Activation of Mitogen-activated Protein Kinases by Arachidonic Acid and Its Metabolites in Vascular Smooth Muscle Cells*

Previous studies from this laboratory and others suggest that arachidonic acid and its metabolites play important roles in a variety of biological processes such as signal transduction, contraction, chemotaxis, and cell growth and differentiation. Here we studied the effect of arachidonic acid on mitogen-activated protein ( M A P ) kinases in vascular smooth muscle cells (VSMC). Arachidonic acid activated MAP kinases in VSMC in a timeand dose-dependent manner. Nordihydroguaiaretic acid (NDGA), a potent inhibitor of the lipoxygenase system, significantly blocked the arachidonic acid-induced activation of MAP kinases, whereas indomethacin, an inhibitor of cyclooxygenase, had no effect. In VSMC, arachidonic acid was converted to 15-hydroxyeicosatetraenoic acid (15-HETE); NDGA inhibited the formation of this HETE. Exogenous addition of 15-HETE to VSMC caused stimulation of MAP kinases. Depletion of protein kinase C attenuated both the arachidonic acidand 15-HETEinduced activation of MAP kinases in VSMC. Together these results suggest that 1) arachidonic acid activates MAP kinases in VSMC; 2) 15-HETE, a 15-lipoxygenase product of arachidonic acid, at least in part, mediates the arachidonic acid effect on MAP kinases; and 3) protein kinase C appears to be important in arachidonic acid activation of MAP kinases. Therefore, MAP kinases may play an important role in arachidonic acid signaling of VSMC growth and function.

acid and its metabolites are critical to a variety of biological processes such as chemotaxis (31, inflammation (1, 21, and signal transduction (4). In addition to these functions, several investigators in recent years have reported that some peptide growth factors such as epidermal growth factor and fibroblast growth factor stimulate the production of arachidonic acid in a number of cell types (5-8). In fact, it is also found that induc-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tion of mitogenesis by these growth factors requires arachidonic acid metabolism (6)(7)(8). Additionally, arachidonic acid metabolites have been found to stimulate growth in many cell types including vascular smooth muscle cells (VSMC) when added exogenously (9)(10)(11)(12). We have previously shown that arachidonic acid induces protooncogene expression in VSMC and mediates oxidant-stimulated expression of these growthresponsive genes in these cells (13,141. Although these findings may suggest an important role for arachidonic acid and its metabolites in cell division, the mitogenic signaling events by these lipid mediators are, however, largely unclear. Mitogen-activated protein ( M A P ) kinases (42""pk, 44""pk) are a group of serinelthreonine kinases which are part of the early activation response to a variety of growth stimuli (15)(16)(17). These kinases are thought to phosphorylate and activate transcription factors such as cjun, c-myc, and p62TcF, which in turn modulate expression of the target genes (18)(19)(20). MAP kinases are activated by phosphorylation on tyrosine and threonine residues by a dual function kinase, MAP kinase kinase (21-23). The MAP kinase kinase is a substrate for Raf-1, a serine/ threonine kinase (24, 25). Raf-1 has been shown to be an integrator of signals received from various pathways including receptor tyrosine kinases (26, 27), G-protein-coupled receptors (28-30), and upstream serinekhreonine kinases such as protein kinase C (PKC) (31, 32).
Proliferation of smooth muscle cells is considered to be a significant factor in the development of atherosclerosis (33). Since arachidonic acid metabolites may be atherogenic (34,351 and arachidonic acid is able to modulate the expression of early growth-responsive genes (13, 141, we hypothesized that arachidonic acid and its metabolites may be involved in the regulation of normal and pathological growth of smooth muscle cells in the vessel wall. Therefore, to understand the mitogenic signaling events by arachidonic acid and its possible role in normal and pathological growth of VSMC, we sought to examine the effect of arachidonic acid on MAP kinases in cultured VSMC. Here, we report for the first time that 1) arachidonic acid activates MAP kinases; 2) X-HETE, a 15-lipoxygenase product of arachidonic acid, appears to account for a portion of the arachidonic acid-induced activation of MAP kinases; and 3) PKC plays a n important role in arachidonic acid activation of MAP kinases in VSMC.
Cell Culture-VSMC from the thoracic aortae of 200-250-8 male Sprague-Dawley rats were isolated and maintained as described previously (13,36). For experiments, cells were plated in 60or 100-mm dishes and made quiescent a t 70430% confluence by incubation in fresh DMEM containing 0.1% calf serum for 48 h. Cells were used between passage numbers 8-18.
Western Blot Analysis-Growth-arrested VSMC (60-mm dishes) were incubated at 37 "C for various times in the presence and absence of 20 p~ arachidonic acid. After incubation, medium was aspirated, and cells were rinsed with cold phosphate-buffered saline and frozen immediately in liquid nitrogen. Cells were then thawed in 250 pl of lysis buffer (50 mM HEPES, pH 7.4, containing 50 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, and 0.01% Triton X-100) and sonicated. Cell lysates were cleared by centrifugation at 14,000 rpm for 20 min in a microcentrifuge. The protein content of the cell lysates was determined using Bio-Rad's Bradford reagent kit. Cell lysates containing equal amounts of proteins (20 pgflane) from each condition were separated by SDSpolyacrylamide gel electrophoresis (37) and transferred electrophoretically to nitrocellulose membranes. The membranes were probed with 1 pg/ml ERK-1 and ERK-2 anti-rabbit primary antibodies. After treating the membrane with peroxidase-conjugated goat anti-rabbit secondary antibodies, peroxidase activity was detected using ECL reagents (Amersham Corp.).
Immunocomplex MAPKinase Assay-cell lysates (250 pl) containing equal amounts of proteins (500 pg) from control and agonist-treated samples were incubated with 10 pl each of ERK-1 ((3-16) and ERK-2 ((2-14) antiserum and 20 p1 of 50% (w/v) protein A-Sepharose beads overnight a t 4 "C. The immunoprecipitates were washed three times with lysis buffer and resuspended in lysis buffer. Reactions were carried out in a final volume of 50 pl containing 50 mM P-glycerophosphate (pH 7.3), 1.5 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol, 10 p~ calmidazolium, 10 mM MgCI,, 10 pg/ml leupeptin, 10 pg/ml aprotinin, 2 pg/ml pepstatin A, 1 mM benzamidine, 0.3 mM [y3'P1ATP, and 0.5 mg/ml MBP for 30 min at 37 "C (16). The reactions were stopped by the addition of an equal volume of 20% cold tricholoroacetic acid. Fifty microliters of the mix were then spotted on a P-81 phosphocellulose filter. The filter was washed four times (5 min each) with 0.5% phosphoric acid and once with absolute ethanol. The filter was dried, and the radioactivity was measured.
Metabolism of Arachidonic Acid-Growth-arrested VSMC (100-mm dishes) were incubated in serum-free DMEM containing 15 pCi/ml [3Hlarachidonic acid (10 JIM final concentration) for 1 h at 37 "C. Where NDGA was used, it was added (10 J~M) 20 min prior to the addition of arachidonic acid. After incubation, radiolabeled arachidonic acid metabolites were extracted from the incubation medium by acidification to pH 3.5 with glacial acetic acid and application of the sample to a CIS-Presep column preconditioned with 10 ml of methanol followed by 10 ml of water. For extraction of cell-associated arachidonic acid metabolites, cells were treated with methanol (2 m1/100-mm dish) after an appropriate period of incubation with ["Hlarachidonic acid. The methanol concentration was then diluted to 10% with water and acidified to pH 3.5 with glacial acetic acid before application to the column. The column was then washed with 10 ml of 1% glacial acetic acid and the fatty acid metabolites were eluted with 5 ml of methanol. Methanol was evaporated to dryness under nitrogen, and samples were reconstituted in 50% methanol (pH 3.5) for analysis by reverse phase HPLC.
HPLC and W Analysis-Reverse phase HPLC analysis was conducted with a C,, Ultrasphere column (5 pm; 4.6 x 250 mm; Altex Scientific, Beckman Instruments, Berkeley, CA) equipped with a Waters (Milford, MA) model U6K injector and a Waters model 6000Apump. Separation of arachidonic acid metabolites was achieved by stepwise elution with a 55-100% methanol gradient (pH 5.05) a t a flow rate of 1.1 mumin as described previously (38). Eluted radioactivity was monitored using a flow-one radioactivity detector (Radiomatic Instruments, Tampa, FL) equipped with a Qume computer (Radiomatic) for data processing. The emuent was also monitored with a Waters model 481 variable wave length detector at 235 nM.
Statistics-Data on kinase activities were presented as means f S.D., and treatment effects were analyzed by Student's t test. p values <0.05 were considered to be significant.

RESULTS AND DISCUSSION
To understand the signal transduction events underlying the arachidonic acid-induced growth-related activities in VSMC, we examined its effect on MAP kinases. Growth-an-ested VSMC were exposed to 20 1.1~ arachidonic acid for varying times and cell extracts were prepared. Cell extracts containing equal amounts of proteins from control and experimental samples were used to test activation of MAP kinases using two approaches. The first approach is based on the slower migration of the phosphorylated and activated form of MAP kinase protein(s) on SDS-polyacrylamide gel electrophoresis compared with the non-phosphorylated inactive form (39). The second approach is the conventional method of determining MAP kinase activity in anti-MAP kinase immunoprecipitates using a specific substrate such as MBP (16). Fig. 1, A and B, shows the rapid and time-dependent activation of MAP kinases by arachidonic acid as determined both by "mobility shift assay" and phosphorylation of MBP. Maximal activation of MAP kinases by arachidonic acid was observed at 5 min, was sustained for 10 min, and then declined thereafter almost reaching basal level at 30 min. Arachidonic acid activated MAP kinases in a dosedependent manner, with a noticeable effect at a concentration of 5 PM and near maximal effect at 20 p~ (Fig. 2, A and B).
To determine if the arachidonic acid effect on MAP kinase activation is specific, we compared the effects of linoleic and stearic acids. Arachidonic and linoleic acid, two commonly occurring polyunsaturated fatty acids which are substrates for the cyclooxygenase and lipoxygenase systems (40-421, were capable of activating MAP kinases, although the former was found to be more potent as compared with the latter (Fig. 3, A and B). As anticipated, stearic acid, a commonly occurring saturated fatty acid which is not a substrate for these enzymes, was found least effective. Use of higher concentrations of these fatty acids (50 p~) also gave results similar to the pattern shown in Fig. 3. This suggests that metabolism of arachidonic acid through the cyclooxygenase or lipoxygenase pathways may be prerequisite for its effect on MAP kinase activation in VSMC.
To address the need for arachidonic acid metabolism, we examined the effects of inhibitors of cyclooxygenase and lipoxygenase on the arachidonic acid activation of MAP kinases.
Growth-arrested VSMC were treated with 20 p~ arachidonic acid for 5 min in the presence and absence of indomethacin ( 10 m) or NDGA (10 w), potent inhibitors of cyclooxygenase and lipoxygenase, respectively (6, 7). Cell lysates were prepared and tested for MAP kinase activation as above. As shown in Fig.  4, A and B, NDGA, which alone had no effect on MAP kinase activation, significantly blocked the arachidonic acid-induced activation of MAP kinases. Indomethacin did not affect arachidonic acid activation of MAP kinases. These results suggest that metabolism of arachidonic acid via the lipoxygenase system, a t least in part, is required for its activation of MAP kinases. To prove that the effect of NDGA on arachidonic acidinduced activation of MAP kinases is due to inhibition of the lipoxygenase system, growth-arrested VSMC were exposed to L3H1arachidonic acid (15 pCi, 10 p~) for 5 min in the presence and absence of NDGA (10 m), and the arachidonic acid me-  (Fig. EA). Earlier studies by others have also shown that the vessel wall can produce these eicosanoids at micromolar levels (35). NDGA significantly inhibited the formation of S H E T E , whereas it did not block the synthesis of PGE, (Fig. 5B).These results indicate that VSMC metabolize arachidonic acid via both the cyclooxygenase and lipoxygenase systems and that NDGA specifically inhibits the lipoxygenase pathway. This finding is also consistent with the above result demonstrating the inhibition of arachidonic acid-induced activation of MAP kinases by NDGA. Note that PGE, synthesis was increased in the presence of NDGA. This could be due to the fact that inhibition of the lipoxygenase pathway results in increased arachidonic acid substrate availability for metabolism by cyclooxygenase. 15-HETE production correlates temporally with that of MAP kinase activation by arachidonic acid. Together these observations suggest that one requirement for activation of MAP kinases by arachidonic acid may be its metabolism through the lipoxygenase system. As 15-HETE appeared to be one of the major lipoxygenase products of arachidonic acid produced in VSMC, we wanted to test whether this product causes activation of MAP kinases.
Growth-arrested VSMC were exposed for 5 min to varying concentrations of 15(S)-HETE and MAP kinase activation was determined. 15(S)-HETE activated MAP kinases in a dosedependent manner (Fig. 6, A and B). The response elicited by 15(S)-HETE was 60% that of arachidonic acid when tested in the same experiment. This suggests that some portion of the arachidonic acid-induced activation of MAP kinases is medi- . a < < < , k k k k k + + 8 5

l-7-[3-hydroxy-4-(4'-iodophenoxy)-l-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic
acid), a thromboxane 4 receptor agonist, activates MAP kinases in pig coronary artery smooth muscle cells. However, a role for thromboxane 4 in arachidonic acid-induced activation of MAP kinases in VSMC in the present scenario appears unlikely because 1) its production was not detected in the cells exposed to [3H]arachidonic acid, and 2) indomethacin had no effect on arachidonic acid-stimulated MAP kinase activation. Therefore, it is possible that some of the MAP kinase effect by arachidonic acid in VSMC may be mediated by arachidonic acid itself or by other metabolites of arachidonic acid whose synthesis is sensitive to NDGA. In fact, in addition to IbHETE, two other NDGA-inhibitable peaks of radioactivity with retention times similar to prostaglandin F, (PGF,)-like compounds (18 min) and Di-HETEs (52 min) were observed in the incubation medium of VSMC exposed to [3H]arachidonic acid. Although an attenuation in the formation of Di-HETEs by NDGA is expected, inhibition of production of PGF,-like compounds by this drug is not clear. Since PGF,-like compounds can be generated by autooxidation of arachidonic acid (44) it is likely that inhibition of their production by NDGA is due to the antioxidant effect of the drug. However, future studies should determine the chemical structure of these compounds and test their ability to activate MAP kinases in VSMC. Earlier studies from several laboratories have demonstrated that arachidonic acid activates PKC (45,46). It has also been shown that PKC agonists such as the tumor promoter, phorbol 12-myristate 13-acetate (PMA), activate MAP kinases in several cell types (31, 47). These observations led us to test whether PKC mediates arachidonic acid-induced activation of MAP kinases in VSMC. PKC was depleted by exposing VSMC to 1 1.1~ phorboll2,13-dibutyrate (PDBu) for 24 h. This regimen was found to be sufficient to deplete cellular PKC levels significantly (>go%), as determined by loss of responsiveness to phorbol ester, in VSMC (48-50). Growth-arrested PKC-depleted VSMC were then exposed to arachidonic acid, PMA (200 nM), or 15-HETE (15 p~) for 5 min, and MAP kinase activity was determined. As shown in Fig. 7, A, B and C, all three agents stimulated MAP kinase activation in VSMC. PDBu pretreatment significantly reduced (70%-80%) the activation of MAP kinases by all three agents. Similar findings were obtained with the PKC inhibitor, staurosporine (data not shown). Together, these results clearly suggest a role for PKC in the activation of MAP kinases by these agents. and that 2) 15-HETE, a 15-lipoxygenase product of arachidonic acid, at least in part, mediates this effect. These results have several implications. First, understanding that MAP kinases are activated by arachidonic acid may strengthen the notion that this fatty acid and its metabolites play an important role in the regulation of VSMC growth. Several findings support this possibility: 1) arachidonic acid stimulates expression of early growth responsive genes such as c-fos and c-jun in VSMC (13,14); 2) arachidonic acid metabolism is required for growth factor-induced cell division in BALB/c 3T3 cells and rat mesangial cells (6, 7); 3) lipoxygenase-dependent metabolites of arachidonic acid induced growth in several cell types including arterial smooth muscle cells and endothelial cells (9-12); 4) inhibitors of the lipoxygenase system blocked growth in several cell types induced by serum (51,52); and 5) MAP kinases appear to be a component common to signaling pathways initiated by a wide range of growth-stimulating factors including mitogens and hormones (15)(16)(17). As arachidonic acid metabolism has been reported to be required for growth factor-induced mitogenesis (6, 7) and arachidonic acid has been shown in the present study to activate MAP kinases, it is likely that arachidonic acid and its metabolites may mediate the growth factorstimulated activation of MAP kinases. Second, MAP kinases may be important in arachidonic acid-modulated vasoactivity. This can be supported by the following facts. Arachidonic acid and its metabolites play a pivotal role in the regulation of vascular smooth muscle cell function such as contraction (53,54); and PKC-dependent MAP kinase activation has been reported to be induced by agonists that cause contraction of smooth muscle cells (55, 56). Third, MAP kinases may be involved in the signaling cascade leading to chemotaxis. This speculation is based on the findings that arachidonic acid and its lipoxygenase-dependent metabolites such as HETEs are chemotactic to VSMC and inflammatory cells (3,571. Fourth, it was recently reported that MAP kinases phosphorylate and activate cytosolic phospholipase 4 in vitro, a rate-limiting en-zyme for arachidonic acid generation (58,591. In this context, it is of interest to note that activation of MAP kinases by arachidonic acid, as demonstrated in the present study, may be involved in the positive feedback regulation of cytosolic phospholipase 4. Regardless of the function of arachidonic acid in which the MAP kinases intervene, the present study demonstrates activation of these signaling kinases by this important fatty acid. It is intriguing that mitogenically responsive lipids such as phosphatidic acid and arachidonic acid inhibit guanosine triphosphatase-activating protein (60). Inhibition of guanosine triphosphatase-activating protein may cause activation of ras by favoring the latter to remain in GTP-bound state. As "ras pathway" is coupled to MAP kinase activation, it is possible that arachidonic acid-induced activation of MAP kinases may be mediated by ras. Some studies have previously shown that PKC activates rus (61), whereas others have demonstrated that rus activates PKC (62). Since the responsiveness of MAP ki-