15-Lipoxygenase-1-enhanced Src-Janus Kinase 2-Signal Transducer and Activator of Transcription 3 Stimulation and Monocyte Chemoattractant Protein-1 Expression Require Redox-sensitive Activation of Epidermal Growth Factor Receptor in Vascular Wall Remodeling*

To understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates signal transducer and activator of transcription 3 (STAT3), we studied the role of epidermal growth factor receptor (EGFR). 15(S)-HETE stimulated tyrosine phosphorylation of EGFR in a time-dependent manner in vascular smooth muscle cells (VSMCs). Interference with EGFR activation blocked 15(S)-HETE-induced Src and STAT3 tyrosine phosphorylation, monocyte chemoattractant protein-1 (MCP-1) expression and VSMC migration. 15(S)-HETE also induced tyrosine phosphorylation of Janus kinase 2 (Jak2) in VSMCs, and its inhibition substantially reduced STAT3 phosphorylation, MCP-1 expression, and VSMC migration. In addition, Src formed a complex with EGFR and Jak2, and its inhibition completely blocked Jak2 and STAT3 phosphorylation, MCP-1 expression, and VSMC migration. 15(S)-HETE induced the production of H2O2 via an NADPH oxidase-dependent manner and its scavengers, N-acetyl cysteine (NAC) and catalase suppressed 15(S)-HETE-stimulated EGFR, Src, Jak2, and STAT3 phosphorylation and MCP-1 expression. Balloon injury (BI) induced EGFR, Src, Jak2, and STAT3 phosphorylation, and inhibition of these signaling molecules attenuated BI-induced MCP-1 expression and smooth muscle cell migration from the medial to the luminal surface resulting in reduced neointima formation. In addition, inhibition of EGFR blocked BI-induced Src, Jak2, and STAT3 phosphorylation. Similarly, interference with Src activation suppressed BI-induced Jak2 and STAT3 phosphorylation. Furthermore, adenovirus-mediated expression of dnJak2 also blocked BI-induced STAT3 phosphorylation. Consistent with the effects of 15(S)-HETE on the activation of EGFR-Src-Jak2-STAT3 signaling in VSMCs in vitro, adenovirus-mediated expression of 15-lipoxygenase 1 (15-Lox1) enhanced BI-induced EGFR, Src, Jak2, and STAT3 phosphorylation leading to enhanced MCP-1 expression in vivo. Blockade of Src or Jak2 suppressed BI-induced 15-Lox1-enhanced STAT3 phosphorylation, MCP-1 expression, and neointima formation. In addition, whereas dominant negative Src blocked BI-induced 15-Lox1-enhanced Jak2 phosphorylation, dnJak2 had no effect on Src phosphorylation. Together, these observations demonstrate for the first time that the 15-Lox1–15(S)-HETE axis activates EGFR via redox-sensitive manner, which in turn mediates Src-Jak2-STAT3-dependent MCP-1 expression leading to vascular wall remodeling.

Vascular smooth muscle cell (VSMC) 2 migration and proliferation contribute to the pathogenesis of atherosclerosis and restenosis (1)(2)(3). It has been shown that 15-lipoxygenase 1 (15-Lox1) and its murine ortholog 12/15-Lox mediate the oxidation of low density lipoprotein, an important factor in the disease process of atherosclerosis (4,5). In addition, using transgenic or knock-out mice models, a number of studies have demonstrated that these lipoxygenases indeed play a role in the pathogenesis of both atherosclerosis and restenosis (6 -8). Lipoxygenases are non-heme iron dioxygenases that stereospecifically introduce molecular oxygen into polyunsaturated fatty acids, resulting in the formation of hydroperoxyeicosatetraenoic acids, which are subsequently converted to hydroxyeicosatetraenoic acids (HETEs) (9). Furthermore, atherosclerotic arteries upon incubation with arachidonic acid (AA) converted it mainly to 15-HETE (10). Despite the importance of eicosanoids, the oxygenated metabolites of AA, in the maintenance of vascular tone (11,12) and their increased production in atherosclerotic arteries (10), relatively little is known in regard to their role in vascular wall remodeling. In this regard, studies from our laboratory as well as others have shown that VSMCs express 15-Lox1 and when exposed to AA, convert it primarily to 15(S)-HETE (13)(14)(15). Furthermore, we have reported that 15(S)-HETE is a potent chemotactic molecule to VSMCs and induces the expression of cytokines such as interleukin-6 (IL- 6) and monocyte chemoattractant protein-1 (MCP-1) (16,17). Our studies have also shown that mitogen-activated protein kinase-mediated cAMP response element binding (CREB) protein and Src-dependent signal transducer and activator of transcription 3 (STAT3) stimulation are required for 15(S)-HETEinduced IL-6 and MCP-1 expression, respectively, in VSMCs (16,17).
Many studies have shown that the Jak family of tyrosine kinases mediates the tyrosine phosphorylation and activation of STATs in the regulation of various cellular responses including cell proliferation and migration in response to a broad range of agonists (18 -22). On the other hand, a few studies have reported that the Src family of non-receptor-tyrosine kinases also plays a role in the activation of STATs in response to some agents such as oxidized lipid molecules (23,24). However, very little is known in regard to the existence of any cross-talk between these two non-receptor-tyrosine kinases in the activation of STAT3. Because our previous studies have indicated that 15(S)-HETE activates STAT3 and it requires the involvement of Src, we asked the question of whether Jak2 has any role in these effects, and if so, is there any cross-talk between these two non-receptor-tyrosine kinases in the activation of STAT3? In this report we show that both the tyrosine kinases interact with each other downstream to EGFR in mediating 15-Lox1-15(S)-HETE-induced STAT3 activation and MCP-1 expression in VSMCs in vitro and in the artery in vivo. Our results also demonstrate that EGFR gets activated acutely in the artery after injury alone and with adenovirus-mediated expression of 15-Lox1 and mediates the Src-Jak2-STAT3-MCP-1 signaling axis leading to vascular wall remodeling.   periods, cell extracts were prepared, equal amounts of protein from control and each treatment were immunoprecipitated (IP) with anti-EGFR antibodies, and the immunocomplexes were analyzed by Western blotting (IB) using anti-PY20 antibodies. B and C, quiescent VSMCs were treated with and without 15(S)-HETE (0.5 M) in the presence and absence of AG1478 (500 nM) for 30 min, and cell extracts were prepared and analyzed by Western blotting for tyrosine phosphorylation of Src (B)and STAT3 (C) using their respective phosphotyrosine-specific antibodies. The blots in panels A-C were reprobed with anti-EGFR, anti-Src, or anti-STAT3 antibodies for normalization. D and E, quiescent VSMCs were treated with and without 15(S)-HETE (0.5 M) in the presence and absence of AG1478 (500 nM) for 2 h, and either total cellular RNA was isolated and analyzed for MCP-1 and ␤-actin mRNA levels by RT-PCR (D), or media were collected and analyzed for MCP-1 release by ELISA (E). F, quiescent VSMCs were treated with and without AG1478 (500 nM) for 30 min and subjected to 15(S)-HETE (0.5 M)-induced migration using a modified Boyden chamber method. G, VSMCs that were transfected with scrambled or EGFR siRNA (100 nM) and growth-arrested were treated with and without 15(S)-HETE (0.5 M) for 30 min, and cell extracts were prepared and analyzed by Western blotting for pSrc and pSTAT3 levels using their respective phosphotyrosine-specific antibodies. The blots were reprobed with anti-Src, anti-STAT3, anti-EGFR, or anti-␤-tubulin antibodies for normalization or to show the effect of EGFR siRNA on its target and non-target molecules. (A7250), and pluronic F127 (P2443) were purchased from Sigma. Diphenyleneiodonium chloride (BML-CN240) was bought from ENZO Life Sciences (Plymouth Meeting, PA). Anti-phospho-STAT3 (9131), anti-phospho-Src (2101), and anti-Src (2109) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-histone H1 (MAB1276), antiphospho-Jak2 (09 -275), anti-Jak2 (06 -255), and anti-phosphotyrosine (05-777) antibodies were obtained from Millipore (Temecula, CA). Anti-STAT3 (SC-482) and anti-EGFR (SC-03) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). AG 1478 (658552), AG 490 (658401), PP1 (529579), and Eosin Y (200-12) were bought from EMD Biosciences (Gibbstown, NJ). The rat MCP-1 ELISA kit (KRC1011) and 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H 2 DCFDA) (C6827) were obtained from Invitrogen. Rat EGFR siRNA (ON-TARGET plus SMARTpool L-080049-01) and scrambled siRNA (ON-TARGET plus Non-targeting Pool) were obtained from Dharmacon RNAi Technologies (Chicago, IL). Hematoxylin (H-3404), biotinylated anti-mouse IgG (BA-9200), ABC kit (PK-6100), and DAB kit (SK-4100) were bought from Vector Laboratories (Burlingame, CA). All primers and oligonucleotides used in this study were synthesized by IDT (Coralville, IA).

Reagents
Cell Culture-Rat VSMCs were isolated and subcultured as described previously (25). VSMCs were used between 6 and 12 passages.
Cell Migration-Cell migration was measured using a modified Boyden chamber method as described previously (29). Wherever adenovirus was used, cells were infected with the respective adenovirus at an m.o.i. of 40 and growth-arrested before they were subjected to stimulus-induced migration. Cell motility is presented as number of migrated cells per field.
RT-PCR-After appropriate treatments, total cellular RNA was isolated from VSMCs using TRIzol reagent as per the man- ufacturer's instructions (Invitrogen). Reverse transcription was carried out with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The cDNA was then used as a template for PCR using specific primers. The primers used are as follows: rat MCP-1 (accession number AF058786) forward (5Ј-CAGAAACCAGCCAACTCTCA-3Ј) and reverse (5Ј-GCTTGAGGTGGTTGTGGAAA-3Ј); rat ␤-actin (accession number EF156276) forward (5Ј-CGTTGACATCCGTA-AAGACC-3Ј) and reverse (5Ј-GATAGAGCCACCAATC-CACA-3Ј). The amplification was carried out on Gene Amp PCR System 2400 (Applied Biosystems). The amplified RT-PCR products were separated on 1.5% (w/v) agarose gels and stained with ethidium bromide, pictures were captured using AlphaEase Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA), and the band intensities were quantified using NIH ImageJ.
ELISA-MCP-1 release into medium was measured by using rat MCP-1 ELISA kit following the manufacturer's instructions, and the data are expressed as pg/ml.
Western Blot Analysis-After appropriate treatments and rinsing with cold PBS, VSMCs were lysed in 500 l of lysis buffer (1ϫ PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM sodium orthovanadate), and the lysates were collected into 1.5-ml microcentrifuge tubes. After 20 min of incubation on ice, the cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. The cell lysates containing an equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were electroblotted to a nitrocellulose membrane (Hybond, GE Healthcare). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (GE Healthcare). The band intensities were quantified using NIH ImageJ.
Carotid Artery Balloon Injury (BI)-All animal protocols were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center, Memphis, TN. BI was performed essentially as described previously (31). At 3 days or 2 weeks after BI, the animals were sacrificed with an overdose of pentobarbital (200 mg/kg), and the carotid arteries were collected and processed for protein extraction, RNA isolation and/or morphometric analysis. For morphometric analysis, carotid arteries were fixed in 10% formalin and embedded in OCT solution (Sakura Finetek USA Inc., Torrance, CA), and 5-m-thick sections were made at equally spaced intervals in the middle of injured and uninjured common carotid artery segments and stained with hematoxylin and eosin. The intimal (I) and medial (M) areas were measured using NIH ImageJ, and the I/M ratios were calculated.
In Vivo SMC Migration Assay-The in vivo SMC migration was determined as described by Bendeck et al. (32). Briefly, 3 days after BI, the carotid arteries were fixed in vivo with 10% buffered formalin at physiological pressure. The middle 1 cm of the denuded (injured) common carotid artery was cut and fixed in cold acetone for 10 min. The artery was then opened longitudinally and pinned down onto an agar plate with the luminal surface facing upward. The arteries were rinsed in PBS and then  placed in 0.3% H 2 O 2 for 30 min to block endogenous peroxidase activity. Nonspecific protein binding was blocked by incubating the arteries in 5% normal goat serum in PBS for 30 min. The arteries were incubated with anti-histone H1 antibodies diluted 1:300 in PBS for 1 h followed by incubation with biotinylated goat anti-mouse IgG for 30 min. Peroxidase labeling was carried out by using ABC kit (Vector Laboratories), and the signals were visualized by using DAB kit (Vector Laboratories). After each step, the slides were rinsed 3 times for 5 min each in PBS. Finally, the opened arteries were placed intimal side up on glass slides with coverslips. As a negative control, samples of the same specimens without the primary antibody incubation were used. The intimal surface of the vessel was examined under a light microscope at X200 magnification, and the total number of positively stained cells per 0.1 mm 2 of the luminal surface area was counted.
Statistics-All the experiments were repeated three times with similar results. Data are presented as the means Ϯ S.D. The treatment effects were analyzed by Student's t test. p values Ͻ0.05 were considered to be statistically significant. In the case of Western blotting, histochemistry, and RT-PCR, one of the representative sets of data is shown.

15(S)-Hydroxyeicosatetraenoic Acid Stimulates Tyrosine
Phosphorylation of Jak2-A large body of data indicates that the Jak family of tyrosine kinases plays a major role in agonistinduced STAT3 phosphorylation (18 -22). Therefore, to find the involvement of the Jak family of tyrosine kinases in 15(S)-HETE-induced STAT3 activation, we studied the role of Jak2. 15(S)-HETE induced tyrosine phosphorylation of Jak2 (Tyr1007/1008) in a time-dependent manner ( Fig. 2A). Next we used both pharmacological and dominant negative mutant approaches to test the role of Jak2 in STAT3 phosphorylation. AG490, a potent inhibitor of Jak2 (34), suppressed 15(S)-HETEinduced STAT3 phosphorylation (Fig. 2B). Similarly, adenovirus-mediated expression of dominant negative Jak2 inhibited 15(S)-HETE-induced STAT3 phosphorylation (Fig. 2C). Blockade of Jak2 activation by either approach also inhibited 15(S)-HETE-induced MCP-1 expression and its release by VSMCs, which in turn affected the migration of these cells (Fig. 2, D-F). 15(S)-Hydroxyeicosatetraenoic Acid-induced STAT3 Phosphorylation Requires Activation of Both Src and Jak2-Because inhibition of either Src or Jak2 attenuated STAT3 phosphorylation, we were intrigued to find whether there is any cross-talk between these two non-receptor-tyrosine kinases. To test this possibility, we studied the role of Src in 15(S)-HETE-induced Jak2 phosphorylation. PP1, a potent inhibitor of Src (24), abolished Jak2 phosphorylation by 15(S)-HETE (Fig. 3A). Consistent with this observation, adenovirus-mediated expression of dnSrc also suppressed 15(S)-HETE-induced Jak2 phosphorylation (Fig. 3B). To find a connection between Jak2 and EGFR, we tested the effect of AG1478 on 15(S)-HETE-induced Jak2 phosphorylation. AG1478 completely inhibited 15(S)-HETEinduced Jak2 phosphorylation (Fig. 3C). Furthermore, downregulation of EGFR levels by its siRNA also attenuated 15(S)-HETE-induced Jak2 phosphorylation (Fig. 3D). To test whether EGFR, Src, and Jak2 form a complex in response to 15(S)-HETE, we performed co-immunoprecipitation assays. Immunoblotting of anti-Src immunoprecipitates of control and 15(S)-HETE-treated VSMCs for EGFR showed a band with a molecular mass of 170 kDa whose intensity was found to be higher in 15(S)-HETE-treated cells compared with control (Fig.  3E). Sequential probing of this membrane with anti-Jak2 antibodies revealed the association of both EGFR and Jak2 with Src. The association of Src and Jak2 with EGFR was further confirmed by immunoblotting of anti-EGFR immunoprecipitates of control and 15(S)-HETE-treated VSMCs with anti-Src and anti-Jak2 antibodies (Fig. 3E).

15(S)-Hydroxyeicosatetraenoic Acid-induced EGFR-Src-Jak2-STAT3 Phosphorylation and MCP-1 Expression Require
Reactive Oxygen Species (ROS) Production-To understand the mechanism(s) of activation of EGFR-Src-Jak2-STAT3 signaling by 15(S)-HETE, we tested its effect on ROS production. 15(S)-HETE induced the production of ROS in a time-dependent manner (Fig. 4A). To find the cellular source of 15(S)-HETEinduced ROS production, we tested the role of NADPH oxi-

DISCUSSION
The important findings of the present study are as follows. 1) 15(S)-HETE induced tyrosine phosphorylation of EGFR in a time-dependent manner. 2) Blockade of EGFR activation inhibited 15(S)-HETE-induced Src and STAT3 tyrosine phosphorylation, MCP-1 expression, and VSMC migration. 3) 15(S)-HETE also induced tyrosine phosphorylation of Jak2 and interference with its activation resulted in down-regulation of STAT3 activation, MCP-1 expression, and VSMC migration. 4) Inhibition of Src suppressed 15(S)-HETE-induced Jak2 tyrosine phosphorylation. 5) 15(S)-HETE induced the production of H 2 O 2 in a time-and NADPH oxidase-dependent manner in VSMCs. 6) H 2 O 2 scavengers, NAC and catalase, blocked 15(S)-HETE-induced EGFR, Src, Jak2, and STAT3 tyrosine phosphorylation and MCP-1 expression. 7) BI induced EGFR, Src, Jak2, and STAT3 tyrosine phosphorylation and MCP-1 expression. 8) Administration of EGFR inhibitor AG1478 attenuated BI-induced Src, Jak2, and STAT3 tyrosine phosphorylation and FIGURE 5. Inhibition of EGFR suppresses BI-induced Src, Jak2, and STAT3 phosphorylation and MCP-1 expression leading to decreased migration of SMC from medial to luminal region and neointima formation. A, 24 h after BI and administration of vehicle or AG1478 (5 M), the rats were sacrificed, and the injured and uninjured common carotid arteries were dissected out and tissue extracts were prepared. AG1478 was delivered via mixing with 30% (w/v) pluronic gel and applied around the injured artery. Upper panel, tissue extracts containing equal amounts of proteins were immunoprecipitated (IP) with anti-EGFR antibodies, and the immunocomplexes were analyzed by Western blotting (IB) with anti-PY20 antibodies. Lower panel, an equal amount of protein from each condition was analyzed for tyrosine phosphorylation of Src, Jak2, and STAT3 by Western blotting using their phosphotyrosine-specific antibodies. The blots in both panels were reprobed with anti-EGFR or anti-STAT3 antibodies for normalization. B, all the conditions were the same as in panel A, except that RNA was isolated and analyzed for MCP-1 and ␤-actin expression by RT-PCR. C, 3 days after BI and administration with vehicle or AG1478 (5 M), injured common carotid arteries were isolated, opened longitudinally, and stained with anti-histone H1 antibodies, and histone H1-positive nuclei were counted. D, 2 weeks after BI and administration with vehicle or AG1478 (5 M), rats were sacrificed, arteries were isolated, MCP-1 expression, resulting in reduced SMC migration from media to intima region and neointima formation. 9) Inhibition of Src via adenovirus-mediated transduction of its dominant negative mutant attenuated BI-induced Jak2 and STAT3 tyrosine phosphorylation, SMC migration from media to intima region, and neointima formation. 10) Interference with activation of Jak2 also reduced STAT3 tyrosine phosphorylation, SMC migration from media to intima region, and neointima formation. 11) Adenovirus-mediated expression of 15-Lox1 enhanced BI-induced EGFR, Src, Jak2, and STAT3 phosphorylation, MCP-1 expression, and neointima formation. 12) Ade-novirus-mediated transduction of either dn-Src or dnJak2 suppressed BI-induced 15-Lox1-enhanced STAT3 tyrosine phosphorylation, MCP-1 expression, and neointima formation. The role of EGFR in mediating intracellular signaling events such as the activation of extracellular signal-regulated kinases in response to a variety of agents, including G protein-coupled receptor (GPCR) agonists and oxidants, has been reported (3, 33, 38 -40). The capacity of 15(S)-HETE in the activation of EGFR and suppression of Src-Jak2-STAT3-MCP-1 signaling by EGFR inhibitor AG1478 or its siRNA suggest that EGFR mediates 15(S)-HETE-induced tyrosine phosphorylation-dependent signaling events leading to MCP-1 expression and VSMC Ad-dnJak2 (10 10 pfu), the uninjured and injured common carotid arteries were isolated, and either tissue extracts were prepared and analyzed for STAT3 phosphorylation using its phosphotyrosine-specific antibodies (A) or RNA was isolated and analyzed for MCP-1 and ␤-actin expression by RT-PCR (B). The blot in panel A was sequentially reprobed with anti-STAT3 and anti-Jak2 antibodies for normalization or to show overexpression of dnJak2. C, all the conditions were the same as in panel A except that after isolation, arteries were opened longitudinally and stained with anti-histone H1 antibodies, and histone H1-positive nuclei were counted. D, 2 weeks after BI and transduction with Ad-GFP (10 10 pfu) or Ad-dnJak2 (10 10 pfu), rats were sacrificed, arteries were isolated, fixed, cross-sections made and stained with H & E. After morphometry analysis, the I/M ratios were calculated. The upper panel shows the representative pictures of balloon-injured Ad-GFP-or Ad-dnJak2-transduced carotid artery cross-sections that were stained with H & E. The bar graph in the bottom panel shows the quantitative analysis of the I/M ratios of the carotid arteries. *, p Ͻ 0.05 versus Ad-GFP-BI (n ϭ 6). migration. In regard to understanding the mechanisms of activation of EGFR-Src-Jak2-STAT3-MCP-1 signaling by 15(S)-HETE, we observed that it induces the production of ROS in NADPH oxidase-dependent manner. Furthermore, the quenching effect of NAC and catalase on 15(S)-HETE-induced ROS production suggests that the ROS species produced by 15(S)-HETE is H 2 O 2 . Because 15(S)-HETE induced the production of H 2 O 2 , and its scavengers NAC and catalase abolished 15(S)-HETE-induced EGFR, Src, Jak2, and STAT3 phosphorylation and MCP-1 expression, it is likely that H 2 O 2 mediates 15(S)-HETE-stimulated EGFR-Src-Jak2-STAT3-MCP-1 signaling in VSMCs. This conclusion can be further supported by our previous observations that oxidants such as H 2 O 2 stimulate tyrosine phosphorylation of EGFR (40). Many lipid molecules such as lysophosphatidic acid and shingosine 1-phosphate that are coupled to GPCRs transactivate EGFR in many cell types (38,41). Although no putative receptor has been identified for HETEs thus far, based on the capacity of 15(S)-HETE to activate EGFR, one cannot rule out a role for a GPCR in the signaling events of this oxidized lipid molecule. Indeed, some reports have shown that it binds to leukotriene B4 low affinity receptor, BLT2, a GPCR (42).
Based on the large body of data, the Jak family of tyrosine kinases was thought to be the major mediator of activation of STATs in response to a variety of stimulants (18,21). However, a few studies have shown that besides the Jak family of tyrosine kinases, other non-receptor-tyrosine kinases such as the Src family of tyrosine kinases can also mediate tyrosine phosphorylation and activation of STATs (43). In this aspect we have previously shown that 15(S)-HETE stimulates STAT3 tyrosine phosphorylation via activation of Src in both VSMCs and endothelial cells (17,44). However, what is not known in this aspect is whether there is any interaction between these two non-receptor-tyrosine kinases in the stimulant-mediated STAT3 FIGURE 8. Overexpression of 15-Lox1 enhances BI-induced EGFR, Src, Jak2 and STAT3 tyrosine phosphorylation, MCP-1 expression, and neointima formation. A-C, 3 days after BI and transduction with Ad-GFP (10 10 pfu) alone or Ad-15-Lox1 (10 10 pfu) with or without Ad-dnSrc (10 10 pfu) or Ad-dnJak2 (10 10 pfu) combination, the uninjured and injured common carotid arteries were isolated, and either tissue extracts were prepared and analyzed for EGFR (panel A) and Src, Jak2, and STAT3 (panel B) tyrosine phosphorylations as described in Fig. 4, panel D, or RNA was isolated and analyzed for MCP-1 and ␤-actin mRNA levels (panel C) as described in Fig. 1, panel D. IP, immunoprecipitation; IB, immunoblot. D, all the conditions were the same as in panel A, except that arteries were isolated 2 weeks after BI and analyzed for neointima as described in Fig. 7, panel D. The bar graph shows the quantitative analysis of the I/M ratios of the carotid arteries. The blot in panel A was reprobed with normal EGFR antibodies, and the blots in panel B were reprobed with normal Src, Jak2, STAT3, or 12-Lox antibodies for normalization or to show the overexpression of 15-lox1. *, p Ͻ 0.05 versus Ad-GFP; **, p Ͻ 0.05 versus Ad-15-Lox1 (n ϭ 6). tyrosine phosphorylation and activation? In this regard, our results provide the first direct evidence for the role of Src in the activation of Jak2, at least in response to 15(S)-HETE, in VSMCs. In addition, both Src and Jak2 form a complex with each other as well as with EGFR and mediate STAT3 tyrosine phosphorylation. In view of these results, it appears that both Src and Jak2 may lie in the same signaling axis downstream to EGFR in the tyrosine phosphorylation and activation of STATs. The interaction between Src and Jak2 can also be seen at the level of STAT3 target gene, MCP-1, as inhibition of either of these non-receptor-tyrosine kinases suppressed its expression and secretion equally.
Although many studies have reported that GPCR agonists such as angiotensin II, thrombin, and sphingosine 1-phosphate transactivate EGFR in VSMCs (17,40,42), the function of EGFR in vascular wall remodeling has not been addressed adequately. In this regard our present results provide compelling evidence for the activation of EGFR in the artery in response to mechanical injury. In addition, activation of EGFR by injury is sufficient in mediating the signaling events such as the Src-Jak2-STAT3 signaling leading to MCP-1 expression, as these events were all sensitive to suppression by its inhibitor AG1478. Furthermore, inhibition of EGFR resulted in the reduction of injury-induced neointima formation. In understanding the role of 15-Lox1-15(S)-HETE axis in vascular wall remodeling, previously we have shown that adenovirus-mediated transduction of 15-Lox1 into artery enhances injury-induced neointima formation. In the present study, our results show that adenovirusmediated transduction of 15-Lox1 into artery also enhances injury-induced EGFR, Src, Jak2, and STAT3 tyrosine phosphorylation as well as MCP-1 expression. Interestingly, EGFR has also been found to be activated in the artery in response to injury in a manner that is dependent on thrombin activity (45). In another study it was reported that blockade of EGFR function by its neutralizing antibodies attenuates injury-induced neointima formation (46). Furthermore, we and others have demonstrated that STAT3 mediates VSMC growth and migration in response to various agents in vitro and neointima formation in response to injury in vivo (30,(47)(48)(49)(50). In addition, the present study demonstrates that 15-Lox1-15(S)-HETE axismediated vascular wall remodeling requires ROS-dependent activation of EGFR, which in turn targets the stimulation of the Src-Jak2-STAT3 signaling leading to MCP-1 expression. Based on these observations, it is conceivable that various cues target EGFR in relaying their signaling events to the effector molecules in the intact artery in response to injury. Thus, EGFR appears to play a central role in vascular wall remodeling after injury and, therefore, could be a potential target for the development of drugs against vascular lesions such as restenosis.