Activation of Galpha s Mediates Induction of Tissue-type Plasminogen Activator Gene Transcription by Epoxyeicosatrienoic Acids*

Koichi NodeDagger , Xiu-Lu Ruan, Jianwu Dai, Shui-Xiang Yang, LeRae Graham§, Darryl C. Zeldin§, and James K. Liao

From the Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, the Dagger  Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan, and the § Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, January 17, 2001, and in revised form, February 19, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 (CYP) epoxygenases that have vasodilatory and anti-inflammatory properties. Here we report that EETs have additional fibrinolytic properties. In vascular endothelial cells, physiological concentrations of EETs, particularly 11,12-EET, or overexpression of the endothelial epoxygenase, CYP2J2, increased tissue plasminogen activator (t-PA) expression by 2.5-fold without affecting plasminogen activator inhibitor-1 expression. This increase in t-PA expression correlated with a 4-fold induction in t-PA gene transcription and a 3-fold increase in t-PA fibrinolytic activity and was blocked by the CYP inhibitor, SKF525A, but not by the calcium-activated potassium channel blocker, charybdotoxin, indicating a mechanism that does not involve endothelial cell hyperpolarization. The t-PA promoter is cAMP-responsive, and induction of t-PA gene transcription by EETs correlated with increases in intracellular cAMP levels and, functionally, with cAMP-driven promoter activity. To determine whether increases in intracellular cAMP levels were due to modulation of guanine nucleotide-binding proteins, we assessed the effects of EETs on Galpha s and Galpha i2. Treatment with EETs increased Galpha s, but not Galpha i2, GTP-binding activity by 3.5-fold. These findings indicate that EETs possess fibrinolytic properties through the induction of t-PA and suggest that endothelial CYP2J2 may play an important role in regulating vascular hemostasis.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The epoxyeicosatrienoic acids (EETs)1 are important vasoactive products of cytochrome P450 epoxygenases (1-3). The EETs have properties similar to those of endothelium-derived hyperpolarizing factor because they hyperpolarize and relax vascular smooth muscle cells by activating calcium-sensitive potassium (KCa2+) channels (4, 5). Recently, two CYP epoxygenases, CYP2C8 and CYP2J2, have been identified in vascular endothelial cells (1, 6). Although both enzymes produce EETs, the regulation of CYP2C8 and CYP2J2 appears to be somewhat different, and their products may exert additional nonvasodilatory effects. For example, Fisslthaler et al. (1) have shown that treatment of porcine coronary artery endothelial cells in vitro with beta -naphthoflavone induces CYP2C8 expression, increases 11,12-EET biosynthesis, and enhances endothelium-derived hyperpolarizing factor-mediated coronary artery relaxation. However, beta -naphthoflavone does not induce the CYP2J subfamily enzymes (7). Furthermore, we have recently reported that EETs possess anti-inflammatory properties through the inhibition of the proinflammatory transcriptional factor, NF-kappa B (6). In contrast to their vasodilatory effects, the anti-inflammatory effects of EETs do not involve endothelial cell hyperpolarization. These studies, therefore, indicate that EETs have multiple homeostatic effects on the vascular wall in addition to their vasodilatory effects.

An interesting observation is that endogenous vasodilators such as nitric oxide (NO) frequently possess anti-inflammatory (8), antithrombotic (9), and antiproliferative properties (10). Indeed, vasoconstriction, smooth muscle cell proliferation, inflammation, and thrombosis are hallmarks of atherosclerotic vascular disease (11). In particular, the rupture of atherosclerotic plaques with ensuing occlusive thrombosis is the predominant feature underlying acute coronary syndromes (12). Thus, it is possible that EETs may play an additional role in the regulation of vascular hemostasis. Vascular thrombosis is regulated, in part, by endogenous mediators such as the proteolytic enzyme, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor (PAI)-1 (13). The function of t-PA is to convert plasminogen to a proteolytic enzyme, plasmin, which digests fibrin-dependent blood clots. Thus, thrombotic vascular complications may be attenuated by agents that increase the secretion of t-PA and/or decrease the expression of PAI-1. Indeed, the plasma level of t-PA has been shown to be inversely correlated with the risk of myocardial infarction (14). The purpose of this study, therefore, was to determine whether EETs could regulate t-PA or PAI-1 expression and, if so, by what mechanism.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Reagents-- All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Ascorbic acid, creatinine phosphate, phosphocreatine kinase, GTPgamma S, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, dithiothreitol, bovine serum albumin, ATP, GDP, GTP, charybdotoxin, H-89, and SKF525A were purchased from Sigma. Triphenylphosphine, alpha -bromo-2,3,4,5,6-pentafluorotoluene, N,N-diisopropylethylamine, and N,N-dimethylformamide were purchased from Aldrich. [1-14C]arachidonic acid, [gamma -32P]GTP (30 Ci/mmol), [35S]GTPgamma S (1250 Ci/mmol), and [alpha -32P]CTP (3000 Ci/mmol), and the polyclonal rabbit antiserum to Galpha s (RM/1) were supplied by PerkinElmer Life Sciences. The polyclonal rabbit antiserum P4 was raised against a decapeptide corresponding to the COOH-terminal region of Galpha i2 and has been characterized previously (15). Protein molecular weight markers were purchased from Life Technologies, Inc. The chemiluminescence detection kit was obtained from Amersham Pharmacia Biotech. The polyvinylidene difluoride transfer membrane (pore size 0.2 µm) was obtained from Bio-Rad. The heterologous 4×-CRE promoter luciferase reporter construct (pCRE-Luc) was purchased from Stratagene (San Diego, CA).

Cell Culture-- Endothelial cells were harvested from human saphenous veins and bovine aortas as described (8, 15). The cells were cultured at 37 °C in a growth medium containing Dulbecco's modified Eagle's medium) supplemented with 5 mM L-glutamine, 10% fetal calf serum (Hyclone, Logan, UT), and an antibiotic mixture of penicillin (100 units/ml), streptomycin (100 mg/ml), and Fungizone (250 ng/ml). Relatively pure (>95%) endothelial cell cultures were confirmed by Nomarski optical microscopy (Olympus IX70, × 40 objective) and by immunofluorescence staining with anti-factor VIII antibodies (Vector Laboratories, Inc., Burlingame, CA). All passages were performed with a disposable cell scraper (Costar), and only endothelial cells of less than six passages were used. Confluent endothelial cells (~2 × 106 for human and ~5 × 106 for bovine) were treated with various concentrations of EETs for the indicated time intervals.

Measurements of EETs-- The method used to quantify endogenous EETs present in bovine aortic endothelial cells was similar to those used to quantify EETs in tissue preparations (7, 16, 17). Briefly, 8 × 106 endothelial cells were frozen in liquid nitrogen and homogenized in 15 ml of phosphate-buffered saline containing triphenylphosphine (10 mg), and the homogenate was extracted under acidic conditions with two volumes of chloroform/methanol (2:1) and twice more with an equal volume of chloroform. The combined organic phases were evaporated in tubes containing mixtures of 1-14C-labeled 8,9-, 11,12-, and 14,15-EET internal standards (57 µCi/µmol; 30 ng each), which were synthesized from [1-14C]arachidonic acid by nonselective epoxidation (18). Saponification to recover phospholipid-bound EETs was followed by silica column purification. The eluent, containing a mixture of radiolabeled internal standards and total endogenous EETs, was resolved into individual regioisomers by HPLC as described (7, 16, 17). For analysis, aliquots of individual EET-PFB esters were dissolved in dodecane and analyzed by gas chromatography/mass spectrometry as described (7, 16, 17). Quantifications were made by selected ion monitoring at m/z 319 (loss of PFB from endogenous EET-PFB) and m/z 321 (loss of PFB from [1-14C]EET-PFB internal standard). The EET-PFB/[1-14C]EET-PFB ratios were calculated from the integrated values of the corresponding ion current intensities.

Measurement of t-PA Expression and Activity-- The amounts of t-PA and PAI-I antigens expressed by endothelial cells were determined using the ImulyseTM reagent kits from Biopool (Umea, Sweden) according to the manufacturer's instructions. The t-PA activity in endothelial cell supernatants was determined by a chromogenic assay (Spectrolyse/Fibrin; Biopool). The t-PA in the supernatant from conditioned cells was bound using a specific t-PA monoclonal antibody (SP-322). Preliminary studies indicate that the SP-322 antibody completely bound all of the t-PA without affecting subsequent t-PA activity. The t-PA substrate consisting of plasminogen, plasmin-sensitive chromogenic substrate, and co-factors for t-PA activity (HEPES buffer, pH 8.5) were added to the bound t-PA, and absorption was read at 405 nm. The standard curve was constructed using human single-chain t-PA, which has been calibrated against an international standard for t-PA (Lot 66570; NIBSC, Hartfordshire, UK).

Northern Blotting-- Equal amounts of total RNA (20 µg) were separated by 1.2% formaldehyde-agarose gel electrophoresis, transferred overnight onto Hybond nylon membranes (Amersham Pharmacia Biotech) by capillary action, and baked for 2 h at 80 °C before prehybridization. Radiolabeling of full-length human t-PA or PAI-1 cDNA was performed using random hexamer priming, [alpha -32P]CTP, and Klenow (Amersham Pharmacia Biotech). The membranes were hybridized with the indicated probes overnight at 45 °C in a solution containing 50% formamide, 5× SSC (1× SSC: 0.15 M NaCl and 0.015 M sodium citrate), 2.5× Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 µg/ml salmon sperm DNA. All Northern blots were subjected to stringent washing conditions (0.2× SSC, 0.1% SDS at 65 °C) before autoradiography with intensifying screen at 80 °C for 24-72 h. Equalization of RNA loading was assessed by ethidium bromide staining of 18 S ribosomal RNA.

[35S]GTPgamma S Binding Assay-- Partially purified membranes were prepared from endothelial cells as previously described (19). Membrane proteins (30 µg) from control and EET-treated endothelial cells were incubated for 30 min at 30 °C in a buffer containing [35S]GTPgamma S (20 nM), GTP (2 µM), MgCl2 (5 mM), EGTA (0.1 mM), NaCl (50 mM), creatine phosphate (4 mM), phosphocreatine kinase (5 units), ATP (0.1 mM), dithiothreitol (1 mM), leupeptin (100 µg/ml), aprotinin (50 µg/ml), bovine serum albumin (0.2%), and triethanolamine HCl (50 mM, pH 7.4). In some samples, membrane was stimulated with the alpha 2-adrenergic agonist, UK14304 (10 nM), for 10 min. The assay was terminated after 30 min with excess unlabeled GTPgamma S (100 µM). Samples were then resuspended in 100 µl of immunoprecipitation buffer containing Triton X-100 (1%), SDS (0.1%), NaCl (150 mM), EDTA (5 mM), Tris-HCl (25 mM, pH 7.4), leupeptin (10 µg), aprotinin (10 µg), and phenylmethylsulfonyl fluoride (2 mM).

The following G protein antisera with their corresponding final dilutions were added to the mixture: Galpha i2 (P4, 1:20) and Galpha s (RM/1, 1:100). The samples were allowed to incubate for 16 h at 4 °C with gentle mixing. The antibody-G protein complexes were then incubated with 50 µl of protein A-Sepharose (1 mg/ml; Amersham Pharmacia Biotech) for 2 h at 4 °C, and the precipitate was collected by centrifugation at 12,000 × g for 10 min. Preliminary studies indicated that all Galpha i2 and Galpha s were completely precipitated by this procedure because Western blot analysis of the supernatant with the P4 and RM/1 antisera did not reveal their presence. The pellets were washed three times in a buffer containing HEPES (50 mM, pH 7.4), NaF (100 µM), sodium phosphate (50 mM), NaCl (100 mM), Triton X-100 (1%), and SDS (0.1%). The final pellet containing the immunoprecipitated [35S]GTPgamma S-labeled G protein was counted in a liquid scintillation counter (LS 1800; Beckman Instruments, Inc.). Nonspecific activity was determined in the presence of excess unlabeled GTPgamma S (10 µM).

Measurement of Intracellular cAMP Levels-- Confluent endothelial cells were pretreated with 3-isobutyl-1-methyl-xanthine (0.5 mM) for 30 min prior to stimulation with the indicated concentrations of EETs for 5 min. Cells were scraped on ice, pelleted, and resuspended in ice-cold 3-isobutyl-1-methyl-xanthine (0.5 mM), boiled for 3 min, and frozen at -70 °C. The intracellular cAMP level was determined using a radioimmunoassay kit with radiolabeled cAMP (Amersham Pharmacia Biotech). A standard curve was constructed using increasing amounts of unlabeled cAMP.

Transfection Studies-- The human t-PA promoter was obtained by the standard PCR method using a human genomic library (Invitrogen) as template. The following paired primers were used: sense, 5'-CGATCGGTACTTTCGGGATGATTCAAGAGGATTAC-3'; antisense, 5'-CGATCAGATCTGAAAGAAGAGGAGACAGACCCCAAG-3'. The PCR product (3.6 kilobase pairs) containing a 3.1-kilobase pair t-PA promoter was obtained using the following PCR conditions (35 cycles): annealing at 48 °C for 2 min, elongation at 68 °C for 4 min, and denaturing at 94 °C for 30 s. The t-PA promoter was verified by sequence analysis and corresponded exactly to the already published 5'-flanking sequence of the t-PA gene (20). The t-PA promoter was then subcloned into a luciferase reporter construct, pGL.2 (Promega, Madison, WI). For cAMP-driven promoter activity, a heterologous promoter (pCRE-Luc, Stratagene) containing four tandem cAMP-response element (4×-CRE) linked to the luciferase reporter gene was used to assess cAMP-dependent gene transcription. The pcDNA3.1/CYP2J2 expression plasmid was prepared as described (6).

Transient transfections in bovine aortic endothelial cells were accomplished using the calcium phosphate precipitation method as described (8). Preliminary studies with beta -galactosidase staining indicate that the transfection efficiency was 12-15%. Bovine endothelial cells (60-70% confluent) were transfected with 5 µg of either pt-PA-Luc or pCRE-Luc and 1 µg of pCMV.beta -galactosidase construct. Approximately 48 h after transfection, endothelial cells were treated with 11,12-EET and the protein kinase A (PKA) inhibitor, H-89, alone or in combination, for 12 h. The luciferase and beta -galactosidase activities were determined by chemiluminescence (Dual-Light, Tropix, Bedford, MA) using a Berthold L9501 luminometer.

Data Analysis-- All values are expressed as mean ± S.E. compared with controls and among separate experiments. Paired and unpaired Student's t tests were employed to determine the significance of changes in GTP binding and luciferase activities. A significant difference was taken for p values of less than 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cell Culture-- There were no observable effects of synthetic EETs or 11,12-DHET, the hydration metabolite of 11,12-EET, on cell number, morphology, or immunofluorescent staining for factor VIII-related antigens. Cellular confluence and viability as determined by trypan blue exclusion was maintained for all treatment conditions described.

Endothelial Arachidonic Acid Metabolism and Detection of Endogenous EETs-- We have previously shown that bovine endothelial cells metabolize radiolabeled arachidonic acid to epoxygenase products (EETs and DHETs) and that endothelial EET biosynthesis is augmented by transfection with an expression vector containing the CYP2J2 cDNA (6). Using a combination of HPLC and gas chromatography/mass spectrometry techniques, we detected EETs as endogenous constituents of cultured bovine aortic endothelial cells (Table I). Endothelial cells contained 7.98 ± 0.90 ng of EET per 107 cells. The 8,9-, 11,12-, and 14,15-EET regioisomers were each present in roughly equal amounts (40, 28, and 32% of total EETs, respectively). The labile 5,6-EET suffers extensive decomposition during the extraction and purification process used and therefore cannot be quantified. Transfection of endothelial cells with the pcDNA3.1 vector containing the CYP2J2 cDNA resulted in a significant 30% increase in total EETs that was due largely due to increases in 11,12- and 8,9-EET (Table I). Treatment of CYP2J2-transfected cells with SKF525A (100 µM) significantly reduced total EETs to control levels. The documentation of EETs in endothelial cells provides direct evidence to support the epoxidation of endogenous arachidonic acid pools by endothelial cytochrome P450s.

                              
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Table I
Endogenous EETs in control and CYP2J2-transfected endothelial cells
Control bovine endothelial cells, endothelial cells transfected with pcDNA3.1/CYP2J2, or endothelial cells transfected with pcDNA3.1/CYP2J2 and treated with SKF525A were used to quantify endogenous 14,15-EET, 11,12-EET, and 8,9-EET as described under "Experimental Procedures." Concentration values (ng of EET/107 cells) are averages of 3-8 determinations and are expressed as mean ± S.E. *, p < 0.05 by analysis of variance.

Effects of EETs on t-PA Expression and Activity-- In a concentration-dependent manner, treatment of endothelial cells with physiologically relevant concentrations of 5,6-, 8,9-, 11,12-, 14,15-EET, or 11,12-DHET (1-1000 nM, 24 h) increased t-PA protein levels (Fig. 1A). Maximal increase was achieved with 11,12-EET at 100 nM. Interestingly, 14,15- and 8,9-EET induced significantly less t-PA expression than 11,12-EET, indicating differential bioactivity of specific EET regioisomers. The increase in t-PA protein levels by 11,12-EET correlated with increases in t-PA steady-state mRNA levels (Fig. 1B). Concentrations of 10 and 100 nM 11,12-EET increased t-PA mRNA levels by 40 ± 5 and 95 ± 6%, respectively (p < 0.05 for both, n = 3). Consistent with effects on t-PA protein levels, a higher concentration of 11,12-EET (i.e. 1000 nM) also did not produce any further increase in t-PA mRNA levels. Interestingly, the increase in t-PA mRNA expression was not due to the membrane hyperpolarizing effects of EETs, since the selective KCa2+ blocker, charybdotoxin (100 nM), was unable to inhibit the increase in t-PA expression by 11,12-EET.


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Fig. 1.   A, cell surface enzyme immunoassay showing the effects of EET regioisomers and 11,12-DHET (1-1000 nM) on t-PA expression (percentage of basal level). The differences between treatment with and without EETs or 11,12-DHET were statistically significant compared with untreated controls (*, p < 0.05; **, p < 0.01). B, representative Northern analyses (20 µg of total RNA/lane) showing the effects of 11,12-EET (10-100 nM) with and without charybdotoxin (100 nM) on endothelial t-PA mRNA levels at 12 h. Equal loading conditions were determined by the corresponding ethidium bromide-stained Northern blot for 18 S ribosomal RNA. Each experiment was performed three times with similar results.

The increase in t-PA expression by 11,12-EET correlated with a 2.8-fold increase in t-PA fibrinolytic activity (Fig. 2A). Maximal t-PA fibrinolytic activity was observed at 100 nM 11,12-EET. However, 10 nM 11,12-EET, which produced a smaller increase in t-PA expression compared with that of 100 nM, did not produce significant increases in t-PA fibrinolytic activity. This may be due to endogenous levels of PAI-1, which is known to bind and inhibit t-PA (13). However, in contrast to t-PA, EETs have no effect on PAI-I protein levels (Fig. 2B). These findings suggest that treatment with EETs would favor fibrinolysis by increasing the ratio of t-PA to PAI-1.


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Fig. 2.   A, effect of 11,12-EET (10-1000 nM) on endothelial t-PA activity (IU/ml). The differences between treatment with and without 11,12-EET were statistically significant (*, p < 0.05). B, PAI-1 expression (percentage of basal level) in human endothelial cells treated with EETs or DHET (1-1000 nM).

In a time-dependent manner, 11,12-EET (100 nM) increased t-PA protein levels with maximal increase occurring after 24 h of exposure (Fig. 3A). This increase in t-PA expression was attenuated by co-treatment with the translational inhibitor, cycloheximide (CHX; 5 µmol/liter), indicating that 11,12-EET does not promote the release of already synthesized t-PA but that de novo protein synthesis was required. Since t-PA expression is known to be induced by agents or conditions that increase cAMP-dependent pathways (21-23), we investigated whether the induction of t-PA expression by EETs was mediated by the cAMP-dependent kinase such as PKA. Co-treatment with the PKA inhibitor, H-89 (30 µM), almost completely inhibited EET-induced t-PA expression (Fig. 3B). Again, the selective KCa2+ blocker, charybdotoxin (100 nM), was unable to inhibit the increase in t-PA expression by 11,12-EET, suggesting a nonhyperpolarizing effect of EETs on t-PA expression.


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Fig. 3.   A, time-dependent effect of 11,12-EET (100 nM) on t-PA expression (percentage of basal level) with or without cycloheximide (CHX; 5 µg/ml). *, p < 0.05 versus control (time 0); #, p < 0.05 versus 11,12-EET. B, effect of PKA inhibitor, H-89 (30 µM) and KCa2+ channel blocker, charybdotoxin (100 nM), on increases in t-PA expression by 11,12-EET. *, p < 0.05 versus control; #, p < 0.05 versus 11,12-EET.

Effects of EETs and CYP2J2 on t-PA Gene Transcription-- Using a 3.1-kilobase pair human t-PA promoter linked to a luciferase reporter (pt-PA.Luc), we found that 11,12-EET and, to a lesser extent, 11,12-DHET increased t-PA promoter activity in a concentration-dependent manner (Fig. 4A). A maximal 4.3-fold increase in t-PA promoter activity by 11,12-EET occurred at a concentration of 100 nM with an EC50 value of ~10 nM. To determine whether overexpression of the endothelial cytochrome P450 isoform, CYP2J2, which produces EETs, can increase t-PA gene transcription, endothelial cells were transiently co-transfected with pcDNA3.1/CYP2J2 cDNA. We have previously shown that, compared with endothelial cells transfected with the empty vector, CYP2J2-transfected endothelial cells produce epoxygenase metabolites at a 2-3-fold higher rate and that this increase was almost completely inhibited by SKF525A (6). Compared with transfection with the empty vector (pcDNA3.1), transfection with CYP2J2 caused a 5.2-fold induction in t-PA promoter activity, which was almost completely blocked by the nonspecific cytochrome P450 epoxygenase inhibitor, SKF525A (Fig. 4B). The KCa2+ blocker charybdotoxin, however, had no effect on EET-induced t-PA promoter activity. These findings suggest that CYP2J2-derived eicosanoids increase t-PA gene transcription.


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Fig. 4.   A, concentration-dependent effect of 11,12-EET and 11,12-DHET (1-1000 nM) on t-PA promoter activity at 12 h. Transfection efficiency was standardized to beta -galactosidase activity, and changes in the ratio of t-PA promoter activity to beta -galactosidase activity were calculated relative to basal activity (-fold induction). (*, p < 0.05; **, p < 0.01 versus basal). B, effects of transfection with empty vector (pcDNA3.1) or pcDNA3.1/CYP2J2 on t-PA promoter activity in the presence or absence of cytochrome P450 epoxygenase inhibitor, SKF-525A (100 µM) or charybdotoxin (100 nM). *, p < 0.05 versus pcDNA3.1; #, p < 0.05 versus CYP2J2-transfected cells.

Effects of EETs and CYP2J2 on cAMP-dependent Gene Transcription-- The induction of t-PA gene transcription is regulated, in part, by increases in intracellular cAMP (21-23). We found that EETs, particularly 11,12-EET, increased intracellular cAMP levels in a concentration-dependent manner (Fig. 5A). Treatment with 11,12-EET (100 nM) produced a 3-fold increase in intracellular cAMP levels (3.1 ± 0.2 to 10.2 ± 0.7 pmol/500,000 cells; p < 0.01, n = 3). To address the question of whether the increase in intracellular cAMP by 11,12-EET can lead to transactivation of cAMP-response element (CRE)-containing promoters, we transfected endothelial cells with a heterologous luciferase reporter construct containing four tandem CREs (pCRE-Luc). Treatment of transfected endothelial cells with 11,12-EET (1-1000 nM, 12 h) increased pCRE-Luc activity by up to 6.8-fold (p < 0.01, n = 4), with maximal effect occurring at a concentration of 100 nM (Fig. 5B). These findings suggest that increases in intracellular cAMP levels and induction of cAMP-responsive trans-acting factors mediate increases in t-PA expression by EETs.


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Fig. 5.   A, effects of EETs and DHET (1-1000 nM) on intracellular cAMP levels at 5 min. *, p < 0.05; **, p < 0.01 versus control. B, concentration-dependent effect of 11,12-EET on CRE promoter activity. Transfection efficiency was standardized to beta -galactosidase activity, and changes in the ratio of t-PA promoter activity to beta -galactosidase activity were calculated relative to basal activity (-fold induction). (*, p < 0.05; **, p < 0.01 versus no treatment).

In support of the role of cAMP in CRE-mediated gene transcription, we found that the increase in pCRE-Luc activity by 11,12-EET (100 nM) was inhibited in a time-dependent manner by inhibition of the cAMP-dependent kinase, PKA (Fig. 6A). Treatment with the PKA inhibitor, H-89, decreased 11,12-EET-induced CRE promoter activity by 70% after 24 h. To determine whether endogenous EETs produced by CYP2J2 can increase CRE promoter activity, we co-transfected endothelial cells with pcDNA3.1/CYP2J2. Compared with transfection with the empty pcDNA3.1 vector, overexpression of pcDNA3.1/CYP2J2 increased CRE promoter activity by 7.3-fold after 12 h (Fig. 6B). This effect was almost completely blocked in the presence of the cytochrome P450 inhibitor, SKF525A. These findings suggest that CYP2J2-derived eicosanoids can functionally increase CRE promoter activity.


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Fig. 6.   Time-dependent effect (0-24 h) of 11,12-EET on CRE promoter activity (-fold induction) in the presence or absence of the PKA inhibitor, H-89 (30 µM). *, p < 0.05; **, p < 0.01 versus control (time 0). #, p < 0.05 versus 11,12-EET. B, the effect of CYP2J2 transfection with and without cytochrome P450 epoxygenase inhibitor, SKF525A, on CRE promoter activity (-fold induction). *, p < 0.01 versus pcDNA3.1; #, p < 0.05 versus 11,12-EET.

Effects of EETs on G Proteins-- To determine the whether the increase in cAMP is due to the effects of EETs on G proteins, we measured Galpha i2 and Galpha s GTP binding activities in membranes from endothelial cells treated with EETs. Treatment with 11,12-EET (10-1000 nM, 5 min) caused a progressive increase in Galpha s GTP-binding activity with a maximal 3-fold increase (6.1 ± 2.1 to 18.5 ± 1.2 fmol/min/mg, p < 0.01, n = 3) occurring at a concentration of 100 nM (Fig. 7A). In contrast, the same membrane from 11,12-EET-treated cells did not demonstrate any change in basal Galpha i2 GTP-binding activity. UK14304 stimulated Galpha i2 GTP-binding activity by ~7-fold (2.8 ± 0.1 to 19 ± 3 fmol/min/mg, p < 0.01, n = 3), which was unaffected by 11,12-EET treatment. Other EET regioisomers such as 5,6-EET, 14,15-EET, and 8,9-EET also increased Galpha s GTP-binding activity, albeit to a lesser extent compared with 11,12-EET, without affecting Galpha i2 activity (Fig. 7B). These findings suggest that the effects of EETs on cAMP levels, CRE promoter activity, and t-PA expression/activity are probably mediated by their stimulatory effects on Galpha s.


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Fig. 7.   Concentration-dependent effect of 11,12-EET (A) or EET regioisomers (B) (100 nM) on specific G protein activity as determined by immunoprecipitation of [35S]GTPgamma S labeling of Galpha s and UK14304 (10 nM)-stimulated [35S]GTPgamma S labeling of Galpha i2 at 5 min. *, p < 0.01 versus control (Cont; untreated).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

We have shown that EETs, particularly 11,12-EET, increase endothelial t-PA expression and activity by a transcriptional mechanism involving activation of Galpha s. The activation of Galpha s leads to the stimulation of adenylyl cyclase, increase in intracellular cAMP, and induction of cAMP-dependent promoter activity. The effect of EETs on Galpha s activity is rather specific, since EETs did not affect basal or UK14304-stimulated Galpha i2 activity. Because Galpha i2 is specifically coupled to UK14304-stimulated alpha 2-adrenergic receptor in vascular endothelial cells (24), these results suggest that EETs selectively modulate Galpha s activity. The net effect of EETs on G proteins, therefore, is to favor receptor-mediated activation of adenylyl cyclase. Indeed, inhibition of cAMP-dependent PKA inhibits EET-induced t-PA expression and CRE promoter activity. These findings are in agreement with previous studies that showed that arteriolar vasodilation by sulfonimide analogs of 11,12-EET analogs is associated with elevation in intracellular cAMP levels and increase in PKA activity (25).

The precise mechanism by which EETs alter membrane signal transduction and increase t-PA expression is not known. Previous studies have shown that 11,12-EET stimulates the ADP-ribosylation of a number of intracellular proteins in vascular smooth muscle including Galpha s (26, 27). Indeed, the binding of 14,15-EET to its putative receptor in U937 cells leads to the increase in cAMP levels and PKA activity and subsequently to a decrease in its receptor binding (28). Thus, it is possible that EET-induced ADP-ribosylation of Galpha s results in the activation of Galpha s similar to that of cholera toxin. Although we cannot exclude the additional possibility that EETs may induce similar changes in other G proteins, these effects of EETs are unlikely given that Galpha i2 activity was not affected. It is also possible that EETs may induce changes in the level of Galpha s and Galpha i2. However, under the conditions of our study, the observed changes in Galpha s activity and cAMP levels occurred within 5 min; which is somewhat rapid for a transcriptional effect on G proteins. Nevertheless, the downstream effect of cAMP is dependent upon de novo protein synthesis, since the protein synthesis inhibitor, cycloheximide, was able to attenuate EET-induced t-PA expression.

The regulation of gene transcription by cAMP is mediated by trans-acting factors, which bind to cAMP-response element (CRE) (5'-ATGACGTCAT-3') of target genes (29). Although the consensus sequence for CRE is not present in the t-PA promoter, a functional CRE-like element has been identified, which acts synergistically with a putative AP-2 binding site to induced t-PA gene transcription by phorbol 12-myristate 13-acetate (22). Indeed, previous studies have shown that cholera toxin and dibutyryl-cAMP can directly induce t-PA gene transcription in rat hepatocytes (30, 31). Furthermore, we have previously shown that low concentrations of alcohol enhance isoproterenol-induced t-PA gene transcription via increase in cAMP levels and induction of CRE promoter activity in vascular endothelial cells (32). These studies, therefore, suggest that the activation of CRE and CRE-binding protein(s) may play an important role in mediating the induction of t-PA by EETs.

The vasodilatory effect of EETs, which resemble endothelium-derived hyperpolarizing factor, is mediated by activation of KCa2+ channels and subsequent hyperpolarization of vascular smooth muscle membranes (4, 5). The anti-inflammatory effect of EETs involves inhibition of cellular adhesion molecule expression via inhibition of NF-kappa B activation (6). The antiproliferative effect of EETs has not been reported, and we do not find that 11,12-EET inhibits platelet-derived growth factor-induced smooth muscle cell or fibroblast proliferation.2 Our study showed that EETs possess anti-fibrinolytic properties through the induction of t-PA expression. By linking EETs to the fibrinolytic pathway, another important nonvasodilatory action of EETs is suggested, and the homeostatic role of EETs in the vascular wall is broadened considerably because thrombosis is implicated in vascular occlusive disease and atherosclerosis (11, 12). In this respect, EETs are similar to endothelium-derived NO, which relaxes vascular smooth muscle (33), inhibits NF-kappa B (8), and prevents platelet aggregation (9). The mechanisms by which EETs exert these effects, however, are quite distinct from that of NO, which suggests that cAMP- and cGMP-dependent pathways may work in concert to regulate vascular hemostasis.

The finding that EET induces t-PA may have important clinical implications, since the etiology of acute coronary syndromes is thought to be due to atherosclerotic plaque rupture with ensuing vascular thrombosis and occlusion (11, 12). Our results, therefore, suggest that EETs may lower the risks of cardiovascular disease by augmenting t-PA levels and enhancing the overall fibrinolytic activity in the vascular wall. However, in contrast to the fibrinolytic effect of EETs, other cytochrome P450-derived eicosanoids have been shown to have opposing effects. For example, 15-hydroxyeicosatrienoic acid, an arachidonic acid metabolite that is produced via oxidation and ketoreduction, has been shown to decrease t-PA expression (34). The concentrations of EETs used in our study are within the range of physiological concentrations of EETs measured in the bloodstream of healthy humans and rats, which is ~30 nM (16). However, it should be noted that EET levels in human and rat heart tissues are estimated to be 6-7-fold higher (35) and may be even higher during vascular injury and inflammation.

In summary, our findings suggest that EETs may play an important role in regulating the fibrinolytic balance in the vessel wall. The potency of EETs' anti-fibrinolytic effects suggests that the biological actions of these cytochrome P450-derived eicosanoids, like those of the prostaglandins and leukotrienes, may be mediated through specific cell surface receptors. The identification and functional characterization of putative EET receptor(s) would greatly aid future research in this field.

    ACKNOWLEDGEMENTS

We thank A. Jetton and T. Murphy for critical reading of the manuscript, J. R. Falck for EET and DHET standards, and S. Degen and D. Luskatoff for human t-PA and PAI-1 cDNAs.

    FOOTNOTES

* Supported by National Institutes of Health (NIH) Grants HL-52233 and HL-48743 and the NIEHS (NIH) Division of Intramural Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Division, Brigham & Women's Hospital, 221 Longwood Ave., LMRC-322, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-264-6336; E-mail: jliao@rics.bwh.harvard.edu.

Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M100439200

2 K. Node, X.-L. Ruan, J. Dai, S.-X. Yang, L. Graham, D. C. Zeldin, and J. K. Liao, unpublished data.

    ABBREVIATIONS

The abbreviations used are: EET, epoxyeicosatrienoic acid; KCa2+, channel, calcium-activated potassium channel; CYP, cytochrome P450; DHET, dihydroxyeicosatrienoic acid; t-PA, tissue-type plasminogen activator; PAI, plasminogen activator inhibitor; CRE, cAMP-response element; GTPgamma S, guanosine 5'-gamma -thiotriphosphate; PKA, protein kinase A; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; PFB, pentafluorobenzyl.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.