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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2008 Dec 18;29(3):387–393. doi: 10.1161/ATVBAHA.108.179150

Role of Src Tyrosine Kinase in the Atherogenic Effects of the 12/15-Lipoxygenase Pathway in Vascular Smooth Muscle Cells

Marpadga A Reddy 1, Saurabh Sahar 1, Louisa M Villeneuve 1, Linda Lanting 1, Rama Natarajan 1
PMCID: PMC2672949  NIHMSID: NIHMS103873  PMID: 19095999

Abstract

Objective

The 12/15-Lipoxygenase (12/15-LO) and its metabolite 12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] mediate proatherogenic responses in vascular smooth muscle cells (VSMCs). We examined the role of the nonreceptor tyrosine kinase Src in the signaling and epigenetic chromatin mechanisms involved in these processes.

Methods and Results

Rat VSMCs (RVSMCs) were stimulated with 12(S)-HETE (0.1 μmol/L) in the presence or absence of the Src inhibitor PP2 (10 μmol/L). Src activation and downstream signaling events including inflammatory gene expression and chromatin histone H3-Lys-9/14 acetylation were examined by immunoblotting, RT-PCR, and chromatin immunoprecipitation assays, respectively. 12(S)-HETE significantly activated Src, focal adhesion kinase, Akt, p38MAPK, and CREB. Expression of monocyte chemoattractant protein-1 and interleukin-6 genes and histone H3-Lys-9/14 acetylation on their promoters were also increased by 12(S)-HETE. PP2 inhibited these responses as well as 12(S)-HETE-induced VSMC migration. Furthermore, dominant negative mutants of Src, CREB, and a histone acetyltransferase p300 significantly blocked 12(S)-HETE–induced inflammatory gene expression. In addition, growth factor induced Src signaling and downstream events including H3-Lys-9/14 acetylation and migration were significantly attenuated in VSMCs derived from 12/15-LO−/− mice relative to WT.

Conclusions

Src kinase signaling plays a central role in the proatherogenic responses mediated by 12/15-LO and its oxidized lipid metabolite 12(S)-HETE in VSMCs.

Keywords: Oxidized lipids, vascular biology, 12/15-lipoxygenase, Src kinase, histone acetylation, atherosclerosis, inflammatory genes

Proatherogenic responses and inflammatory gene expression in vascular smooth muscle cells (VSMCs) play important roles in the development of atherosclerosis.1 Several lines of evidence have demonstrated the key role of murine leukocyte 12/15-lipoxygenase (12/15-LO) in the pathology of atherosclerosis, diabetes, and its complications.2-8 In VSMCs, 12/15-LO and its oxidized lipid metabolites such as 12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] mediate growth factor–induced proliferation, migration, and gene expression.2 12(S)-HETE directly activates and induces potent proatherogenic and proinflammatory responses in VSMCs.3,9-13 VSMCs derived from 12/15-LO−/− mice produce significantly reduced 12(S)-HETE levels and exhibit attenuated growth factor–induced responses such as growth, migration, and inflammatory gene expression relative to those derived from genetic control mice.14 12/15-LO gene silencing with specific siRNAs could inhibit NF-κB and inflammatory gene expression in VSMCs.15 Conversely, overexpression of 12/15-LO using viral vectors increased 12(S)-HETE levels and enhanced inflammatory genes as well as VSMC migration.16 Recent studies also identified the role of 12/15-LO metabolites in growth factor–induced VSMC–monocyte binding in VSMCs.11 These studies demonstrate the key role of signaling by 12/15-LO and its product 12(S)-HETE leading to proatherogenic responses in VSMCs. Although evidence shows that 12(S)-HETE stimulates specific signaling pathways such as protein kinase C, ras, p38mitogen-activated protein kinase (p38MAPK), and cAMP Response binding element protein (CREB) transcription factor,10,13,17-20 it is not clear whether 12(S)-HETE directly induces inflammatory cytokine and chemokine genes in VSMCs. Furthermore, the upstream signaling kinases and nuclear events involved in 12(S)-HETE–induced inflammatory gene expression are also not known.

The nonreceptor tyrosine kinase Src plays a central role in the signaling events leading to cell proliferation, migration, and gene expression via interaction with multiple signaling pathways through their SH2 and SH3 domains.21 Src kinases mediate oxidant stress, migration, cell proliferation, and inflammatory gene expression induced by growth factors, bioactive lipids like lysophosphatidic acid and ligands of the receptor for advanced glycation end products in VSMCs.22-28 However, the role of Src kinase in inflammatory gene expression and atherogenic responses mediated by 12/15-LO and its products is not known. Here we examined whether 12(S)-HETE activates Src kinase and whether Src activation plays a key role in 12(S)-HETE and 12/15-LO–mediated atherogenic responses in VSMCs. Our results demonstrate that 12(S)-HETE can directly induce Src activation and that Src inhibitors can block 12(S)-HETE–induced signaling leading to inflammatory gene expression, chromatin histone acetylation, and migration in VSMCs. Furthermore, Src activation and its downstream signaling were significantly attenuated in VSMCs from 12/15-LO−/− mice demonstrating the key role of Src kinase in 12/15-LO–mediated proatherogenic responses in VSMCs.

Materials and Methods

A detailed description of the methods is provided in the supplemental materials (available online at http://atvb.ahajournals.org). Rat VSMCs (RVSMCs) were isolated from male Sprague-Dawley rats (Charles River, Hollister, Calif), whereas mouse VSMCs (MVSMCs) were isolated from leukocyte 12/15-LO−/− mice on a C57BL/6 background (B6.129S2-Alox15tm1Fun; stock No: 002778) and control (WT) mice (Jackson Laboratories, Bar Harbor, Maine) as described.14,29 VSMCs were serum-depleted for 48 hours before stimulation with 12(S)-HETE (0.1 μmol/L) or PDGF (1 nmol/L) and where indicated pretreated with Src inhibitor PP2 (10 μmol/L). VSMC migration assays were performed as described14 and with an Oris cell migration assay kit (Platypus Technologies). VSMCs were transfected using Amaxa's Nucleofection kit. Chromatin immunoprecipitation (ChIP) assays, RT-PCRs, and Western blots were performed as described previously.29,13 Data are presented as means±SEM.

Results

Activation of Src Kinase by 12(S)-HETE in VSMCs

Evidence shows that 12(S)-HETE can induce atherogenic responses in VSMCs via activation of specific pathways such as p38MAPK and CREB in VSMCs.13 Here, we examined whether 12(S)-HETE activates Src kinase and whether this plays a key role in atherogenic responses including inflammatory gene expression and migration. Immunoblotting of cell lysates from 12(S)-HETE (0.1 μmol/L)–treated RVSMCs with phospho(Tyr416)-Src antibody showed that 12(S)-HETE increased phospho-Src levels as early as 5 minutes and this was sustained up to 1 hour (Figure 1A). Quantification of data from multiple experiments showed significant Src activation by 12(S)-HETE at 5 minutes (Figure 1B). In contrast, neither the vehicle control Ethanol (Et), nor a stereoisomer 12(R)-HETE (RH) which is not a 12/15-LO product, had any effect (Figure 1C). These results demonstrate that 12(S)-HETE specifically activates Src kinase in VSMCs.

Figure 1.

Figure 1

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Src kinase Activation by 12(S)-HETE. A, Cell lysates from RVSMCs stimulated with 12(S)-HETE (0.1 μmol/L) were immunoblotted with indicated antibodies. B, Levels of phospho(Tyr416)-Src (pSrc) expressed as fold over control (*P<0.003, n=6). C, Cell lysates from RVSMCs treated with vehicle ethanol (Et), or 12(S)-HETE (SH), or 12 (R)-HETE (RH) for 5 minutes were immunoblotted with indicated antibodies.

Role of Src Kinase in 12(S)-HETE Induced Signals Leading to VSMC Migration

Next, we examined the role of Src activity in 12(S)-HETE–induced signaling including activation of Focal Adhesion Kinase (FAK) and Akt kinase, which play important roles in gene expression and cell migration.24 VSMCs were pretreated with vehicle (DMSO) or a Src inhibitor PP2 (10 μmol/L) for 30 minutes and then stimulated with 12(S)-HETE for 10 to 30 minutes. Immunoblotting of cell lysates with phospho(Y397) FAK and phospho(Ser473)-Akt antibodies showed that 12(S)-HETE induced the activation of both FAK and Akt (Figure 2A and 2B). Furthermore, pretreatment of VSMCs with PP2 significantly blocked 12(S)-HETE induced FAK and Akt activation relative to vehicle DMSO (Figure 2A and 2B).

Figure 2.

Figure 2

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Src involvement in 12(S)-HETE–induced activation of FAK and Akt and VSMC migration. A, Cell lysates from RVSMCs stimulated with 12(S)-HETE in the presence of DMSO (DM) or PP2 (10 μmol/L) were immunoblotted with indicated antibodies. B, Signal intensities of phospho(Tyr397)-FAK (pFAK) and phospho(Ser473)-Akt (pAkt) expressed as fold stimulaion (*P<0.001, n=3). C, VSMCs were pretreated for 1 hour with DMSO or PP2, and migration assays were performed in the presence of vehicle (Ctrl) or 12(S)-HETE for 24 hours. Results were expressed as % of Control (*P<0.05, n=4).

To determine the functional significance of Src activation, VSMCs were pretreated with vehicle (DMSO) or PP2 (10 μmol/L), and a VSMC migration assay was performed in the presence of either the vehicle (EtOH) or 12(S)-HETE (0.1 μmol/L) for 24 hours as described under Methods. As shown in Figure 2C, 12(S)-HETE treatment significantly increased VSMC migration and furthermore, this effect of 12(S)-HETE was abrogated by pretreatment with PP2, demonstrating the key role of Src activation in 12(S)-HETE induced VSMC migration.

Src Involvement in 12(S)-HETE–Induced Inflammatory Gene Expression

Inflammatory gene expression plays a key role in VSMC dysfunction.1,30 However, it is not known whether 12(S)-HETE induces inflammatory genes in VSMCs. To test this, serum depleted VSMCs were stimulated with 12(S)-HETE for 0 to 24 hours, and total RNA analyzed by RT-PCR using interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) gene specific primers. 12(S)-HETE significantly increased both IL-6 and MCP-1 mRNA expression levels with a peak at 1 hour (Figure 3A and 3B). Next, we examined the role of Src kinases in 12(S)-HETE–induced inflammatory gene expression. Pretreatment of VSMCs with the Src inhibitor PP2 abolished 12(S)-HETE induced IL-6 and MCP-1 expression (Figure 3C and supplemental Figure I). To specifically establish the role of Src, VSMCs were transfected with a plasmid vector expressing SrcRF, a dominant negative mutant of Src kinase,31 or a control vector expressing EGFP. Transfected cells were serum depleted and stimulated with 12(S)-HETE. Results showed that 12(S)-HETE–induced IL-6 and MCP-1 mRNA levels were significantly blocked in cells transfected with SrcRF mutant compared to control EGFP (Figure 3D). These results clearly demonstrate that Src kinase can regulate 12(S)-HETE–induced inflammatory gene expression in VSMCs.

Figure 3.

Figure 3

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Role of Src in 12(S)-HETE-induced inflammatory gene expression. A, Time course of 12(S)-HETE (0.1 μmol/L) induced IL-6 and MCP-1 mRNA levels determined by RT-PCR. B, Quantification of gene expression at 1 hour expressed as fold over control cells (*P<0.01, **P<0.05, vs Control, n=6). C, RT-PCRs of indicated genes using RNA from RVSMCs pretreated with DMSO (DM) or PP2 and stimulated with 12(S)-HETE. Results were expressed as fold stimulation. (*P<0.005, **P<0.01, vs DM, n=3). D, RT-QPCR using indicated primers with RNA from control or 12(S)-HETE–stimulated RVSMCs transfected with either EGFP or SrcRF expression plasmids. Results expressed as fold stimulation over respective control (*P<0.01, **P<0.002 vs EGFP, n=3).

Involvement of CREB in 12(S)-HETE–Induced Inflammatory Gene Expression

We previously showed that 12(S)-HETE activates the transcription factor CREB via p38MAPK in VSMCs, and this leads to fibronectin gene expression.13 However, it is not known whether CREB activation is downstream of Src kinase and whether it regulates 12(S)-HETE–induced inflammatory gene expression. We first examined the effect of PP2 on p38MAPK and CREB activation by 12(S)-HETE, using immunoblotting with phospho-specific antibodies. PP2 significantly blocked both p38MAPK and CREB activation (Figure 4 A and 4B), demonstrating that Src is upstream of these events in VSMCs stimulated with 12(S)-HETE. ChIP assays using CREB antibody showed that 12(S)-HETE increased recruitment of CREB at both IL-6 and MCP-1 promoters (Figure 4C). Furthermore, transfection of RVSMCs with KCREB,29 a CREB-dominant negative mutant, significantly inhibited 12(S)-HETE–induced IL-6 and MCP-1 mRNA expression, compared to those transfected with control vector expressing EGFP (Figure 4D). Thus, 12(S)-HETE stimulated Src signaling in VSMCs can lead to inflammatory gene expression via CREB activation.

Figure 4.

Figure 4

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Src kinase involvement in 12(S)-HETE–induced p38MAPK and CREB activation. A, RVSMCs pretreated with DMSO (DM) or PP2 were stimulated with 12(S)-HETE for 10 minutes and cell lysates were immunoblotted with indicated antibodies. B, Quantification of p38MAPK and CREB activation expressed as fold stimulation over respective controls (*P<0.007, **P<0.002, vs DM, n=3). C, PCR of DNA samples from ChIP assays performed with CREB antibody to determine occupancy of CREB at IL-6 and MCP-1 promoters in response to 12(S)-HETE. Results are representative of 2 experiments. D, RT-QPCR using indicated gene primers with RNA from control or 12(S)-HETE–stimulated RVSMCs transfected with either EGFP or KCREB expression plasmids. Results expressed as fold stimulation over respective control (*P<0.01, **P<0.001 vs EGFP, n=3).

Src Activation Is Attenuated in 12/15-LO−/− VSMCs

Our previous results demonstrated that MVSMCs derived from 12/15-LO−/− mice display reduced proatherogenic responses, including migration and gene expression.14 However, the mechanisms involved were not completely understood. Because our current data show that Src kinase plays an important role in 12(S)-HETE signaling, we tested the hypothesis that reduced Src activation may be responsible for attenuated responses in 12/15-LO−/− cells. MVSMCs derived from WT or 12/15-LO−/− mice were stimulated with PDGF (1 nmol/L), and cell lysates immunoblotted with phosho-Src (pY416) antibody. Results showed that, although the time course of PDGF-induced Src activation was similar in MVSMCs derived from both WT and 12/15-LO−/− mice (Figure 5A), the magnitude of Src activation was greatly reduced in MVSMCs from 12/15-LO−/− animals compared to WT (Figure 5B). Results from multiple experiments showed that PDGF-induced Src activation at the 5-minute time point was significantly reduced in MVSMCs from 12/15-LO−/− mice relative to WT cells (Figure 5C).

Figure 5.

Figure 5

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Src Kinase activation and its downstream signaling are attenuated in MVSMCs derived from 12/15-LO−/− mice. A, Cell lysates from MVSMCs stimulated with PDGF (1 nmol/L) for various time periods were immunoblotted with indicated antibodies. B, Time course of phospho(Tyr416)-Src (pSrc) levels expressed as fold over control. C, Quantification of PDGF-induced Src (pSrc), Akt (pAkt), and FAK (pFAK) activation in WT and LO−/− MVSMCs determined by immunoblotting with phospho-specific antibodies and expressed as fold stimulation (*P<0.02, **P<0.001, #P<0.04 vs WT, n=3). D, PDGF-induced IL-6 and MCP-1 mRNA levels were determined by RT-PCR and results expressed as percent of WT cells (*P<0.04, **P<0.003 vs WT, n=3). E, PDGF-induced VSMC migration assays were performed and results expressed as % of WT cells (*P<0.01, vs WT, n=6). F and G, 12(S)-HETE partially restores the migratory phenotype of 12/15-LO−/− MVSMCs. MVSMC migration assays were performed using Oris cell migration assay kit under basal (F) or PDGF treated conditions (G) and with or without 12(S)-HETE treatment. The number of migrated cells was expressed as % of WT (F) or % of respective control (G). *P<0.05 vs WT, † P<0.05 vs 12/15-LO−/− without HETE treatment, n=4. HT-12(S)-HETE; PD-PDGF.

Next, we examined key signals downstream of Src kinase in these cells. Immunoblotting of cell lysates with phospho-specific antibodies showed that PDGF-induced activation of Akt and FAK were also significantly reduced in MVSMCs derived from 12/15-LO−/− mice compared to WT (Figure 5C). Furthermore, PDGF induced inflammatory gene expression (Figure 5D) and VSMC migration (Figure 5E) was also significantly reduced in 12/15-LO−/− MVSMCs compared to WT cells. These results demonstrate that impaired Src signaling may be a key underlying mechanism for the reduced proatherogenic responses in VSMCs derived from 12/15-LO−/− mice.

We also examined whether 12(S)-HETE can rescue the impaired migratory phenotype of 12/15-LO−/− VSMCs by compensating for the 12/15-LO deficiency. Migration assays were performed with or without 12(S)-HETE. Results showed that 12(S)-HETE addition could significantly reverse the attenuated basal as well as PDGF-induced migration of 12/15-LO−/− MVSMCs compared to control (Figure 5F and 5G). However, this reversal was only partial, suggesting that 12(S)-HETE deficiency is not the only reason for the reduced migratory response of 12/15-LO−/− MVSMCs and that other 12/15-LO metabolites such as 15(S)-HETE or hydroxyoctadecadienoic acids (HODEs) may also be involved.

Role of Src in 12(S)-HETE Induced Chromatin Histone Acetylation

Having demonstrated the role of Src kinase in 12(S)-HETE mediated inflammatory gene expression, we next examined its involvement in chromatin histone modifications. Evidence shows that chromatin histone modifications such as histone H3 lysine 9/14 acetylation (H3K9/14Ac) are increased at active gene promoters.32 However, it is not known if 12(S)-HETE can induce such nuclear chromatin events. To examine this, we performed ChIP assays using H3K9/14Ac antibody, and ChIP enriched DNA samples were analyzed by real time QPCR using IL-6 and MCP-1 promoter primers. As shown in Figure 6, 12(S)-HETE induced H3K9/14Ac at the promoters of IL-6 (A) and MCP-1 (B). Transfection of VSMCs with a histone acetyl transferase (HAT) p300 mutant lacking HAT activity (p300-DN)29 significantly inhibited 12(S)-HETE induced IL-6 (C) and MCP-1 (D) expression confirming the important role of histone acetylation in 12(S)-HETE induced gene expression. Furthermore, Src inhibitor PP2 significantly inhibited 12(S)-HETE induced H3K9/14Ac at IL-6 (E) and MCP-1 (F) promoters. PDGF induced H3K9/14Ac levels were also significantly reduced at the IL-6 (G) and MCP-1 (H) promoters in 12/15-LO−/− cells, which also exhibit reduced Src signaling, compared to WT cells. These results demonstrate the key role of Src in 12(S)-HETE mediated histone acetylation leading to gene activation in VSMCs.

Figure 6.

Figure 6

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Role of Src in 12(S)-HETE–induced H3K9/14Ac at inflammatory gene promoters. A and B, QPCRs of ChIP DNA samples using IL-6 (A) and MCP-1 (B) promoter primers. ChIP assays were performed with H3K9/14Ac antibody using cell lysates from control or 12(S)-HETE–stimulated RVSMCs. Results were expressed as fold over control after normalization with input samples (*P<0.05 vs Control, n=3). C and D, RT-QPCR showed significant inhibition of 12(S)-HETE–induced IL-6 (C) and MCP-1 (D) gene expression in RVSMCs transfected with p300 lacking HAT activity (p300-DN) relative to pEGFP transfected cells (*P<0.05 vs Control, n=3). E and F, QPCRs of ChIP samples showing inhibition of 12(S)-HETE–induced H3K9/14Ac by PP2 at the IL-6 (E) and MCP-1 (F) promoters (*P<0.05 vs Control, n=3). G and H, QPCRs of ChIP DNA samples showing attenuation of PDGF-induced H3K9/14Ac at IL-6 (G) and MCP-1 (H) promoters in 12/15-LO−/− MVSMCs relative to WT cells. (*P<0.05 vs WT, n=3).

Discussion

In this report, we have demonstrated for the first time the role of nonreceptor Src tyrosine kinase in signaling and nuclear chromatin events triggered by 12/15-LO and its oxidized lipid product 12(S)-HETE leading to migration and inflammatory gene expression in VSMCs. Src kinases integrate multiple signaling pathways essential for diverse biological processes via interaction with several intracellular signaling molecules through the SH2 and SH3 domains.21 We noted that Src kinase stimulated by 12(S)-HETE can mediate the activation of FAK, a key signal involved in VSMC migration.33 Src can activate FAK via phosphorylation of Tyr397 leading to autophosphorylation of other tyrosine residues. These phospho-tyrosine residues can recruit several other signaling molecules to focal adhesion sites including paxillin, Grb2, p130CAS, and phosphatidylinositol 3-kinase (PI3K) that are involved in cytoskeleton rearrangement, migration, and gene expression. In some instances, integrins can also activate FAK, which can in turn activate Src kinases by dissociating the inhibitory intramolecular association of C-terminal Tyr527 with Src SH2-domain.33,34 Our data showing inhibition of 12(S)-HETE–induced FAK-Tyr397 phosphorylation by Src inhibitor PP2 demonstrated that FAK is downstream of Src under these conditions. PP2 also blocked 12(S)-HETE–induced VSMC migration and activation of p38MAPK and Akt, key signals involved in cell migration.35,36 Together, these results demonstrate that Src kinase signaling plays an essential role in 12(S)-HETE–induced VSMC migration.

We previously demonstrated that the p38MAPK-mediated CREB activation leads to fibronectin gene expression in 12(S)-HETE–treated VSMCs.13 Our current results show that 12(S)-HETE can directly induce inflammatory genes IL-6 and MCP-1 in VSMCs, and this was significantly blocked by Src pharmacological inhibitor PP2 as well as a dominant negative mutant SrcRF, clearly demonstrating the role of Src in inflammatory gene expression. Both IL-6 and MCP-1 promoters contain CREB binding sites, and mutation of these sites blocked induction of these genes.37,38 We showed that 12(S)-HETE increased recruitment of CREB at the IL-6 and MCP-1 gene promoters and that KCREB, which prevents DNA binding of endogenous CREB,29 significantly blocked 12(S)-HETE–induced inflammatory gene expression, demonstrating an important role for CREB. Recent reports showed that the 15-LO and cyclooxygenase-2 metabolites of arachidonic acid, including 15(S)-HETE, could increase VSMC migration via CREB,39 but the upstream mediators were not identified. Our results clearly demonstrated that Src signaling leading to p38MAPK is upstream of CREB activation leading to inflammatory cytokine and chemokine gene expression in 12(S)-HETE stimulated VSMCs. These genes can induce VSMC proliferation and migration and also activate endothelial cells or monocytes, thereby promoting vessel wall inflammation.30

Our novel results also showed for the first time that an oxidized lipid like 12(S)-HETE can induce chromatin histone modifications such as H3K9/14Ac, and this occurs in a Src-dependent fashion. Furthermore, 12(S)-HETE–induced gene expression was blocked by p300-DN, a dominant negative mutant of the coactivator HAT p300, and thereby implicating p300 in 12(S)-HETE induced H3K9/14Ac and gene expression. Because p300 interacts with CREB,32 inhibition of CREB activation by Src inhibitors might prevent p300 recruitment to inflammatory gene promoters leading to reduced H3K9/14Ac and gene expression. Alternately, the Src downstream kinase p38MAPK may be required for the phosphorylation and activation of p300. Further studies are needed to identify these potential epigenetic mechanisms.

Another important finding of these studies is that Src kinase activation and its downstream signaling, including FAK, p38MAPK, and Akt activation was significantly reduced in MVSMCs derived from 12/15-LO−/− mice. Our previous reports showed that CREB activation, another key transcription factor activated by Src, was also reduced in 12/15-LO−/− MVSMCs.11 This was associated with reduced expression of inflammatory genes and decreased VSMC migration demonstrating a key role for Src in 12/15-LO signaling leading to VSMC dysfunction. Although 12(S)-HETE addition could reverse the defective basal and PDGF-induced migration of 12/15-LO−/− cells, this effect was only partial suggesting that although 12(S)-HETE is a key 12/15-LO product inducing the VSMC responses, other 12/15-LO metabolites such as 15(S)-HETE and HODEs may also be involved. Evidence shows that HODEs can induce the expression of chemokines such as MCP-1 in VSMCs,12 and 15(S)-HETE can also induce VSMC migration.39 PDGF-induced signaling activates phospholipases, which produce arachidonic acid from membrane phospholipids. 12/15-LO produces 12(S)-HETE from arachidonic acid, which in turn can mediate optimal PDGF induced responses via activation of Src kinases. Interestingly, our results also demonstrated for the first time, a key role for 12/15-LO and Src signaling in chromatin histone modifications associated with active gene expression, such as H3K9/14Ac, and inflammation in VSMCs. Thus, impaired Src kinase signaling attributable to lack of 12/15-LO may be the underlying mechanism involved in attenuated growth factor–induced responses and inflammatory gene expression in MVSMCs from 12/15-LO−/− mice.

In summary, our results reveal new mechanisms by which proatherogenic 12/15-LO and its oxidized lipid products contribute to vascular inflammation via Src activation. This is particularly significant because 12/15-LO can induce LDL oxidation, and 12/15-LO products act as seeding molecules for LDL oxidation.40,41 These studies also reinforce the evidence that Src kinase can regulate the proinflammatory responses induced by oxidized LDL and its components.25,42,43 Thus, Src kinase activation induced by oxidized lipids can integrate multiple signaling pathways and nuclear chromatin events and lead to vascular inflammation.

Acknowledgments

Sources of Funding

This work was supported by National Institutes of Health Grant RO1HL87864 (to R.N.), a Junior Faculty Award (1-08-JF-42) from the American Diabetes Association (to M.A.R.), and a predoctoral fellowship from the American Heart Association, Western States Affiliate (to S.S.).

Footnotes

Disclosures

None.

References

  • 1.Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008;28:812–819. doi: 10.1161/ATVBAHA.107.159327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Zhao L, Funk CD. Lipoxygenase pathways in atherogenesis. Trends Cardiovasc Med. 2004;14:191–195. doi: 10.1016/j.tcm.2004.04.003. [DOI] [PubMed] [Google Scholar]
  • 3.Natarajan R, Nadler JL. Lipid inflammatory mediators in diabetic vascular disease. Arterioscler Thromb Vasc Biol. 2004;24:1542–1548. doi: 10.1161/01.ATV.0000133606.69732.4c. [DOI] [PubMed] [Google Scholar]
  • 4.Reilly KB, Srinivasan S, Hatley ME, Patricia MK, Lannigan J, Bolick DT, Vandenhoff G, Pei H, Natarajan R, Nadler JL, Hedrick CC. 12/15-Lipoxygenase activity mediates inflammatory monocyte/endothelial interactions and atherosclerosis in vivo. J Biol Chem. 2004;279:9440–9450. doi: 10.1074/jbc.M303857200. [DOI] [PubMed] [Google Scholar]
  • 5.Antonipillai I, Nadler J, Vu EJ, Bughi S, Natarajan R, Horton R. A 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid, is increased in diabetics with incipient and early renal disease. J Clin Endocrinol Metab. 1996;81:1940–1945. doi: 10.1210/jcem.81.5.8626861. [DOI] [PubMed] [Google Scholar]
  • 6.Natarajan R, Gerrity RG, Gu JL, Lanting L, Thomas L, Nadler JL. Role of 12-lipoxygenase and oxidant stress in hyperglycaemia-induced acceleration of atherosclerosis in a diabetic pig model. Diabetologia. 2002;45:125–133. doi: 10.1007/s125-002-8253-x. [DOI] [PubMed] [Google Scholar]
  • 7.Hatley ME, Srinivasan S, Reilly KB, Bolick DT, Hedrick CC. Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice. J Biol Chem. 2003;278:25369–25375. doi: 10.1074/jbc.M301175200. [DOI] [PubMed] [Google Scholar]
  • 8.Li SL, Reddy MA, Cai Q, Meng L, Yuan H, Lanting L, Natarajan R. Enhanced proatherogenic responses in macrophages and vascular smooth muscle cells derived from diabetic db/db mice. Diabetes. 2006;55:2611–2619. doi: 10.2337/db06-0164. [DOI] [PubMed] [Google Scholar]
  • 9.Natarajan R, Gonzales N, Lanting L, Nadler J. Role of the lipoxygenase pathway in angiotensin II-induced vascular smooth muscle cell hypertrophy. Hypertension. 1994;23:I142–I147. doi: 10.1161/01.hyp.23.1_suppl.i142. [DOI] [PubMed] [Google Scholar]
  • 10.Natarajan R, Bai W, Rangarajan V, Gonzales N, Gu JL, Lanting L, Nadler JL. Platelet-derived growth factor BB mediated regulation of 12-lipoxygenase in porcine aortic smooth muscle cells. J Cell Physiol. 1996;169:391–400. doi: 10.1002/(SICI)1097-4652(199611)169:2<391::AID-JCP19>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  • 11.Cai Q, Lanting L, Natarajan R. Growth factors induce monocyte binding to vascular smooth muscle cells: implications for monocyte retention in atherosclerosis. Am J Physiol Cell Physiol. 2004;287:C707–C714. doi: 10.1152/ajpcell.00170.2004. [DOI] [PubMed] [Google Scholar]
  • 12.Dwarakanath RS, Sahar S, Reddy MA, Castanotto D, Rossi JJ, Natarajan R. Regulation of monocyte chemoattractant protein-1 by the oxidized lipid, 13-hydroperoxyoctadecadienoic acid, in vascular smooth muscle cells via nuclear factor-kappa B (NF-kappa B) J Mol Cell Cardiol. 2004;36:585–595. doi: 10.1016/j.yjmcc.2004.02.007. [DOI] [PubMed] [Google Scholar]
  • 13.Reddy MA, Thimmalapura PR, Lanting L, Nadler JL, Fatima S, Natarajan R. The oxidized lipid and lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid induces hypertrophy and fibronectin transcription in vascular smooth muscle cells via p38 MAPK and cAMP response element-binding protein activation. Mediation of angiotensin II effects. J Biol Chem. 2002;277:9920–9928. doi: 10.1074/jbc.M111305200. [DOI] [PubMed] [Google Scholar]
  • 14.Reddy MA, Kim YS, Lanting L, Natarajan R. Reduced growth factor responses in vascular smooth muscle cells derived from 12/15-lipoxygenase-deficient mice. Hypertension. 2003;41:1294–1300. doi: 10.1161/01.HYP.0000069011.18333.08. [DOI] [PubMed] [Google Scholar]
  • 15.Li SL, Dwarakanath RS, Cai Q, Lanting L, Natarajan R. Effects of silencing leukocyte-type 12/15-lipoxygenase using short interfering RNAs. J Lipid Res. 2005;46:220–229. doi: 10.1194/jlr.M400328-JLR200. [DOI] [PubMed] [Google Scholar]
  • 16.Dwarakanath RS, Sahar S, Lanting L, Wang N, Stemerman MB, Natarajan R, Reddy MA. Viral vector-mediated 12/15-lipoxygenase overexpression in vascular smooth muscle cells enhances inflammatory gene expression and migration. J Vasc Res. 2008;45:132–142. doi: 10.1159/000109966. [DOI] [PubMed] [Google Scholar]
  • 17.Natarajan R, Lanting L, Xu L, Nadler J. Role of specific isoforms of protein kinase C in angiotensin II and lipoxygenase action in rat adrenal glomerulosa cells. Mol Cell Endocrinol. 1994;101:59–66. doi: 10.1016/0303-7207(94)90219-4. [DOI] [PubMed] [Google Scholar]
  • 18.Szekeres CK, Trikha M, Honn KV. 12(S)-HETE, pleiotropic functions, multiple signaling pathways. Adv Exp Med Biol. 2002;507:509–515. doi: 10.1007/978-1-4615-0193-0_78. [DOI] [PubMed] [Google Scholar]
  • 19.Kalyankrishna S, Malik KU. Norepinephrine-induced stimulation of p38 mitogen-activated protein kinase is mediated by arachidonic acid metabolites generated by activation of cytosolic phospholipase A(2) in vascular smooth muscle cells. J Pharmacol Exp Ther. 2003;304:761–772. doi: 10.1124/jpet.102.040949. [DOI] [PubMed] [Google Scholar]
  • 20.Rao GN, Baas AS, Glasgow WC, Eling TE, Runge MS, Alexander RW. Activation of mitogen-activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells. J Biol Chem. 1994;269:32586–32591. [PubMed] [Google Scholar]
  • 21.Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol. 1997;13:513–609. doi: 10.1146/annurev.cellbio.13.1.513. [DOI] [PubMed] [Google Scholar]
  • 22.Berk BC, Corson MA. Angiotensin II signal transduction in vascular smooth muscle: role of tyrosine kinases. Circ Res. 1997;80:607–616. doi: 10.1161/01.res.80.5.607. [DOI] [PubMed] [Google Scholar]
  • 23.Weber DS, Taniyama Y, Rocic P, Seshiah PN, Dechert MA, Gerthoffer WT, Griendling KK. Phosphoinositide-dependent kinase 1 and p21-activated protein kinase mediate reactive oxygen species-dependent regulation of platelet-derived growth factor-induced smooth muscle cell migration. Circ Res. 2004;94:1219–1226. doi: 10.1161/01.RES.0000126848.54740.4A. [DOI] [PubMed] [Google Scholar]
  • 24.Cospedal R, Abedi H, Zachary I. Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Cardiovasc Res. 1999;41:708–721. doi: 10.1016/s0008-6363(98)00232-6. [DOI] [PubMed] [Google Scholar]
  • 25.Schmitz U, Thommes K, Beier I, Vetter H. Lysophosphatidic acid stimulates p21-activated kinase in vascular smooth muscle cells. Biochem Biophys Res Commun. 2002;291:687–691. doi: 10.1006/bbrc.2002.6493. [DOI] [PubMed] [Google Scholar]
  • 26.Waltenberger J, Uecker A, Kroll J, Frank H, Mayr U, Bjorge JD, Fujita D, Gazit A, Hombach V, Levitzki A, Bohmer FD. A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling. Circ Res. 1999;85:12–22. doi: 10.1161/01.res.85.1.12. [DOI] [PubMed] [Google Scholar]
  • 27.Yamboliev IA, Chen J, Gerthoffer WT. PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs. Am J Physiol Cell Physiol. 2001;281:C709–C718. doi: 10.1152/ajpcell.2001.281.2.C709. [DOI] [PubMed] [Google Scholar]
  • 28.Reddy MA, Li SL, Sahar S, Kim YS, Xu ZG, Lanting L, Natarajan R. Key role of Src kinase in S100B-induced activation of the receptor for advanced glycation end products in vascular smooth muscle cells. J Biol Chem. 2006;281:13685–13693. doi: 10.1074/jbc.M511425200. [DOI] [PubMed] [Google Scholar]
  • 29.Sahar S, Reddy MA, Wong C, Meng L, Wang M, Natarajan R. Cooperation of SRC-1 and p300 with NF-kappaB and CREB in angiotensin II-induced IL-6 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2007;27:1528–1534. doi: 10.1161/ATVBAHA.107.145862. [DOI] [PubMed] [Google Scholar]
  • 30.Raines EW, Ferri N. Thematic review series: The immune system and atherogenesis. Cytokines affecting endothelial and smooth muscle cells in vascular disease. J Lipid Res. 2005;46:1081–1092. doi: 10.1194/jlr.R500004-JLR200. [DOI] [PubMed] [Google Scholar]
  • 31.Mukhopadhyay D, Tsiokas L, Zhou XM, Foster D, Brugge JS, Sukhatme VP. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 1995;375:577–581. doi: 10.1038/375577a0. [DOI] [PubMed] [Google Scholar]
  • 32.Berger SL. Histone modifications in transcriptional regulation. Curr Opin Genet Dev. 2002;12:142–148. doi: 10.1016/s0959-437x(02)00279-4. [DOI] [PubMed] [Google Scholar]
  • 33.Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 2005;6:56–68. doi: 10.1038/nrm1549. [DOI] [PubMed] [Google Scholar]
  • 34.Shattil SJ. Integrins and Src: dynamic duo of adhesion signaling. Trends Cell Biol. 2005;15:399–403. doi: 10.1016/j.tcb.2005.06.005. [DOI] [PubMed] [Google Scholar]
  • 35.Huang C, Jacobson K, Schaller MD. MAP kinases and cell migration. J Cell Sci. 2004;117:4619–4628. doi: 10.1242/jcs.01481. [DOI] [PubMed] [Google Scholar]
  • 36.Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM, Backx PH. The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. J Mol Cell Cardiol. 2004;37:449–471. doi: 10.1016/j.yjmcc.2004.05.015. [DOI] [PubMed] [Google Scholar]
  • 37.Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, Haegeman G. The nuclear factor-kappaB engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J Biol Chem. 1999;274:32091–32098. doi: 10.1074/jbc.274.45.32091. [DOI] [PubMed] [Google Scholar]
  • 38.Bogdanov VY, Poon M, Taubman MB. Platelet-derived growth factor-specific regulation of the JE promoter in rat aortic smooth muscle cells. J Biol Chem. 1998;273:24932–24938. doi: 10.1074/jbc.273.38.24932. [DOI] [PubMed] [Google Scholar]
  • 39.Dronadula N, Rizvi F, Blaskova E, Li Q, Rao GN. Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility. J Lipid Res. 2006;47:767–777. doi: 10.1194/jlr.M500369-JLR200. [DOI] [PubMed] [Google Scholar]
  • 40.Navab M, Hama SY, Cooke CJ, Anantharamaiah GM, Chaddha M, Jin L, Subbanagounder G, Faull KF, Reddy ST, Miller NE, Fogelman AM. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1. J Lipid Res. 2000;41:1481–1494. [PubMed] [Google Scholar]
  • 41.Parthasarathy S, Wieland E, Steinberg D. A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein. Proc Natl Acad Sci U S A. 1989;86:1046–1050. doi: 10.1073/pnas.86.3.1046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Yeh M, Gharavi NM, Choi J, Hsieh X, Reed E, Mouillesseaux KP, Cole AL, Reddy ST, Berliner JA. Oxidized phospholipids increase interleukin 8 (IL-8) synthesis by activation of the c-src/signal transducers and activators of transcription (STAT)3 pathway. J Biol Chem. 2004;279:30175–30181. doi: 10.1074/jbc.M312198200. [DOI] [PubMed] [Google Scholar]
  • 43.Velarde V, Jenkins AJ, Christopher J, Lyons TJ, Jaffa AA. Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells. J Appl Physiol. 2001;91:1412–1420. doi: 10.1152/jappl.2001.91.3.1412. [DOI] [PubMed] [Google Scholar]

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