Src-dependent STAT-3-mediated expression of monocyte chemoattractant protein-1 is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.

To understand the role of human 15-lipoxygenase 1 (15-LOX1) in vascular wall remodeling, we have studied the effect of the major 15-LOX1 metabolite of arachidonic acid, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), on vascular smooth muscle cell (VSMC) migration both in vitro and in vivo. Among 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, 15(S)-HETE potentially stimulated more vascular smooth muscle cell (VSMC) migration. In addition, 15(S)-HETE-induced VSMC migration was dependent on Src-mediated activation of signal transducer and activator of transcription-3 (STAT-3). 15(S)-HETE also induced monocyte chemoattractant protein-1 (MCP-1) expression via Src-STAT-3 signaling, and neutralizing anti-MCP-1 antibodies completely negated 15(S)-HETE-induced VSMC migration. Cloning and characterization of a 2.6-kb MCP-1 promoter revealed the presence of four putative STAT-binding sites, and the site that is proximal to the transcription start site was found to be essential for 15(S)-HETE-induced Src-STAT-3-mediated MCP-1 expression. Rat carotid arteries that were subjected to balloon injury and transduced with Ad-15-LOX1 upon exposure to [(3)H]arachidonic acid ex vivo produced 15-HETE as a major eicosanoid and enhanced balloon injury-induced expression of MCP-1 in smooth muscle cells in Src and STAT-3-dependent manner in vivo. Adenovirus-mediated delivery of 15-LOX1 into rat carotid artery also led to recruitment and homing of macrophages to medial region in response to injury. In addition, transduction of Ad-15-LOX1 into arteries enhanced balloon injury-induced smooth muscle cell migration from media to intima and neointima formation. These results show for the first time that 15-LOX1-15(S)-HETE axis plays a major role in vascular wall remodeling after balloon angioplasty.

CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.

OBJECTIVE: 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. cAMP 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

15(S)-HETE production in human retinal microvascular endothelial cells by hypoxia: Novel role for MEK1 in 15(S)-HETE induced angiogenesis.

PURPOSE: To examine for the expression of 15-lipoxygenase 1 (15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis. METHODS: Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis. RESULTS: HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 15(S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 (FGF-2). In addition, 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. CONCLUSIONS: These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15(S)-HETE in HRMVECs. In addition, 15(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.

Inhibition of myoblast migration by prostacyclin is associated with enhanced cell fusion.

Satellite cells are stem cells that are critical for the formation and growth of skeletal muscle during myogenesis. To differentiate and fuse, proliferating satellite cells or myoblasts must migrate and establish stable cell-cell contacts. However, the factors that regulate myoblast migration and fusion are not understood completely. We have identified PGI2 as a novel regulator of myogenesis in vitro. PGI2 is a member of the family of prostaglandins (PG), autocrine/paracrine signaling molecules synthesized via the cyclooxygenase-1 and -2 pathways. Primary mouse muscle cells both secrete PGI2 and express the PGI2 receptor, IP, at various stages of myogenesis. Using genetic and pharmacological approaches, we show that PGI2 is a negative regulator of myoblast migration that also enhances cell fusion. Thus, PGI2 may act as a "brake" on migrating cells to facilitate cell-cell contact and fusion. Together, our results highlight the importance of the balance between positive and negative regulators in cell migration and myogenesis. This work may have implications for migration of other populations of adult stem cells and/or cells that undergo fusion.

VKORC1 haplotypes are associated with arterial vascular diseases (stroke, coronary heart disease, and aortic dissection).

BACKGROUND: The haplotypes in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) have been found to affect warfarin dose response through effects on the formation of reduced-form vitamin K, a cofactor for gamma-carboxylation of vitamin K-dependent proteins, which is involved in the coagulation cascade and has a potential impact on atherosclerosis. We hypothesized that VKORC1-dependent effects on the coagulation cascade and atherosclerosis would contribute to susceptibility for vascular diseases. METHODS AND RESULTS: To test the hypothesis, we studied the association of polymorphisms of VKORC1 with stroke (1811 patients), coronary heart disease (740 patients), and aortic dissection (253 patients) compared with matched controls (n=1811, 740, and 416, respectively). Five common noncoding single-nucleotide polymorphisms of VKORC1 were identified in a natural haplotype block with strong linkage disequilibrium (D'>0.9, r2>0.9), then single-nucleotide polymorphism (SNP) +2255 in the block was selected for the association study. We found that the presence of the C allele of the +2255 locus conferred almost twice the risk of vascular disease (odds ratio [OR] 1.95, 95% confidence interval [CI] .58 to 2.41, P<0.001 for stroke; OR 1.72, 95% CI 1.24 to 2.38, P<0.01 for coronary heart disease; and OR 1.90, 95% CI 1.04 to 3.48, P<0.05 for aortic dissection). We also observed that subjects with the CC and CT genotypes had lower levels of undercarboxylated osteocalcin (a regulator for the bone), probably vascular calcification, and lower levels of protein induced in vitamin K absence or antagonism II (PIVKA-II, a des-gamma-carboxy prothrombin) than those with TT genotypes. CONCLUSIONS: The haplotype of VKORC1 may serve as a novel genetic marker for the risk of stroke, coronary heart disease, and aortic dissection.

Curcumin suppresses interleukin 1beta-mediated microsomal prostaglandin E synthase 1 by altering early growth response gene 1 and other signaling pathways.

Curcumin (diferuloylmethane) is one of the phytophenolic compounds found in the turmeric plant with anti-inflammatory and anticarcinogenic activities. One possible mechanism for these activities is the inhibition of prostaglandin (PG) E(2) formation. In this study and other reports, curcumin suppresses interleukin-1beta-induced formation of prostaglandin E(2) in a concentration-dependent manner. Interleukin-1beta-induced microsomal prostaglandin E synthase 1 (mPGES-1) and cyclooxygenase-2 were attenuated by curcumin at the protein and mRNA levels, but a more dramatic inhibition of mPGES-1 expression was observed at lower concentrations of curcumin in A549 human lung epithelial cells. The inhibition of mPGES-1 expression by curcumin shifted the arachidonic acid profile from PGE(2) to PGF(2alpha) and 6-keto-PGF(1alpha) as major metabolites. The expression of early growth response gene 1 (EGR-1), a key transcription factor of cytokine-induced mPGES-1, was inhibited by curcumin. Incubation with siRNA for EGR-1 inhibited interleukin (IL)-1beta-induced mPGES-1, and the controlled expression of EGR-1 increased the mPGES-1 expression. Several proinflammatory signaling molecules, such as nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinases, are also known to affect curcumin-regulated gene expression. Curcumin inhibited IkappaBalpha phosphorylation and degradation and thus reduced the expression of mPGES-1. Curcumin suppressed cytokine-induced mPGES-1 by inhibiting phosphorylation of Jun N-terminal kinase (JNK)1/2. However, EGR-1 expression was suppressed by lower concentrations of curcumin, as compared with JNK1/2 and IkappaBalpha. These results indicate that curcumin inhibits IL-1beta-induced PGE(2) formation by inhibiting the expression of mPGES-1 that is mediated by suppression of EGR-1 expression as well as NF-kappaB and JNK1/2.

15S-Lipoxygenase-2 mediates arachidonic acid-stimulated adhesion of human breast carcinoma cells through the activation of TAK1, MKK6, and p38 MAPK.

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.