicine, 715 Albany St, Boston, MA 02118. E-mail pbrecher{at}bu.edu

	Abstract

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Abstract—The anti-inflammatory effects of salicylate are well known, but the intracellular mechanisms underlying those effects remain to be clarified and are not explained solely by an influence on cyclooxygenase activity. In the present study, we have used cardiac fibroblasts stimulated by either angiotensin II (Ang II) or platelet-derived growth factor (PDGF) to demonstrate an inhibitory effect of salicylate on the phosphorylation of the nonreceptor tyrosine kinases, proline-rich tyrosine kinase 2 (PYK2) and c-Src, by immunoprecipitation and immunoblotting methods. This inhibition was dose dependent, with a clear effect observed at concentrations between 5 and 20 mmol/L salicylate. Intracellular Ca²⁺ chelation and protein kinase C (PKC) inhibition reduced Ang II and PDGF-induced PYK2 and c-Src phosphorylation. Salicylate significantly inhibited the phosphorylation of both of the tyrosine kinases activated by either ionophore A23187 or thapsigargin treatment, which led to an elevation of cytosolic Ca²⁺. Activation of PKC by phorbol ester phosphorylated both PYK2 and Src, and this effect also was attenuated by salicylate. In contrast, salicylate had no effect on either the transactivation of the epidermal growth factor receptor by Ang II or the phosphorylation of phospholipase C- by PDGF. These studies indicate a novel site of action for salicylate on PYK2 and c-Src phosphorylation and suggest that this inhibitory effect on these important signaling intermediates may be through a Ca²⁺- and PKC-dependent mechanism.

Key Words: NSAIDs • kinase • signal transduction • fibroblasts

	Introduction

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The ability of NSAIDs to reduce complications associated with inflammatory diseases has historically been associated with the ability of these drugs to suppress prostaglandin biosynthesis by inhibiting the enzymes cyclooxygenase I or cyclooxygenase II (COX 1 and 2). However, in recent years many studies have suggested alternative mechanisms for the anti-inflammatory action of drugs such as aspirin or its metabolite, salicylate. Both aspirin and salicylate inhibit neutrophil activation by interfering with signal transduction pathways independently of prostaglandin synthesis.^{1 2} Sodium salicylate and aspirin, but not several other anti-inflammatory agents, including indomethacin, were shown to disrupt activation of the nuclear factor-B pathway by preventing inhibitor of B (IB) degradation and the nuclear translocation of nuclear factor-B.³ Therapeutic serum concentrations of salicylate, the major metabolite of aspirin and a relatively weak inhibitor of COX 1 and COX 2, correlated better with clinical signs of anti-inflammation than did serum concentrations of aspirin.⁴ Salicylate was recently shown to promote apoptosis via activation of p38 mitogen-activated protein kinase (p38 MAPK) in human FS-4 fibroblasts.⁵ Such studies suggest that signal transduction pathways are implicated in the anti-inflammatory effects of salicylate.

In several previous studies, we have examined the role of salicylate in modulating the cytokine-induced production of inducible NO synthase (iNOS) by using cardiac fibroblasts as an experimental model, because both cytokines and iNOS may have an important role in the progression of cardiac fibrosis. We described the effects of cytokine treatment on iNOS expression in cardiac fibroblasts and showed that sodium salicylate inhibited cytokine-induced nitrite accumulation.⁶ We further investigated iNOS mRNA expression by cytokines and showed that the effects of salicylate were to inhibit iNOS transcription.⁷ Those studies^{6 7} implicated cardiac fibroblasts as a source of NO in inflammatory cardiac diseases and suggested a possible therapeutic role for salicylate and aspirin in diminishing the steady-state levels of iNOS mRNA. We recently examined the role of the mitogen-activated protein kinase (MAPK) cascades in the control of iNOS expression and found not only that MAPK cascades were involved but also that salicylate selectively inhibited the phosphorylation of extracellular signal–regulated kinase (ERK) but had no obvious effect on the activation of either p38 MAPK or c-Jun N-terminal kinase.⁸

Studies have implicated MAPK pathways in the action of angiotensin II (Ang II) on cardiac fibroblasts, and we have used Ang II in several studies showing that either NO or the antioxidant N-acetylcysteine could modify the activation of MAPK by Ang II.^{9 10 11} In those studies, we characterized several intermediates required for the activation of ERK by Ang II in the cardiac fibroblasts. In the present study, we have used this experiment model to evaluate the possible sites of action of salicylate on components of the signaling system involved in Ang II or platelet-derived growth factor (PDGF) activity, and we have found that salicylate suppressed the phosphorylation of the Ca²⁺-dependent nonreceptor tyrosine kinases, proline-rich tyrosine kinase 2 (PYK2) and c-Src. These signaling intermediates are known to have widespread effects on diverse signaling pathways.^{10 11 12 13 14} The present findings establish a novel site of action for salicylate in hormonal signaling and may offer new insight into how this drug modulates the signal transduction in a Ca²⁺-dependent and protein kinase C (PKC)-dependent manner.

	Methods

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Materials
All tissue culture media and antibiotics were from Life Technologies, Inc; Ang II, sodium salicylate, thapsigargin, phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF) were purchased from Sigma Chemical Co; ionophore A23187 was from Alexis Corp; BAPTA-AM and Gö 6983 were from Calbiochem; and PDGF-BB was from Upstate Biotechnology, Inc. Anti-phosphotyrosine (PY20) and anti-PYK2 monoclonal antibodies were obtained from Transduction Laboratories; EGF receptor antibody was from Santa Cruz Biotechnology, Inc; anti–phospholipase C- (PLC-) antibody was from Upstate Biotechnology; anti–phospho-specific PYK2 polyclonal antibodies (pY402, pY579, pY580, and pY881) were purchased from BioSource, Inc; anti–phospho-Src antibody (pY416) was a kind gift from Dr Xiqiang Hong (New England BioLabs, Beverly, Mass).

Cell Culture and Treatment
Primary cultures of neonatal rat cardiac fibroblasts were obtained and maintained in culture as described previously.⁶ Cells in the sixth passage were used for all experiments. Ang II was routinely added to the cells at a final concentration of 10 nmol/L, and PDGF was added at a final concentration of 10 ng/mL. Sodium salicylate was added to the culture medium from a 500 mmol/L solution and had no effect on the pH of the culture medium. Salicylate was routinely added 30 minutes before the addition of agonists. Salicylate (20 mmol/L) alone had no effect on the phosphorylation of either PYK2, c-Src, EGF receptor, or PLC-. All experiments are representative of multiple experiments using separate cell preparations.

Immunoprecipitation and Immunoblotting
For the determination of PYK2, c-Src, and PLC-, tyrosine phosphorylation, immunoprecipitation, and immunoblotting were performed as described previously.^{9 10 11} For direct measurement of different phosphorylation sites of PYK2, the aliquots of whole-cell lysates were resolved by 10% SDS-PAGE, and the immunoblotting was performed by anti–selective phospho-PYK2 polyclonal antibodies (pY402, pY579, pY580, and pY881).

Statistical Analysis
Data are presented as mean±SE of 3 experiments unless designated otherwise. Statistical analysis was performed by ANOVA, as appropriate. A value of P<0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Salicylate Inhibits PYK2 Phosphorylation in Response to Ang II and PDGF
Treatment of quiescent rat cardiac fibroblasts with Ang II (10 nmol/L) or PDGF-BB (10 ng/mL) showed a rapid and transient increase in phosphorylated PYK2 as early as 1 minute, a maximal increase within 5 minutes, and a subsequent decrease thereafter (Figure 1A). To determine whether salicylate influenced PYK2 phosphorylation, cells were pretreated with different concentrations of salicylate ranging from 5 to 20 mmol/L for 30 minutes and then incubated with either Ang II or PDGF for 5 minutes. Figure 1 shows that salicylate caused a dose-dependent inhibition of PYK2 phosphorylation by either Ang II (Figure 1B) or PDGF (Figure 1C) stimulation.

View larger version (37K): [in this window] [in a new window]   Figure 1. Effect of salicylate (Sal) on PYK2 tyrosine phosphorylation. Whole-cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and analyzed by immunoblotting with anti-PYK2 antibody. A, Cells were incubated with Ang II (10 nmol/L) and PDGF (10 ng/mL) for the indicated times, and then PYK2 phosphorylation was determined. B and C, Cells were pretreated with the indicated concentration of Sal for 30 minutes, followed by 10 nmol/L Ang II (B) or 10 ng/mL PDGF (C) for 5 minutes. The graphs in panels B and C represent the mean±SE from densitometric analyses of 3 independent experiments, and values are expressed as a fold increase relative to control (Ctl), arbitrarily defined as 1 U. *P<0.05 vs Ang II (B) or PDGF (C) treatment.

Different Phosphorylation Sites of PYK2 Are Suppressed by Salicylate
A panel of specific phosphorylation state–selective antibodies of PYK2 was used to determine whether specific tyrosine sites within PYK2 were phosphorylated in response to agonists and to examine whether salicylate selectively reduced individual tyrosine phosphorylation. As shown in Figure 2, Ang II treatment resulted in the phosphorylation of each of 4 phosphorylated forms of PYK2 tested (pY402, pY579, pY580, and pY881). PDGF treatment also increased the PYK2 isoforms, although the response was reproducibly less than that seen with Ang II. After 10 mmol/L salicylate pretreatment, cells treated with Ang II or PDGF had decreased PYK2 phosphorylation at all sites, with no suggestion of a specific effect on one phosphorylated form over another.

View larger version (83K): [in this window] [in a new window]   Figure 2. Effect of Sal on different phosphorylation sites of PYK2. Cells were pretreated with 10 mmol/L Sal for 30 minutes or were left untreated and then incubated with Ang II (10 nmol/L) or PDGF (10 ng/mL) for 5 minutes. Different phosphorylation sites of PYK2 were assayed with anti–phospho-specific PYK2 polyclonal antibodies (pY402, pY579, pY580, and pY881) by immunoblotting from whole-cell lysates. pY indicates phosphotyrosine.

Salicylate Inhibits c-Src Phosphorylation in Response to Ang II and PDGF
Both PYK2 and c-Src are nonreceptor tyrosine kinases that are activated by Ang II, and PDGF has been implicated in the signaling response.^{10 11 12} Figure 3A shows that Ang II or PDGF elicited tyrosine phosphorylation of c-Src within 1 minute, and this response was sustained for at least 15 minutes. Salicylate had a similar inhibitory effect on c-Src phosphorylation induced by Ang II (Figure 3B) or PDGF (Figure 3C), and that inhibition was comparable to that shown above for PYK2 phosphorylation.

View larger version (38K): [in this window] [in a new window]   Figure 3. Effect of Sal on c-Src tyrosine phosphorylation. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and assayed by immunoblotting with anti–c-Src antibody. A, Cells were incubated with Ang II (10 nmol/L) and PDGF (10 ng/mL) for the indicated times, and then c-Src phosphorylation was determined. B and C, Cells were pretreated with the indicated concentrations of Sal for 30 minutes, followed by 10 nmol/L Ang II (B) or 10 ng/mL PDGF (C) for 5 minutes. The graphs in panels B and C show the densitometric analyses of 3 separate experiments. *P<0.05 vs Ang II (B) or PDGF (C).

Effect of Salicylate on the Association of PYK2 With c-Src
To determine whether c-Src plays a role in PYK2 signaling activated by Ang II or PDGF, cell lysates obtained from either Ang II or PDGF treatment were immunoprecipitated with anti–c-Src antibody and then analyzed by immunoblotting with anti-PYK2 monoclonal antibody. As shown in Figure 4, in unstimulated cells, the association between PYK2 and c-Src was relatively weak. Both Ang II and PDGF increased complex formation between PYK2 and c-Src, although the complex induced by Ang II was stronger than that induced by PDGF. Because the results from Figures 1, 2, and 3 show that salicylate decreased the phosphorylation of either PYK2 or c-Src when cells were treated with Ang II or PDGF, we next determined whether salicylate affected formation of the PYK2-Src complex. Salicylate treatment significantly suppressed the association of PYK2 with c-Src stimulated by either Ang II or PDGF, providing further evidence that salicylate influences signaling pathways that involve PYK2 and c-Src.

View larger version (31K): [in this window] [in a new window]   Figure 4. Association of PYK2 with c-Src was suppressed by Sal. Cardiac fibroblasts were stimulated with Ang II (10 nmol/L) or PDGF (10 ng/mL) for 5 minutes with or without Sal pretreatment for 30 minutes. Cell lysates were immunoprecipitated (IP) with anti–c-Src antibody, followed by immunoblotting (IB) with anti-PYK2 monoclonal antibody. The graph shows the densitometric analyses of 3 separate experiments. *P<0.05 indicates the significant differences between the cells with and without Sal pretreatment.

Salicylate Mediates PYK2 and c-Src Phosphorylation Through Ca²⁺ and PKC-Dependent Mechanisms
The effects of intracellular Ca²⁺ and PKC on the agonist-induced phosphorylation of both PYK2 and c-Src were studied in the experiments shown in Figure 5. The increased PYK2 and Src phosphorylation induced by either Ang II or PDGF was almost completely abolished by pretreatment with the intracellular Ca²⁺ chelator BAPTA-AM and partially blocked by the PKC inhibitor Gö 6983, respectively (Figure 5A). To determine whether direct activation of PKC or increased intracellular Ca²⁺ could alter PYK2 and Src phosphorylation, the fibroblasts were treated with PMA, the calcium ionophore A23187, or thapsigargin, an agent that specifically inhibits the endoplasmic reticulum Ca²⁺-ATPase, with the net effect of increasing intracellular Ca²⁺. As shown in Figure 5B, treatment of the cells with either A23187, thapsigargin or PMA caused PYK2 and Src phosphorylation, and the inhibitory effect of salicylate on the phosphorylation of these signaling intermediates was obvious in all cases.

View larger version (56K): [in this window] [in a new window]   Figure 5. Effect of Ca2+ and PKC on PYK2 and c-Src phosphorylation. A, After pretreatment for 30 minutes with 50 µmol/L BAPTA-AM (BA) or 1 µmol/L Gö 6983 (Gö), the cells were treated with Ang II (10 nmol/L) or PDGF (10 ng/mL) for 5 minutes. Graph shows the results from densitometric analyses of 3 experiments. *P<0.05 vs Ang II or PDGF treatment. B, Cells were incubated for 15 minutes with 10 µmol/L A23187, 10 µmol/L thapsigargin (TG), or 2 µmol/L PMA in the absence or presence of a 30-minute pretreatment with 10 mmol/L Sal. To examine PYK2 and c-Src phosphorylation, cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and assayed by immunoblotting with either anti-PYK2 monoclonal antibody or anti–c-Src antibody. Graph shows the densitometric analyses of 3 separate experiments. *P<0.05 indicates the significant differences between the cells with and without Sal pretreatment.

Salicylate Does Not Affect PLC- Phosphorylation by PDGF
Experiments were performed to determine whether PLC- phosphorylation in response to PDGF was affected by salicylate. As we had noted recently,¹¹ PLC- was strongly phosphorylated by PDGF but not by Ang II. A23187 treatment also did not cause PLC- phosphorylation in the cardiac fibroblasts (Figure 6A). Salicylate pretreatment did not significantly influence the phosphorylation of PLC- induced by PDGF under conditions in which either PDGF (Figure 6B) or salicylate (Figure 6C) concentrations were varied.

View larger version (40K): [in this window] [in a new window]   Figure 6. Effect of salicylate on PLC- phosphorylation. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and subsequently analyzed by immunoblotting with anti–PLC- antibody. A, Cells were treated with either 10 nmol/L Ang II (AII) or 10 ng/mL PDGF for 5 minutes or 10 µmol/L A23187 for 15 minutes. Salicylate pretreatment (10 mmol/L) was for 30 minutes where designated. B, Cells were pretreated for 30 minutes in the absence or presence of 10 mmol/L salicylate and then incubated for a further 5 minutes with designated concentrations of PDGF. C, Cells were preincubated for 30 minutes with 5, 10, or 20 mmol/L salicylate and then stimulated for 5 minutes with 10 ng/mL PDGF.

Salicylate Does Not Affect EGF Receptor Phosphorylation by Ang II
Recently, transactivation of the EGF receptor by Ang II has been documented in vascular smooth muscle cells¹⁵ and cardiac fibroblasts.^{10 16} Figure 7A indicated that the increasing amounts of salicylate did not decrease the phosphorylation of the EGF receptor caused by Ang II. Figure 7B shows that EGF receptor phosphorylation by EGF was much stronger than that by Ang II. Salicylate had no inhibitory effect on EGF receptor phosphorylation by either Ang II or EGF. Equal loading of samples was confirmed by reprobing the identical membrane with an anti–EGF receptor antibody (Figure 7A and 7B, bottom).

View larger version (55K): [in this window] [in a new window]   Figure 7. Effect of salicylate on EGF receptor (EGFR) phosphorylation. A, Cells were pretreated with the indicated concentration of salicylate for 30 minutes, followed by 10 nmol/L Ang II for 3 minutes, and cell extracts were immunoprecipitated (IP) with anti-EGFR antibody. Immunoblotting (IB) was performed with an anti-phosphotyrosine antibody (PY20). B, Cells were pretreated with or without 10 mmol/L salicylate for 30 minutes and then treated with 10 nmol/L Ang II or 2 ng/mL EGF for 3 minutes. EGFR phosphorylation (pEGFR) was examined as described above. Total EGFR was detected by reprobing the membrane with an anti-EGFR antibody..

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of the present study document the inhibitory effects of salicylate on the phosphorylation of both PYK2 and c-Src during the initial response of cardiac fibroblasts to Ang II or PDGF. PYK2 and Src have been implicated in early signaling responses of G protein–coupled receptors, including the angiotensin type 1 receptor and also tyrosine kinase–linked receptors.^{17 18 19} Consistent with those reports, Ang II or PDGF did cause transient PYK2 and Src phosphorylation in the cardiac fibroblasts and therefore could be used as an experimental model to determine what effect salicylate might have on those intracellular tyrosine kinases. These Ca²⁺-dependent nonreceptor tyrosine kinases are thought to be involved in the activation of ERK by agonists such as Ang II and PDGF. Salicylate inhibition of PYK2 and Src was independent of the activation of PLC- (a rapid response to the addition of PDGF) but not Ang II in the cardiac fibroblasts and was also independent of the transactivation of the EGF receptor by Ang II (a response required for the activation of ERK by Ang II but not for PDGF).¹⁶

PYK2 is part of a distinct family of nonreceptor tyrosine kinases that are regulated by a variety of extracellular stimuli. Several phosphorylation sites on PYK2 have been identified, including the binding site for the SH2 domains of Src (at Tyr402 of PYK2) and the putative binding site for the SH2 domain of Grb2 (at Tyr881 of PYK2).^{14 20 21} We used selective antibodies against 4 different phosphorylation sites on PYK2 (at Tyr402, Tyr579, Tyr580, and Tyr881), recently made available commercially, to show that each of those sites was phosphorylated in response to either Ang II or PDGF, and we showed that salicylate suppressed each of those phosphorylation events. These data suggest that salicylate does not selectively influence unique tyrosine phosphorylation sites within PYK2, but the findings do correlate with other data indicating that the Tyr402 site is required for interaction with c-Src.¹⁴ Recent studies suggest that c-Src has an important role in Ang II signal transduction and has been reported to form an active complex with PYK2, presumably dependent on increases in intracellular Ca²⁺ and involving the SH2 domain of Src, and then to bind to the appropriate region of PYK2.^{13 19} We found that c-Src, like PYK2, was rapidly phosphorylated in response to either Ang II or PDGF and that salicylate reduced Src phosphorylation in a manner comparable to that observed for PYK2. Furthermore, salicylate not only decreased the phosphorylation of either PYK2 or c-Src but also inhibited the formation of a complex between PYK2 and Src induced by Ang II or PDGF.

Both Ang II and PDGF are known to mediate their effects through increases in either intracellular Ca²⁺ or PKC activity. It was recently reported that Ca²⁺ and PKC are independent and potentially additive activators of PYK2 when stimulated by Ang II or PDGF in vascular smooth muscle cells.¹⁷ The present study in the cardiac fibroblast showed that pretreatment with BAPTA-AM resulted in a significant inhibition of Ang II or PDGF-induced PYK2 and Src activity, whereas a PKC inhibitor (Gö 6983) partially inhibited both PYK2 and Src activity. These findings suggest that Ca²⁺ and/or PKC has a regulatory role in PYK2 and Src phosphorylation. Salicylate pretreatment inhibited both A23187- and thapsigargin-induced PYK2 and Src phosphorylation, implicating an effect on responses secondary to increased Ca²⁺ transients, and salicylate also reduced PMA-induced phosphorylation of both PYK2 and Src, an effect not necessarily due to changes in Ca²⁺. The results suggested that salicylate seems to regulate PYK2 and c-Src phosphorylation through both Ca²⁺- and PKC-dependent mechanisms.

We also investigated the effects of salicylate on other intermediates involved in Ang II and PDGF signal transduction. An important role for PLC-1 is well established in PDGF signal transduction cascades. When PLC- becomes phosphorylated, it results in the formation of diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol activates PKC, and inositol 1,4,5-trisphosphate induces Ca²⁺ release from intracellular stores.²² Thus, PLC- may be an upstream mediator of PYK2 and Src, because salicylate did not affect PDGF-induced PLC- phosphorylation, whereas PDGF stimulated PYK2, and Src tyrosine phosphorylation was dependent on the activation of both downstream effects (Ca²⁺ and PKC) of PLC-.

The relationship between Ang II action and EGF receptor phosphorylation has been documented.^{10 11 16} Our findings (ie, that salicylate pretreatment, by use of conditions that effectively suppressed Ang II phosphorylation of PYK2, had no influence on the transactivation of the EGF receptor by Ang II or on the phosphorylation of the receptor by EGF) are consistent with the possibility that PYK2 and c-Src activation proceed through a pathway independent of EGF receptor transactivation in the cardiac fibroblasts.

The concentrations of salicylate used in the present study are slightly higher than the concentrations found in the serum of patients treated with salicylate for inflammatory diseases, which range from 1 to 5 mmol/L.^{23 24} However, local concentrations within specific tissue sites or accumulation of salicylate or active metabolites within a target cell type could reach the levels used in our model system. Other studies have suggested that salicylate potentially influences the intracellular redox state,²⁵ and we have published several studies showing that Ang II signaling in the cardiac fibroblast is influenced by antioxidants such as N-acetylcysteine.^{10 11} Thus, whether salicylate inhibits the phosphorylation of PYK2 and c-Src by altering the intracellular redox state requires further investigation. Both Ang II and PDGF are known to activate multiple signaling pathways, and Ang II has been shown to play a critical role in the pathogenesis of hypertension, inflammation, atherosclerosis, and congestive heart failure.¹² The expanding role of salicylate on signaling by Ang II and PDGF may provide new insight for understanding the anti-inflammatory effect of this drug.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-53471.

Received April 20, 2000; first decision May 18, 2000; accepted July 10, 2000.

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