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Circulation Research. 2004;94:1050-1058
Published online before print March 25, 2004, doi: 10.1161/01.RES.0000126404.41421.BE
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(Circulation Research. 2004;94:1050.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Sphingosine 1-Phosphate Transactivates the Platelet-Derived Growth Factor ß Receptor and Epidermal Growth Factor Receptor in Vascular Smooth Muscle Cells

Tatsuo Tanimoto, Andreea O. Lungu, Bradford C. Berk

From the Center for Cardiovascular Research, University of Rochester, Rochester, NY.

Correspondence to Bradford C. Berk, Center for Cardiovascular Research, University of Rochester, 601 Elmwood Ave, Box 679, Rochester, NY 14642. E-mail bradford_berk{at}urmc.rochester.edu


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
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Sphingosine 1-phosphate (S1P) is a bioactive lipid generated during vascular injury that regulates cell growth, differentiation, survival, and motility via endothelial differentiation gene (EDG) family G protein–coupled receptors. Although several G protein–coupled receptor ligands transactivate receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), S1P-stimulated receptor tyrosine kinase transactivation has not been well studied. We show that platelet-derived growth factor ß receptor (PDGFßR) and EGFR are tyrosine phosphorylated in response to S1P in rat aortic vascular smooth muscle cells (VSMCs). S1P-stimulated transactivation of PDGFßR and EGFR was mediated via Gi-coupled EDG receptors. S1P-stimulated transactivation of EGFR and PDGFßR was dependent on Src, reactive oxygen species, and cholesterol-rich membranes. A phosphoinositide 3-kinase–Akt pathway was activated by S1P and blocked by AG1296 and AG1478. Activation of extracellular signal–regulated kinase (ERK) 1 and ERK2 pathway by S1P was blocked only by AG1478. In Chinese hamster ovary cells that expressed exogenous EDG-1, activation of Akt and ERK1/2 in response to S1P was observed and was enhanced by coexpression of PDGFßR or EGFR. S1P-mediated VSMC proliferation was shown to be secondary to transactivation, because it was suppressed by AG1296 and AG1478. These data establish S1P as an important stimulus for EGFR and PDGFßR activation in VSMCs that may have important implications for the vessel response to injury.


Key Words: signal transduction • epidermal growth factor • sphingosine 1-phosphate


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
S1P, a bioactive lipid released by activated platelets, induces many biological responses, including cell proliferation, differentiation, survival, and motility.1–4 S1P is a ligand for the EDG family of G protein–coupled receptors (GPCRs). These receptors, which include EDG-1/S1P1, EDG-3/S1P3, EDG-5/S1P2, EDG-6/S1P4, and EDG-8/S1P5, all bind S1P and dihydro-S1P with high affinity but couple to different G-proteins and thus regulate diverse processes.5 Whereas EDG-1, EDG-6, and EDG-8 couple mainly to Gi, both EDG-3 and EDG-5 activate Gi, Gq, and G12/13. These receptors are highly regulated and differentially expressed in various tissues. In vascular smooth muscle cells (VSMCs), the expression levels of EDG-3 and -5 are high whereas EDG-1 is relatively low.6–11 However, it has been suggested that EDG-1 is the major receptor for S1P-stimulated VSMC proliferation and migration.7 Conversely, EDG-5 seems to suppress VSMC motility.6

Transactivation of receptor tyrosine kinase (RTK) by binding of ligand to GPCRs has been shown to have important physiological consequences.12–16 Activation of EGFR and platelet-derived growth factor ß receptor (PDGFßR) by several GPCR ligands, including thrombin, angiotensin II (Ang II), lysophosphatidic acid (LPA), and endothelin-1, has been well studied.17–19 EGFR is also transactivated by PDGF20 and insulin-like growth factor I.21 Because S1P is a relatively newly identified GPCR ligand, S1P-stimulated transactivation of RTK has not been well studied. Recently, our group and another reported that S1P transactivates the vascular endothelial growth factor (VEGF) receptor 2/Flk-1/KDR in endothelial cells, which contributed to endothelial NO synthase activation and cell motility.22,23 Of great interest, Hobson et al24 and Alderton et al25 reported a novel type of reverse transactivation from the PDGFßR to EDG-1. Specifically, in response to PDGF-BB, sphingosine kinase was activated, S1P was formed and secreted, and EDG-1 was activated.

In this report, we show that S1P stimulates transactivation in VSMCs of both PDGFßR and EGFR, which is important for stimulation of phosphoinositide 3-kinase (PI3K), extracellular signal–regulated kinase (ERK) 1/2, and VSMC proliferation.


*    Materials and Methods
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 *Materials and Methods
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Reagents
Sources of reagents are listed in the online data supplement, available at http://circres.ahajournals.org.

Cell Culture and Proliferation
VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) and maintained in DMEM with 10% FBS, as described.15,17 (Animals were maintained by the University of Rochester Vivarium and Division of Laboratory Animal Medicine of the School of Medicine and Dentistry following National Institutes of Health guidelines.) VSMCs at 70% to 80% confluence were growth arrested by incubation in DMEM without serum for 48 hours. Adenoviral dominant-negative c-Src or LacZ were infected (1000 MOI) to VSMCs, as described previously.26 To assay proliferation, VSMCs (25 000 cells) were seeded in 24-well plates in DMEM with 10% FBS. The next day, medium was changed to DMEM without FBS-containing agonists and incubated for 2 or 4 days. Then cells were trypsinized and cell number was counted. Chinese hamster ovary (CHO) cells were maintained in Ham’s F12 medium supplemented with 10% FBS. CHO cells stably expressing mouse PDGFßR were a gift from Dr Harlan Ives27 and were maintained in 600 µg/mL G418. pcDNA3.1-FLAG-EDG-1 and pcDNA3.1-EGFR were gifts from Drs Timothy Hla7 and Hamid Band,28 respectively.

Immunoblot Analysis
Western blot analyses were performed from >=3 experiments, as described.20,21 Densitometric analyses were performed by NIH imaging. For analysis of data from several experiments, background intensity (lane 1) was subtracted from the intensity of each lane. Next, the value of the total pixel intensity for each protein on its Western blot was set to 100%, and the relative densitometry of each lane was expressed as percent of total. This approach was used because basal phosphorylation was so low in some experiments that fold increase was not a meaningful measurement.

Immunoprecipitation
Lysates containing equal amounts (300 µg) of protein were precleared with protein A/G PLUS-agarose and immunoprecipitated as described previously.20,21

Detergent-Free Purification of Cholesterol-Rich Membrane Fractions
Cholesterol-rich membrane (CRM) fractions were prepared as reported by Song et al.29 Details are available in the online data supplement.

Superoxide Measurement
The redox-sensitive fluorescent dye hydroethidine was used to evaluate in situ production of superoxide.30 Details are available in the online data supplement.

Statistical Analysis
Data are presented as mean±SEM for all experiments that were performed at least three times. Data were evaluated by ANOVA followed by Dunnett’s multiple-comparison tests.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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S1P Stimulates Tyrosine Phosphorylation of PDGFßR and EGFR
In response to 1 µmol/L S1P, tyrosine phosphorylation of PDGFßR and EGFR increased in a time-dependent manner (Figures 1A and 1B). Both PDGFßR and EGFR tyrosine phosphorylation peaked at 5 minutes, and PDGFßR phosphorylation decreased to basal levels by 120 minutes. EGFR tyrosine phosphorylation decreased but still remained at 120 minutes. c-Src, Akt, and ERK1/2 were also phosphorylated with peaks at 2, 5, and 10 minutes, respectively. PI3K and Shc, which contribute to growth factor–stimulated activation of Akt and ERK, were also tyrosine phosphorylated in response to S1P. Coimmunoprecipitation of PI3K with the PDGFßR and EGFR was observed in a time-dependent manner. Interestingly, Shc was associated with EGFR but not with PDGFßR. The time course for PI3K binding to the PDGFR and EGFR and phosphorylation of PI3K were similar, suggesting that after binding to the EGFR or PDGFR, PI3K is activated.



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  		Figure 1. S1P transactivates PDGFßR and EGFR. A, VSMCs were stimulated with 1 µmol/L S1P for the indicated times, and cell lysates were prepared. PDGFßR and EGFR were immunoprecipitated and immunoblotted with antibodies to phosphotyrosine (4G10), PI3K, Shc, and PDGFßR and EGFR. Phosphorylation of PI3K and Shc was analyzed by immunoprecipitation of each protein and immunoblotting with anti-phosphotyrosine antibody. Tyrosine phosphorylation of p85 PI3K has been shown to correlate with increased PI3K activity, likely via release of inhibition of p110 PI3K.44 Phosphorylation of c-Src, Akt, and ERK1/2 was analyzed by phospho-specific antibodies. B, Summary of phosphorylation time course. The density values at time 0 were subtracted from the value of each lane, and the data were expressed by setting the total densitometry of all time points as 100%. Thus, each blot was expressed as percent of total (shown is the mean±SE, n>=3). Shc bound to PDGFßR in the top panel was expressed as relative density compared with corresponding PDGFßR level, because the density level was very low.

The concentration response for S1P showed an approximate EC50 of 1 nmol/L for phosphorylation of PDGFßR, EGFR, and Akt, and maximum phosphorylation was observed at {approx}100 nmol/L (not shown). In contrast, ERK1/2 phosphorylation did not saturate >=1 µmol/L S1P. Because 1 µmol/L is considered the physiological highest concentration of S1P, we did not examine higher concentrations. The EC50 for ERK1/2 phosphorylation was {approx}30 nmol/L S1P based on the assumption that phosphorylation at 1 µmol/L was maximal. These data suggested that S1P-stimulated activation of Akt and ERK is secondary to transactivation of RTK, although there may be differences in downstream events given the concentration-response differences for Akt versus ERK1/2.

VSMCs also express LPA receptors, and S1P can cross-react with LPA receptors. In our experiments, 10 µmol/L LPA transactivated the EGFR to an extent similar to 1 µmol/L S1P, but 1 µmol/L LPA transactivated EGFR weakly and 10 µmol/L LPA transactivated the PDGFßR very weakly (data not shown). These results suggest that the effect of S1P at 1 µmol/L observed in VSMCs is mainly mediated by S1P receptors. Based on these data, cells were treated with 1 µmol/L S1P to assay the effect of several inhibitors, because phosphorylation levels of PDGFßR and EGFR were {approx}5-fold increased at that concentration.

S1P-Stimulated Transactivation of PDGFßR and EGFR Is Inhibited by AG1296 and AG1478
Transactivation of RTK in response to GPCR ligands depends on the tyrosine kinase activity of RTK.17,31 We examined whether S1P-induced PDGFßR and EGFR transactivation was dependent on tyrosine kinase activity using specific inhibitors. As shown in Figure 2A and the Table, AG1296, a PDGFßR tyrosine kinase inhibitor, abolished phosphorylation of PDGFßR in response to S1P (compare lanes 5 and 7, IP: PDGFßR) and partially inhibited phosphorylation of Akt (lanes 5 and 7, IB: pAkt). AG1478, a specific EGFR tyrosine kinase inhibitor, abolished EGFR phosphorylation (lanes 5 and 6, IP: EGFR) and inhibited the phosphorylation of ERK1/2 (lanes 5 and 6, IB: pERK) and Akt (lanes 5 and 6, IB: pAkt) {approx}60% to 70%. Pretreatment of VSMCs with both AG1296 and AG1478 caused additional inhibition of the phosphorylation of Akt in response to S1P (lane 8). Specificity of the inhibitors was demonstrated by complete but selective inhibition of PDGF-BB–mediated and EGF-mediated tyrosine phosphorylation of PDGFßR and EGFR, respectively (data not shown).



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  		Figure 2. Effect of PDGFßR tyrosine kinase inhibitor AG1296 and EGFR tyrosine kinase inhibitor AG1478 on signal transduction and cell proliferation. A, VSMCs were pretreated with vehicle (DMSO), 10 µmol/L AG1296, or 300 nmol/L AG1478 for 30 minutes and treated with 1 µmol/L S1P for 3 minutes. Samples immunoprecipitated with PDGFR or EGFR or nonimmunoprecipitated lysate were analyzed by Western blotting. The extent of phosphorylation was quantified as in Figure 1. B, VSMCs were seeded in 24-well plates in DMEM supplemented with 10% FBS. The next day, medium was changed to DMEM without FBS containing 1 µmol/L S1P, 10 ng/mL EGF, or 20 ng/mL PDGF-BB for up to 4 days. Medium was changed and cells were counted every 2 days. Data are mean±SEM. *P<0.05. C, Using the protocol in Figure 2C, VSMC proliferation was measured with or without AG1296 or AG1478.


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  		Table 1. Summary of Inhibitors on S1P-Stimulated Protein Phosphorylation



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  		Figure 4. Effect of cholesterol depletion on S1P-stimulated transactivation of PDGFßR and EGFR. A, VSMCs were pretreated with or without 10 mmol/L ß-cyclodextrin or 20 µmol/L filipin for 1 hour and stimulated with 1 µmol/L S1P for 3 minutes, 20 ng/mL PDGF-BB for 5 minutes, or 10 ng/mL EGF for 2 minutes. Samples were immunoprecipitated and analyzed by Western blotting. For p-PDGFßR and p-EGFR, relative phosphorylation levels are expressed as percent of total.

AG Compounds Suppress VSMC Proliferation in Response to S1P
To show the physiological significance of S1P-mediated RTK transactivation, we studied VSMC growth. In response to 1 µmol/L S1P, VSMC proliferation increased significantly compared with control at day 4 (Figure 2B). This was {approx}50% of the response observed with PDGF-BB or EGF treatment. AG1478 and AG1296 alone inhibited S1P-mediated proliferation by 30% and 31%, respectively, and together inhibited proliferation by 49% (Figure 2C), indicating an important role for RTK transactivation in S1P-stimulated growth. PDGF-BB–stimulated cell proliferation was blocked by AG1296 but not by AG1478, and EGF-stimulated growth was blocked only by AG1478.

Gi-Dependent Transactivation of PDGFßR and EGFR
Because EDG receptor signaling requires G proteins, we examined the effect of pertussis toxin (PTx), a Gi inhibitor. PTx significantly inhibited both PDGFßR and EGFR transactivation in response to S1P (Table and online Figure S1). Phosphorylation of Akt and ERK1/2 was also blocked significantly. In contrast, PDGF-BB–stimulated phosphorylation of PDGFßR and EGF-stimulated phosphorylation of EGFR were not inhibited by PTx (online Figure S1). Because Gi is common to all three S1P receptors (EDG-1, -3, and -5) reported to be expressed in VSMCs,6,7,9,11 it is possible that any one of these receptors can mediate transactivation. However, according to Kluk and Hla, EDG-1 is most important for VSMC proliferation and migration.7

Transactivation of PDGFßR and EGFR in CHO Cells Overexpressing EDG-1, PDGFßR, and EGFR
To strengthen the evidence that the S1P receptor (EDG-1) mediates S1P transactivation, we transfected EDG-1 alone or with the PDGFßR and EGFR in CHO cells. Expression of EDG receptors in CHO cells has been reported previously32 to be very low, and expression of EDG-1, -3, or -5 could not be detected by Western blotting. Endogenous expression of Akt and ERK1/2 was observed (Figure 3C), but PDGFßR and EGFR were not detected by immunoblotting (data not shown). In CHO cells cotransfected with EDG-1 and PDGFßR, PDGFßR was transactivated in response to S1P (Figure 3A, lane 2) to a level {approx}50% of that observed with PDGF itself (Figure 3A, lane 3). The same results were observed in CHO cells cotransfected with EDG-1 and EGFR (Figure 3B, lanes 2 and 3). When all three constructs were cotransfected (Figures 3A and 3B, right side), both PDGFßR and EGFR transactivation in response to S1P was observed, but there was no synergism. Of interest, PDGF transactivation of the EGFR (Figure 3B, lane 6) was significantly greater than EGF transactivation of PDGFßR (Figure 3A, lane 7).



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  		Figure 3. S1P stimulates PDGFßR and EGFR transactivation in CHO cells. CHO cells were transfected with EDG-1, EGFR, or PDGFßR alone or together by LipofectAMINE 2000. Cells were serum-starved for 1 day and stimulated with 1 µmol/L S1P, 10 ng/mL EGF, or 20 ng/mL PDGF-BB. Cell lysates were prepared, and immunoprecipitation and immunoblotting were performed. A, PDGFßR was immunoprecipitated. B, EGFR was immunoprecipitated. C, Total cell lysates were probed for AKT, ERK1/2 EDG-1, and p-Akt and p-ERK1/2, as indicated. Graphs show the extent of phosphorylation quantified by densitometry as in Figure 1.

We next determined the effect of EDG-1 and RTK transactivation of activation of Akt and ERK1/2 (Figure 3C). In the absence of transfection, no activation of Akt or ERK1/2 in response to S1P was observed (Figure 3C, lane 2). Normal cell function was confirmed by Akt and ERK1/2 phosphorylation in CHO cells that overexpressed EDG-1 when stimulated with S1P (Figure 3C, lane 4). This low level of ERK1/2 and Akt activation by S1P in EDG-1–transfected cells (lane 4) likely represents an RTK-independent signaling pathway for S1P-EDG-1 signaling. In CHO cells cotransfected with EDG-1 and PDGFßR, Akt and ERK1/2 phosphorylation in response to S1P was significantly enhanced compared with CHO cells transfected only with EDG-1 (lane 6 versus lane 4). The same results were observed in CHO cells cotransfected with EDG-1 and EGFR (lane 9 versus lane 4). When all three receptors were cotransfected, there was no additional enhancement of Akt and ERK1/2 activation in response to any agonist (Figure 3C, lanes 11 through 14), consistent with the RTK activation (Figures 3A and 3B). These data suggest that there are direct signaling pathways from EDG-1 to Akt and ERK1/2 as well as indirect pathways via PDGFßR and EGFR transactivation.

S1P Transactivation of PDGFßR and EGFR Requires Cholesterol-Rich Membranes
It has been previously suggested that CRMs, including lipid rafts and caveolae, are important sites for GPCR-mediated transactivation of RTKs and signal transduction.33–35 To evaluate the role of CRM in S1P transactivation, we disrupted CRM by depleting cholesterol with ß-cyclodextrin or filipin (Figure 4 and the Table). These agents strongly inhibited S1P-mediated transactivation of both PDGFßR (Figure 4A, lanes 4 through 6, IP: PDGFßR) and EGFR (Figure 4B, lanes 4 through 6, IP: EGFR). In contrast, PDGF-BB–stimulated and EGF-stimulated phosphorylation of their receptors was not blocked by cholesterol depletion (Figures 4A and 4B, lanes 7 through 9). These findings suggest an important role for CRM in S1P-mediated RTK transactivation.

To evaluate the role of CRM additionally, we examined changes in subcellular location of PDGFßR, EGFR, caveolin-1, PI3K, and Shc induced by S1P using sucrose gradient fractionation (Figures 5A and 5B). Caveolin-1, PDGFßR, and EGFR were primarily localized to CRM (fractions 4 through 6) and did not change when cells were stimulated with S1P, PDGF, or EGF (Figures 5A and 5B). In contrast, PI3K and Shc were primarily located in non-CRM heavy fractions (fractions 9 through 12) basally and translocated to CRM in response to S1P in a time-dependent manner (Figures 5A and 5B, fractions 4 through 6). Both P13K and Shc showed clear translocation at 5 minutes of S1P stimulation (P13K, 7% to 14%; Shc, 3% to 12%; Figure 5B), with a peak at 20 minutes for Shc. PI3K translocation was blocked by both AG1478 and AG1296, but Shc translocation was inhibited only by AG1478. These data are consistent with findings that Shc associated only with the EGFR (Figure 1) and the inhibitor studies in Figure 2.



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  		Figure 5. Translocation of PI3K and Shc to CRM fractions in response to S1P stimulation. A, VSMCs were pretreated with vehicle (DMSO), 10 µmol/L AG1296, or 300 nmol/L AG1478 for 30 minutes and exposed to 1 µmol/L S1P for 2, 5, or 10 minutes, 20 ng/mL PDGF-BB for 5 minutes, or 10 ng/mL EGF for 2 minutes. Sucrose gradient fractionation was performed and fractions separated by SDS-PAGE and analyzed by Western blotting. B, Distribution in 12 fractions of each protein were quantified and expressed as percent of total. Results are mean±SEM of 3 determinations. To normalize the results for different experiments and blots, we made multiple exposures and used exposures that provided a similar absolute density for caveolin-1 in the CRM fraction. This exposure was then used to determine the total pixel intensity for each protein on its Western blot, and the value was set to 100%.

Characterization of S1P Transactivation Pathway
To elucidate the mechanism by which S1P transactivates the EGFR and PDGFR, we used multiple inhibitors (Table and online Figures S2 through S6). Because S1P has been shown to increase calcium in VSMCs,9 we chelated calcium with BAPTA/AM (online Figure S2). This completely inhibited transactivation of PDGFßR and EGFR in response to S1P and also inhibited downstream Akt and ERK1/2 phosphorylation (online Figure S2 and the Table). Wortmannin and PD98059, specific inhibitors of PI3K and MEK-1, respectively, did not inhibit S1P-mediated RTK phosphorylation but inhibited Akt and ERK1/2, respectively, as expected (online Figure S2 and the Table). These results suggest no feedback by PI3K and MEK-1 on RTK transactivation.

Because transactivation of RTK in VSMC by Ang II is reactive oxygen species (ROS) dependent,36 we measured ROS in response to S1P. S1P stimulated superoxide generation to an extent comparable to Ang II (Figure 6). Next we examined the effect of antioxidants on the transactivation of RTK by S1P. N-acetyl cysteine, Tiron, and ebselen inhibited S1P transactivation of PDGFßR and EGFR (Table and online Figure S3). Thus, in VSMCs, S1P-stimulated transactivation of RTK is ROS dependent.



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  		Figure 6. S1P stimulates ROS production in VSMCs. Superoxide generation by S1P (100 nmol/L for 2 minutes) or Ang II (100 nmol/L for 1 minute) was determined by fluorescence microscopy. Methanol (MeOH) and PBS were used as vehicle controls for S1P and Ang II, respectively.

Src-dependent transactivation of EGFR in response to GPCR ligands has been well studied.13,15 To evaluate the role of Src, we treated VSMCs with the Src inhibitor PP2 and adenoviral dominant-negative c-Src. Both treatments blocked PDGFßR and EGFR transactivation in response to S1P (Table and online Figures S4 and S5). Interestingly, PP2 partially inhibited PDGF-BB–induced and EGF-induced phosphorylation of PDGFßR and EGFR, respectively. As reported previously, some phosphorylation sites in RTK are Src dependent.37,38

EGFR transactivation by several GPCR ligands depends on matrix metalloprotease (MMP) activation, release of HB-EGF, and binding of HB-EGF to EGFR.12,13,31 Therefore, we examined whether S1P-mediated EGFR and PDGFßR transactivation was MMP dependent. EGFR transactivation by S1P was blocked by the MMP inhibitors GM 6001 and o-phenanthrolene (Table and online Figure S6A). In contrast, S1P-stimulated transactivation of PDGFßR was not attenuated by MMP inhibitors or by PDGF-BB–neutralizing antibody (Table and online Figure S6B). These data suggest that PDGFßR transactivation by S1P does not require MMP activation and that the pathway differs from EGFR transactivation.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The major finding of this study is that S1P transactivates PDGFßR and EGFR in VSMCs via an EDG receptor, probably EDG-1. Both EGFR and PDGFßR transactivation required Gi activation, receptor tyrosine kinase activity, Src, ROS, and intact cholesterol-rich membrane domains, as shown in Figure 7. Based on the time course in Figure 1, a sequential pathway of Src-RTK-Akt/ERK is plausible, although given the limitations imposed by subcellular location and signal amplification, the time course is merely suggestive. Novel findings for S1P transactivation were the requirement for ROS generation and translocation of p85PI3K and Shc to cholesterol-rich membranes (caveolae in Figure 7). Transactivation is critical for S1P signaling, because >70% inhibition of Akt and ERK1/2 activation occurred in the presence of AG1478 and AG1296 (Figure 2 and the Table). S1P transactivation requires EDG receptors (as opposed to intracellular metabolism), because sphingosine did not stimulate EGFR and PDGFßR phosphorylation (data not shown). Importantly, because both EGFR and PDGFßR contributed to VSMC growth in response to S1P, we propose that these pathways are important for S1P-mediated VSMC effects, such as proliferation and migration. Because many of the conclusions are based on the use of inhibitors, additional investigation using different approaches will be needed to confirm our findings.



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  		Figure 7. Model for S1P-mediated transactivation of PDGFßR and EGFR.

Transactivation of RTK in response to activation of many GPCRs has been reported.12–16 EGFR transactivation by ligands such as thrombin, Ang II, lysophosphatidic acid, and endothelin-1 has been well studied.18,19,31 Because S1P is a relatively newly identified GPCR ligand, S1P-stimulated transactivation of RTK has not been well studied. One report suggested involvement of EGFR transactivation in response to S1P because ERK1/2 activation was inhibited by AG1478.39 Boguslawski et al10 reported tyrosine phosphorylation of a 175- to 185-kDa protein in response to S1P stimulation, consistent with the PDGFR. In this study we report that transactivation of EGFR by S1P likely depends on MMP, similar to other GPCR ligands. However, we have not observed the activation of specific MMP or production of HB-EGF. Additional investigations are needed to confirm the involvement of MMP in EGFR transactivation. In contrast, PDGFßR transactivation was not MMP dependent, similar to results we have previously reported for S1P-mediated transactivation of VEGFR2/Flk-1/KDR in endothelial cells.

The present study is consistent with previous data for transactivation of PDGFßR and EGFR by Ang II in VSMCs that showed key roles for ROS40 and caveolae.36 We found that S1P increased superoxide generation to a similar extent as Ang II, and transactivation of both PDGFßR and EGFR was inhibited by antioxidants. S1P transactivation of VEGFR2/Flk-1/KDR in endothelial cells was not ROS dependent,22 which is logical, because S1P does not generate ROS in endothelial cells.41 Thus, ROS generation in response to S1P is a specific feature of VSMCs. A novel finding in our study was translocation of p85PI3K and Shc to CRM and association of these molecules with PDGFßR and EGFR. These results strongly support the involvement of caveolae or lipid rafts as the subcellular location where transactivation of PDGFßR and EGFR occurs.

The present study suggests that there is a hierarchy in the transactivation of EGFR and PDGFßR when they are expressed together, as in VSMCs. Specifically, we found for Akt activation that both EGFR and PDGFßR were involved but for ERK1/2 activation only EGFR transactivation was involved (Figure 2A). We speculate that this is a consequence of Shc being recruited only to EGFR or crosstalk between EGFR and the PDGFßR. This may occur if the EGFR is more easily transactivated and inhibits signaling of the PDGFßR. This concept is also supported by the finding that when the PDGFßR was expressed alone in CHO cells, activation of both Akt and ERK was observed. Also, in CHO cells transfected only with EGFR but without EDG-1, EGFR was phosphorylated by S1P. CHO cells do not express EDG-1, -3, or -5, but there is a possibility that another S1P receptor might be expressed in this cell, and overexpressed EGFR was transactivated slightly by S1P stimulation. In fact, highly overexpressed EGFR usually autophosphorylated without stimulation. Under our conditions, expression levels might not be sufficient for autophosphorylation without stimulation, but because expression is higher than the level in VSMCs, EGFR may be activated by a weak stimulation. We have not examined the actual sites phosphorylated in EGFR and PDGFßR in response to S1P, but we speculate that the PI3K binding sites are phosphorylated in both PDGFßR and EGFR whereas the Shc binding sites are tyrosine phosphorylated only in the EGFR.

An interesting model of reverse transactivation was demonstrated for the PDGFR and EDG-1.24 One study showed that PDGF binding to the PDGFR activated sphingosine kinase, which then generated S1P. S1P was secreted and activated EDG-1 on the cell surface. This report and our present data suggest a sequential loop of transactivation between EDG receptors and PDGFR, which may potentiate signal transduction and play an important role in S1P and PDGF signaling. It should be noted that other recent studies question the existence of reverse transactivation.42 Our data that PTx did not block PDGF-BB–stimulated or EGF-stimulated ERK and Akt phosphorylation also argue against reverse transactivation (online Figure S1). Additional investigations are needed to clarify the possibility of a sequential loop of transactivation.

There is increasing evidence for an important role for S1P and its receptors in vessel growth and development. Both endothelial cells and VSMCs express multiple S1P receptors, and S1P stimulates proliferation and migration of these cells. We have shown that S1P induced RTK transactivation via EDG-1 in CHO cells cotransfected with EDG-1 and RTK. Two other EDG receptors (EDG-3 and EDG-5) expressed in VSMCs may also participate in RTK transactivation. The expression levels of these receptors are high in VSMCs from several rat strains6,7,9 as well as in human VSMCs and airway SMCs.10,11,43 Based on present data, we believe that EDG-1 mainly contributes to RTK transactivation in VSMCs, but additional study is needed to confirm the role of EDG-1. S1P is released by activated platelets and is also synthesized in endothelial cells and VSMCs. We have now demonstrated that S1P transactivates three RTKs (VEGFR2/Flk-1/KDR, PDGFßR, and EGFR) that are highly expressed in endothelial cells22 and VSMCs. These observations suggest that RTK transactivation in response to S1P plays an important role in vascular physiology, especially at sites of arterial injury and thrombosis.


*    Acknowledgments
 
This work was supported by grants HL49192 and HL64839 to B.C.B. We thank Drs Hojo, Saito, Yin, Maekawa, and Cavet for useful discussions.


*    Footnotes
 
Original received May 28, 2003; resubmission received December 30, 2003; revised resubmission received March 11, 2004; accepted March 11, 2004.


*    References
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 *References
 

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