|Home | About | Journals | Submit | Contact Us | Français|
Migration of vascular smooth muscle cells (VSMCs) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. The lipoxygenase products of polyunsaturated fatty acids (PUFA) were shown to play a role in these diseases. Cyclic AMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. The aim of the present study was to test the role of CREB in an oxidized lipid molecule, 15(S)-HETE-induced VSMC migration and neointima formation.
15(S)-HETE stimulated VSMC migration in CREB-dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1 or p38MAPK inhibited 15(S)-HETE-induced CREB phosphorylation and VSMC migration. 15(S)-HETE induced expression and secretion of interleukin-6 (IL-6), as analyzed by RT-PCR and ELISA, respectively. Neutralizing anti-IL-6 antibodies blocked 15(S)-HETE-induced VSMC migration. Dominant-negative mutant-mediated blockade of ERK1/2, JNK1, p38MAPK or CREB suppressed 15(S)-HETE-induced IL-6 expression in VSMCs. Serial 5′ deletions and site-directed mutagenesis of IL-6 promoter along with chromatin immunoprecipitation using anti-CREB antibodies showed that cAMP response element is essential for 15(S)-HETE-induced IL-6 expression. Dominant-negative CREB also suppressed balloon injury-induced IL-6 expression, SMC migration from media to intimal region and neointima formation. Adenovirus-mediated transduction of 15-lipoxygenase 2 (15-LOX2) caused increased production of 15-HETE in VSMCs and enhanced IL-6 expression, SMC migration from media to intimal region and neointima formation in response to arterial injury.
The above results suggest a role for 15-LOX2-15-HETE in the regulation of VSMC migration and neointima formation involving CREB-mediated IL-6 expression.
VSMC migration from media to intima plays a determinant role in atherosclerosis and restenosis (1-3). Arachidonic acid (AA) and its oxygenative metabolites, known as eicosanoids, are involved in the maintenance of vascular tone (4, 5). Lipoxygenases (LOXs) are non-heme iron dioxygenases that stereospecifically introduce molecular oxygen into polyunsaturated fatty acids (PUFA) such as AA, resulting in the formation of hydroperoxyeicosatetraenoic acids (HPETEs) which are further converted to hydroxyeicosatetraenoic acids HETEs. Two LOXs, in particular, 15- LOX1 in humans and its closely related ortholog, 12/15-LOX, in mice, as well as 5-LOX that convert AA to HETEs are the prime candidates implicated in atherosclerosis and restenosis (6-8). It is known that oxidation of low-density lipoprotein (LDL) is a contributing factor in the pathogenesis of atherosclerosis (9-11). Many studies have shown that 15-LOX1 and 12/15-LOX are involved in the oxidation of LDL, and thereby in the pathogenesis of atherosclerosis (10, 11). It was also demonstrated that atherosclerotic arteries express increased levels of 15-LOX1 and its AA product, 15-HETE in rabbits (12, 13). In addition, recently LOX products of PUFA have also been shown to be potent chemoattractants for residential and invading immune cells recruited to lesion areas (14). Though the association of LOX products of PUFA with the pathophysiology of vessel wall diseases was documented, the precise mechanisms by which these lipid molecules act on VSMCs is not well understood.
Cyclic AMP-response element-binding protein (CREB) belongs to the basic leucine-zipper family of transcriptional factors that were shown to play an important role in gene regulation, particularly in response to cAMP (15). This transcription factor is activated by phosphorylation of Ser133 residue, which is typically performed by protein kinase A (16). However, other protein kinases such as extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), calmodulin kinase (CaMK), and protein kinase B (PKB) have also been shown to phosphorylate and activate CREB (15, 17). CREB forms homo- or heterodimers with members of either the CREB/activating transcriptional factor (ATF) or the activator protein-1 (AP-1) family of transcriptional factors (18, 19). A number of VSMC chemotactic molecules such as platelet-derived growth factor-BB (PDGF-BB), angiotensin II (AngII), thrombin and tumor necrosis factor-α (TNF-α), have been shown to stimulate phosphorylation of CREB in the modulation of VSMC migration and/or proliferation (20-22). However, some studies have demonstrated a negative correlation between CREB levels and VSMC migration as well as proliferation (23).
Previously, we have reported that AA induces VSMC motility via activation of CREB (24). To understand the role of eicosanoids in the pathogenesis of vessel wall diseases, we performed a systematic study to identify eicosanoids with potent chemotactic activities and elucidate the underlying signaling mechanisms. In the present communication, we report for the first time that 15(S)-HETE, a major 15-LOX1/2 metabolite of AA, stimulates VSMC migration and this phenomenon requires MAPK-dependent CREB-mediated IL-6 expression. Furthermore, our results show that balloon injury-induced IL-6 expression and neointima formation were dependent on CREB activation.
For a detailed Materials and Methods section, please see www.ahajournals.org.
Towards understanding the role of eicosanoids in the pathophysiology of VSMC responses, we focused on studying the effects of various LOX metabolites of AA (i.e., 5(S)-HETE, 12(S)-HETE and 15(S)-HETE) in stimulating VSMC migration using modified Boyden chamber method. 5(S)-HETE, 12(S)-HETE and 15(S)-HETE) were found to stimulate VSMC migration more than 2-fold compared to control (Figure 1). Among the three HETEs, 15(S)-HETE was found to be more potent in stimulating VSMC migration. Given the more effectiveness of 15(S)-HETE in stimulating VSMC migration than 5(S)-HETE or 12(S)-HETE, we further focused on investigating the mechanisms involved in its chemotactic actions.
Several lines of evidence suggest a role for CREB in the regulation of cell migration, and proliferation (21, 22, 24). Activation of CREB is mediated by its phosphorylation at Ser133 (15-17). To examine whether CREB is activated by 15(S)-HETE, we performed Western blot analysis using its phosphospecific antibodies. We observed that CREB phosphorylation was increased in 15(S)-HETE-treated VSMCs compared to untreated cells, while the level of total CREB remained unchanged (Figure 2A). 15(S)-HETE-induced increases in CREB phosphorylation occurred at 1 min and peaked at 10 min (Figure 2A). To examine the role of CREB in 15(S)-HETE-induced VSMC migration, we applied a dominant-negative mutant approach. Dominant-negative CREB (KCREB) lacks DNA binding capacity due to replacement of lysine 287 with leucine in the DNA binding domain (25). VSMCs were transduced with Ad-GFP or Ad-dnCREB at 40 moi, quiesced and tested for 15(S)-HETE-induced migration. 15(S)-HETE induced migration of VSMCs that were transduced with Ad-GFP by 3-fold compared to its corresponding control. On the other hand, 15(S)-HETE-induced migration was inhibited in VSMCs that were transduced with Ad-dnCREB (Figure 2B). Ad-dnCREB transduced VSMCs showed slightly blunted migration. These results suggest the involvement of CREB in 15(S)-HETE-induced VSMC migration.
Several protein kinases are reported to phosphorylate CREB at Ser133 (15-17). From our earlier studies it was evident that AA-induced CREB Ser133 phopsphorylation is mediated by MAPKs (24). To investigate whether MAPKs are also involved in 15(S)-HETE-enhanced CREB Ser133 phosphorylation, we first tested the effects of 15(S)-HETE on phosphorylation of ERK1/2, JNK1/2 and p38MAPK by Western blot analysis using their respective phosphospecific antibodies. 15(S)-HETE stimulated phosphorylation of all three MAPKs in a time-dependent manner (Figure 3A). 15(S)-HETE-induced increases in the phosphorylation of ERK1/2, JNK1/2 and p38MAPK were observed at 1 min, peaked at 10 min and declined subsequently as compared to control. However, in the case of ERK1/2, a second peak of phosphorylation was observed at 2 hrs of 15(S)-HETE treatment. We then used a dominant-negative mutant approach to test the role of MAPKs in CREB phosphorylation. Transduction of VSMCs with dominant-negative mutants of MEK1, JNK1 and p38MAPK adenovirus inhibited 15(S)-HETE-induced ERK1/2, JNK1 and p38MAPK phosphorylation, respectively (Figure 3B). Adenovirus-mediated expression of dnMEK1, dn-JNK1 and dnp38MAPK also blocked 15(S)-HETE-induced VSMC migration (Figure 3C). Similarly, adenovirus-mediated expression of dnMEK1, dnJNK1 or dnp38MAPK blocked 15(S)-HETE-induced CREB Ser133 phosphorylation (Figure 3D).
A Large body of data shows that IL-6 influences VSMC migration (26, 27). In addition, IL-6 has been shown to be a CREB target gene in response to several stimulants (28). So we next asked whether IL-6 plays a role in 15(S)-HETE-induced VSMC migration. 15(S)-HETE induced IL-6 expression and secretion as measured by RT-PCR and ELISA, respectively (Figure 4A & B). In addition, neutralizing anti-IL-6 antibodies completely abrogated 15(S)-HETE-induced VSMC migration (Figure 4C).
To test the role of MAPKs and CREB in 15(S)-HETE-induced IL-6 expression, VSMCs were transduced with Ad-GFP, Ad-dnMEK1, Ad-dnJNK1, Ad-dnp38MAPK or Ad-dnCREB, quiesced and treated with and without 15(S)-HETE. RNA was isolated at 2 hrs and medium was collected 4 hrs of treatment and analyzed for IL-6 mRNA levels and IL-6 secretion by RT-PCR and ELISA, respectively. Transduction of Ad-dnMEK1, Ad-dnJNK1, Ad-dnp38MAPK or Ad-dnCREB abolished 15(S)-HETE-induced IL-6 expression and secretion (Figure 5A-D). The IL-6 promoter contains several cis-DNA binding regulatory elements, including physical binding sites for the AP-1, CRE and NF-κB transcription factors (28). To determine the promoter element responsible for the IL-6 expression by 15(S)-HETE, we performed promoter deletion analysis. As compared to vector control, 15(S)-HETE induced IL-6 promoter-luciferase activity by 2- to 3-fold with all but -150 nt and -27 nt constructs (Figure 5E). Mutation of CRE binding element (TGACGTCA to TCGATCCA) in the -1039 nt IL-6 promoter-luciferase construct also abolished 15(S)-HETE-induced increases in luciferase activity (Figure 5F). As determined by EMSA, 15(S)-HETE induced CREB DNA binding activity in a time-dependent manner with [32P]-labeled CRE element of the IL-6 promoter as a probe and this response was substantially attenuated by adenovirus-mediated expression of dnMEK1, dnJNK1, dnp38MAPK or dnCREB (Figure 5G). The specificity of CREB DNA binding activity was confirmed by competition with molar excess cold probe and supershift with anti-CREB antibodies. In addition, ChIP analysis revealed that CREB binds to IL-6 promoter in VSMCs in response to 15(S)-HETE and interference with CREB activation via adenovirus-mediated expression of its dominant negative mutant suppressed this effect (Figure 5H).
Smooth muscle cell migration from media to intima is a key event in neointima formation (3). To validate the in vitro observations in vivo, we have constructed an adenoviral vector for expression of 15-LOX2, whose function was demonstrated to convert AA mainly to 15-HETE (29). Exposure of Ad-15-LOX2 but not Ad-GFP-transduced VSMCs converted AA selectively to 15-HETE (Figure 6A upper panel). In order to test the role of 15(S)-HETE-CREB signaling in vascular wall remodeling, soon after balloon injury, arteries were transduced with Ad-GFP, Ad-dnCREB or Ad-15-LOX2 and three days later the arteries were dissected out. Either RNA was isolated and analyzed for IL-6 mRNA levels by RT-PCR, or fixed, opened longitudinally, immunostained with anti-histone H1 antibodies and positive cells were counted as a measure of SMC migration from medial to intimal region. Balloon injury induced the expression of IL-6 by about 2-fold in the arteries that were transduced with Ad-GFP. Similarly, balloon injury triggered the migration of SMC from medial to intimal region in Ad-GFP-transduced arteries. In contrast, transduction of Ad-dnCREB resulted in marked suppression in balloon injury-induced IL-6 expression as well SMC migration from medial to intimal region (Figure 6A bottom panel & B). Adenovirus-mediated expression of 15-LOX2 enhanced both balloon injury-induced IL-6 expression and SMC migration from medial to intimal region (Figure 6A bottom panel & B). To test the role of 15(S)-HETE-CREB signaling in balloon injury-induced neointima formation, 2 weeks after balloon injury and transduction with Ad-GFP Ad-dnCREB, or Ad-15-LOX2, arteries were isolated, fixed, cross-sections were cut, stained with H & E and morphometric analysis was performed. Balloon injury, with or without Ad-GFP-transduction, induced neointima formation almost to the same extent (Figure 6C). However, infusion of adenovirus harboring dnCREB into injured arteries inhibited neointima formation by about 35% (p<0.05) resulting in an increase in lumen size (Figure 6C). Adenovirus-mediated expression of 15-LOX2 enhanced neointima formation resulting reduced lumen size (Figure 6C).
LOXs form a heterogeneous family of lipid-peroxidizing enzymes that introduce molecular oxygen into PUFA such as AA and linoleic acid (LA). Based on their ability to oxidize the corresponding carbon atom of AA they are classified as 5-, 8-, 12-, and 15-LOXs (8). A large body of literature suggests that LOXs, including 15-LOX play a role in the pathogenesis of vessel wall diseases (10, 11, 14). However, the mechanism(s) of their action is not very clear. Therefore, here we studied the effect of 15(S)-HETE, a product of 15-LOX1/2, on VSMC migration and explored the underlying mechanisms. We observed that: 1. Among the three LOX products of AA tested, 15(S)-HETE was found to be most potent in inducing VSMC migration; 2. 15(S)-HETE induced CREB phosphorylation in a time-dependent manner and adenovirus-mediated expression of its dominant-negative mutant inhibited 15(S)-HETE-induced VSMC migration; 3. 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1/2 and p38MAPK in a time-dependent manner; 4. Adenovirus-mediated expression of dominant-negative mutants of MEK1, JNK1 or p38MAPK inhibited 15(S)-HETE-induced CREB Ser133 phosphorylation as well as VSMC migration. 5. 15(S)-HETE induced IL-6 expression and secretion and neutralizing anti-IL-6 antibodies inhibited 15(S)-HETE-induced VSMC migration. 6. Transducion of dn-MEK1, dnJNK1, dnp38MAPK or dnCREB suppressed 15(S)-HETE-induced IL-6 expression and secretion; 7. Deletion and mutational analysis of IL-6 promoter as well as ChIP analysis showed that cAMP-response element (CRE) is important for 15(S)-HETE-induced IL-6 expression and secretion; 8. Dominant-negative CREB also inhibited balloon injury-induced IL-6 expression, SMC migration from media to intimal region and neointima formation; 9. Adenovirus-mediated expression of 15-LOX2 produced 15-HETE as a major AA metabolite in VSMCs and enhanced balloon injury-induced IL-6 expression, SMC migration from media to intimal region and neointima formation. Together these observations reveal that 15(S)-HETE stimulates VSMC migration and this phenomenon requires MAPK-dependent CREB-mediated IL-6 expression.
Stimulus induced migration and proliferation of VSMCs is an essential factor in vascular wall remodeling (30). The role of CREB in the vessel wall remodeling appears complex. Various stimulants such as PDGF-BB and AngII, have been shown to stimulate CREB in VSMCs (20, 28). In fact, CREB has been shown to mediate VSMC migration and proliferation in response to TNF-α and thrombin, respectively (21, 22). Our results suggest that CREB plays a role in VSMC migration stimulated by 15(S)-HETE. However discrepancy prevails on CREB’s role in VSMC migration and proliferation. It has been demonstrated that adenovirus-mediated gene transfer of a mutant CREB promotes VSMC apoptosis and inhibits intimal lesions following balloon angioplasty (31), suggesting a role for CREB in VSMC proliferation. Investigations by Klemm et al (23) revealed that forced expression of constitutively active CREB decreases mitogen-stimulated VSMC proliferation and migration indicating its anti-mitogenic and motogenic effects. It was postulated that the transient exposure to agonists results in phosphorylation of CREB, which in turn promotes VSMC differentiation and survival in the healthy vessel wall, however, in the diseased vessel, sustained stimulation causes downregulation of CREB phosphorylation, which leads to cell death (32). Since blockade of CREB inhibits neointima formation, it is likely that prolonged availability of stimulants such as occurs by injury may activate CREB in a sustained manner mediating VSMC migration, proliferation or both. Various protein kinases, including MAPK are reported to phosphorylate CREB at Ser133 (11, 17). In our previous study AA-stimulated CREB phosphorylation was mediated by ERK1/2 and p38MAPK, but was not dependent on JNK1/2 (24). However, our present results show that all three MAPKs, including JNK1 are involved in 15(S)-HETE-induced CREB phosphorylation and activation. The discrepancy in regard to the role of JNK1 in CREB phosphorylation by AA and 15(S)-HETE could be due to non-specific effects of the inhibitor employed previously. It is noteworthy that interference with activation of any one of the three groups of MAPKs inhibited CREB phosphorylation partially and VSMC migration substantially. These findings suggest that in addition to their role in CREB activation, ERK1/2, JNK1 and p38MAPK may be involved in the regulation of other signaling molecules whose functions are also required for cell migration. Previously, we have shown that 15(S)-HETE via Rac1 activates MEK1, which in turn leads to stimulation of both ERK1/2 and JNK1 in endothelial cells (33, 34). Based on these findings, we anticipate that a common upstream signaling molecule such as Rac1 may be involved in ERK1/2, JNK1 and p38MAPK activation by 15(S)-HETE in VSMC.
The role of IL-6, a pleiotropic cytokine, in vessel wall remodeling is supported by the findings that its production was increased in VSMCs in response to a variety of agonists such as PDGF-BB, thrombin, and AngII (26, 35, 36). In addition, IL-6 has been shown to stimulate VSMC migration and proliferation (26, 27). IL-6 gene promoter contains several transcription factor-binding sites and CRE was found to be important in agonist-induced expression of IL-6 in VSMCs (28). From the present results, it is evident that 15(S)-HETE stimulates production of IL-6 via activation of CREB. Furthermore, inhibition of 15(S)-HETE-induced VSMC migration by neutralizing anti-IL-6 antibodies supports a role for IL-6 in VSMC migration by this lipid molecule. In addition, increased expression of IL-6 in the balloon-injured artery further suggests its role in vascular wall remodeling. The most convincing evidence for the role of 15-HETE in vascular wall remodeling is that transduction of 15-LOX2 that converts AA only to 15-HETE, enhanced balloon injury-induced IL-6 expression and SMC migration from media to intimal region forming neointima. Previously we reported that both PDGF-BB and thrombin induce IL-6 expression via NFAT-dependent manner in VSMCs (26). Based on these observations as well as present findings, it appears that IL-6 is a downstream effector molecule for regulation by various stimulants, although via different mechanisms, mediating the mitogenic and motogenic responses. IL-6 signaling mechanisms have also been implicated in the regulation of key genes associated with cell migration such as MMPs and cytoskeletal proteins (27, 37). Therefore, IL-6 released in response to various chemoattractants can act in an autocrine or paracrine manner through cell surface receptors and regulate cell migration and/or proliferation and thereby enhancing the disease progression. In fact, inhibition of GP130, a signal transducer of IL-6, blocked VSMC migration both in vitro and in vivo and neointima formation in vivo (38).
This work was supported by a grant from the National Heart, Lung and Blood Institute/National Institutes of Health (HL064165). We are thankful to Dr. Alan R. Brash of the Vanderbilt University Medical Center for providing us with expression vector for 15-LOX2.