20-Hydroxyeicosatetraenoic acid mediates ... - Semantic Scholar

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12701–12706, October 1998 Pharmacology

20-Hydroxyeicosatetraenoic acid mediates calciumycalmodulindependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells M. M. MUTHALIF, I. F. BENTER, N. KARZOUN, S. FATIMA, J. HARPER, M. R. UDDIN,

AND

K. U. MALIK*

Department of Pharmacology, College of Medicine, The University of Tennessee Center for Health Sciences, Memphis, TN 38163

Communicated by Philip Needleman, Monsanto Company, St. Louis, MO, August 19, 1998 (received for review February 18, 1998)

subunits of heterotrimeric G proteins (18, 19). However, the mechanism by which CaMK activates MAPK is not known. AA is metabolized by cyclooxygenase into prostaglandins and thromboxane A2, by lipoxygenase into leukotrienes and hydroxyeicosatetraenoic acids (HETEs) [5-, 12(S)-, and 15-HETE], and by cytochrome P450 (CYP450) into epoxyeicosatrienoic acid and 12(R),19- and 20-HETE (1, 3). AA and some of its lipoxygenase products (12- and 15-HETE) stimulate MAPK activity (20–22). Moreover, 5-HETE has been shown to increase cPLA2 activity in human neutrophils (23). These observations led us to hypothesize that the NE-induced increase in MAPK activity is caused by AA or its metabolites generated through activation of cPLA2 by CaMKII. To test this hypothesis, we investigated the effects of NE, Ang II, and epidermal growth factor (EGF) on Ras, MAPK, CaMKII, and cPLA2 activity in the presence and absence of inhibitors of CYP450, lipoxygenase, and cyclooxygenase in rabbit aortic VSMCs. We have found that NE, Ang II, and EGF activate the RasyMAPK pathway through generation of a CYP450 metabolite of AA, 20-HETE, after initial activation of cPLA2 by CaMKII. Activation of MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism.

ABSTRACT Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca21 influx, increase Ca21ycalmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA2 were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA2 and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA2 by CaMKII. Activation of RasyMAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism. This mechanism of RasyMAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

MATERIALS AND METHODS Preparation of VSMCs. Aortae were rapidly removed from male New Zealand White rabbits and the VSMCs were isolated as described (24). Cells between passages 2 and 8 were plated in 12- or 24-well plates or 100-mm plates. Cells were maintained under 5% CO2 in M-199 medium (Sigma) with penicillin, streptomycin, and 10% fetal bovine serum. Experimental Protocol. VSMCs that were arrested for 48 h with medium containing 0.05% fetal bovine serum were used for all studies. Cells were incubated with inhibitors of cPLA2 (methylarachidonylfluorophosphonate, MAFP) (25); MEK (PD-98059) (26); CaMKII (KN-93) (27); cyclooxygenase (indomethacin) (28); lipoxygenase (baicalein, BACL) (29); CYP450 (17octadecynoic acid, 17-ODYA) (30); farnesyltransferase (FPT III) (31); or their respective vehicles and exposed to NE (10 mM); AA (1–20 mM); Ang II (100 nM); EGF (100 nM); 5-, 12(R)-, 12(S)-, 15-, or 20-HETEs (1–250 nM); or their vehicles for an additional 5–15 min. The concentrations of various inhibitors used in our study have been reported to be effective in blocking the activity of these enzymes in other cell systems (25–31). MAFP and HETEs were obtained from Cayman Chemicals (Ann Arbor, MI). NE, myelin basic protein, and indomethacin were from Sigma. Ang II was from Bachem. AA was from Nu Chek Prep

Activation of phospholipase A2 (PLA2) liberates arachidonic acid (AA) from phospholipids. AA metabolites, including prostaglandins, leukotrienes, lipoxins, and hydroxy derivatives, have been implicated in numerous physiological and pathophysiological processes (1–5). Recent studies using ‘‘knock-out’’ mice or inhibitors indicate that AA-generating cytosolic PLA2 (cPLA2) plays a role in macrophage production of inflammatory mediators, reproductive physiology, allergic responses, postischemic brain injury, cell proliferation, and cancer (6–9). Neurotransmitters, hormones, and growth factors activate cPLA2 and protein kinases in many cell types, including vascular smooth muscle cells (VSMCs) (10–14). The adrenergic transmitter norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca21 influx, increases Ca21ycalmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cPLA2, resulting in release of AA for prostacyclin synthesis (15, 16). This pathway of MAPK activation by CaMKII is mediated through stimulation of MAPK kinase (MEK). CaMKIV expressed in PC-12 cells has also been shown to activate MAPKs (17). MAPKs are also stimulated by bg

Abbreviations: Ang II, angiotensin II; AA, arachidonic acid; BACL, baicalein; CaM, calmodulin; CaMKII, Ca21yCaM-dependent protein kinase II; cPLA2, cytosolic phospholipase A2; CYP450, cytochrome P450; EGF, epidermal growth factor; HETE, hydroxyeicosatetraenoic acid; ERK, extracellular regulated kinase; MAFP, methylarachidonylfluorophosphonate; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; NE, norepinephrine; 17-ODYA, 17-octadecynoic acid; VSMC, vascular smooth muscle cell. *To whom reprint requests should be addressed. e-mail: kmalik@ utmem1.utmem.edu.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9512701-6$2.00y0 PNAS is available online at www.pnas.org.

12701

12702

Pharmacology: Muthalif et al.

(Elysian, MN). PD-98059 was from New England Biolabs. Indomethacin, 17-ODYA, and BACL were from Biomol (Plymouth Meeting, PA). FPT III and KN-93 were from Calbiochem. Enzyme Assays. In vitro MAPK assays: The activity of MAPK was determined in VSMC lysates with a Biotrak kit (Amersham) using a peptide substrate KRELVEPLTPAGEAPNQALLR as directed by the manufacturer’s instructions.

FIG. 1. NE-induced activation of MAPK and cPLA2 but not CaMKII is attenuated by inhibitors of cPLA2, CYP450, and lipoxygenase. Rabbit VSMCs were treated with the vehicle dimethyl sulfoxide (VEH) alone or with inhibitors of cPLA2 (MAFP, 50 mM), CaMKII (KN-93, 20 mM), or MEK (PD-98059, 20 mM) for 4 h or with inhibitors of CYP450 (17-ODYA, 5 mM), lipoxygenase (BACL, 5 mM), or cyclooxygenase (indomethacin, IND, 5 mM) for 30 min and then stimulated with NE (10 mM) for 10 min. Cells were harvested, and cell lysates were analyzed. (A) Effect of MAFP on MAPK activity measured by the in-gel MAPK assay. Arrows indicate 44- and 42-kDa bands identified as ERK1 and ERK2. (B) Effect of MAFP on cPLA2, MAPK, and CaMKII activities as measured by in vitro kinase assay. (C–F) NE-induced activation of MAPK and cPLA2 but not CaMKII is attenuated by inhibitors of CYP450 and lipoxygenase but not cyclooxygenase. MAPK activity was determined by in vitro kinase assay (C) and in-gel kinase assay method (D). As a control for protein loading, an unidentified myelin basic protein kinase band is indicated by an arrow. (G) Effect of NE on AA release and cPLA2 activity during blockade of MAPK activity and AA metabolism. Cells were incubated with a combination of 17-ODYA, BACL, and PD-98059 and then stimulated with NE (10 mM) for 10 min. Basal values of cPLA2, MAPK, and CaMKII activities and AA release are given in text. The data shown represent the mean 6 SEM of three to six experiments on three batches of cells. p, Value significantly different from basal; †, value significantly different from that obtained with VEH of inhibitors; ††, value significantly different from that obtained with either of the inhibitors alone.

Proc. Natl. Acad. Sci. USA 95 (1998) In-Gel MAPK Assay. MAPK activity was measured in VSMC homogenates by renaturation assay in polyacrylamide gels containing myelin basic protein as described (32). In Vitro CaMKII Assay. CaMKII activity was assayed in cell lysates by using CaMKII assay kits (Upstate Biotechnology) with a peptide substrate KKALRRQETVDAL by the manufacturer’s recommendations. cPLA2 Assay. cPLA2 activity in VSMC lysates (20–30 mg of protein per assay) was measured by using L-[1-14C]phosphatidylcholine (57 mCiymmol, American Radiolabeled Chemicals; 1 Ci 5 37 GBq) as substrate as described (33). [3H]AA Release. VSMCs were labeled with [3H]AA (0.25 mCiyml; 100 Ciymmol; Dupont-New England Nuclear) for 16 h, and AA release was measured as described (15). CYP450 Metabolism of AA in Rabbit VSMCs. VSMC (passages 2–4), cultured in 150-mm plates, were permeabilized with Tween 40 (0.1%) for 15 min, washed twice with Krebs buffer, and incubated with [14C]AA (40–60 mCiymmol; 0.5 mCiyml; NEN) at 37°C for 1 h in 0.1 M potassium phosphate (pH 7.4) containing 10 mM MgCl2, 1 mM EDTA, 1 mM NADPH, and an NADPHregenerating system containing 20 mM isocitrate and 0.1 M isocitrate dehydrogenase. Cells were incubated with 20 mM AA for 5 min and then stimulated with NE (10 mM) for 30 min. The aqueous medium was collected, and AA metabolites were extracted and separated with a binary gradient HPLC system (Waters Associates) using a Nucleosil C18 column (5 mM) with a two-solvent gradient elution as described (34). The column eluate was monitored at 235 nm, and radioactivity in the samples was measured by liquid scintillation spectrometry. Western Blotting of CYP450 4A. Lysate and microsomal proteins of VSMCs were separated on an SDSypolyacrylamide gel (10%), transferred to a nitrocellulose membrane, and incubated

FIG. 2. Production of 20-HETE and expression of CYP450 in VSMCs. (A) Representative reverse-phase HPLC of products formed by VSMCs incubated with [14C]AA. Solid line, absorbance units full scale (AUFS); dashed line, radioactivity; arrow, position of authentic standards. (B) Detection of CYP450 4A in microsomes and lysates of VSMCs. Approximately 100–200 mg of proteins from microsomes and lysates were subjected to SDSyPAGE (10% gel) and detected by Western blotting using a rat CYP450 4A polyclonal antibody raised in goats. Lanes (from left to right) are clofibrate-treated liver microsomes as a standard, VSMC lysates (two lanes), and microsomes isolated from rabbit VSMCs.


Proc. Natl. Acad. Sci. USA 95 (1998)

12703

FIG. 3. Effects of 20-HETE and other HETEs on MAPK (A) and cPLA2 (B) activities and AA release (C) in rabbit VSMCs. VSMCs were preincubated with 20 mM PD-98059 (PD) for 4 h and exposed to 20-, 15-, 12(S)-, and 12(R)-HETEs (each at 0.25 mM) for 10 min. To measure AA release, VSMCs were labeled with [3H]AA for 18 h and then exposed to HETEs for 15 min. The data shown represent the mean 6 SEM of triplicate measurements of MAPK and cPLA2 activities and AA release in three batches of cells. Basal values of cPLA2, MAPK activity, and AA release are given in text. p, Value significantly different from basal; †, value significantly different from that obtained with VEH of PD-98059. (D) Effect of 20-HETE on MAPK activity. Cells were incubated with 20-HETEs (0.25 mM) for 10 min after pretreatment of cells with or without 5 mM PD-98059 for 4 h or 5 mM17-ODYA and 5 mM BACL for 30 min. This figure is a representation of two experiments.

for 16 h with rat CYP450 4A antibody (Gentest, Woburn, MA; 1:500 dilution) raised in goats. The immunoblots were developed by using an enhanced chemiluminescence kit (Amersham). Raf Phosphorylation. Raf-1 kinase threonine phosphorylation was determined in cell lysates. The cell lysates were immunoprecipitated with anti-phosphothreonine antibody (Sigma). The immunoprecipitates were separated in a 7.5% SDSypolyacrylamide gel and transferred to nitrocellulose membrane; Raf-1 kinase was detected by the enhanced chemiluminescence method with antiRaf-1 kinase antibody (Upstate Biotechnology, Lake Placid, NY). Translocation of Ras. Cells were fixed in 4% paraformaldehyde in buffer (10 mM Pipesy5 mM EGTAy2 mM MgCl2y0.2% Triton) for 3 min and postfixed in 95% ethanol for 5 min at 220°C. Cells were visualized with anti-Ras (Santa Cruz Biotechnology) and Texas red-conjugated horse anti-mouse IgG (Vector Laboratories) by confocal microscopy (Bio-Rad, MRC-1000, Laser Scanning Confocal Imaging system using an argonykrypton lamp) as described (15). Analysis of Data. The basal values ranged between 916 and 2,387 cpm of [14C]AA per 25 mg of protein per 60 min for cPLA2, 11,300 and 26,345 cpm of 32P per 15 mg of protein per 10 min for MAPK, 11,340 and 22,986 cpm of 32P per 15 mg of protein per 30 min for CaMKII, and 0.93 and 1.72% fractional AA release in different batches of cells. The 3H released into the medium is expressed as percentage of the total cellular activity and referred to as fractional AA release. Although the basal values of cPLA2, MAPK, and CaMKII activity and AA release were variable in different batches of cells, the effect of agonists and inhibitors on the activity of these enzymes and AA release was consistent within each batch of cells. Therefore, the changes produced by agonists and inhibitors have been presented as percent above basal. The results are expressed as mean 6 SEM. Data were analyzed with one-way ANOVA. The Newman–Keuls multiple range test was applied to determine the difference among multiple groups, and an unpaired Student’s t test was used to determine the difference between two groups. Differences were considered significant at P , 0.05.

RESULTS CYP450 and Lipoxygenase Metabolites of Arachidonic Acid Mediate NE-Induced MAPK Activity in Rabbit VSMCs. MAFP,

an inhibitor of cPLA2 (25), attenuated the NE-induced increase in cPLA2 and MAPK activity but not in CaMKII activity (Fig. 1 A and B). This raises the possibility that the NE-induced increase in MAPK activity is caused by AA or its metabolites generated through activation of cPLA2 by CaMKII. That an inhibitor of AA metabolism, 5,8,11,14-eicosatetraynoic acid (35), decreased MAPK but not CaMKII activity (data not shown) supports this hypothesis. If AA metabolites activate MAPK in VSMCs, then inhibition of the AA-metabolizing enzymes CYP450, lipoxygenase, andyor cyclooxygenase should attenuate the NE-induced increase in MAPK and cPLA2 activities. The inhibitors of CYP450 (17ODYA) and, to a lesser degree, lipoxygenase (BACL) attenuated the NE-induced increase in MAPK and cPLA2 activity but not CaMKII activity (Fig. 1C–F). Indomethacin did not affect NEinduced activation of MAPK or cPLA2 activity. Exogenous AA increased MAPK and cPLA2 activity, and this effect was also inhibited by 17-ODYA and by BACL. AA also increased CaMKII activity, but this was not inhibited by 17-ODYA or BACL (data not shown). The inhibitors of CYP450 (17-ODYA), lipoxygenase (BACL), and MEK (PD-98059) (Fig. 1G), which diminished the activities of extracellular regulated kinase (ERK) 1 and ERK2 (Fig. 1D and ref. 15), did not completely block the NE- and AA-induced increases in cPLA2 activity. Metabolism of AA by CYP450 and Lipoxygenase in Rabbit VSMCs. In VSMCs, AA is metabolized by cyclooxygenase to prostaglandins and thromboxane A2 and by lipoxygenase to 5-, 12(S)-, or 15-HETE (36). Since the NE-induced increase in MAPK activity was not only attenuated by a lipoxygenase inhibitor but also by a CYP450 inhibitor, we investigated the production of CYP450 metabolites of AA in rabbit VSMCs. We found by reverse-phase HPLC that AA is metabolized by CYP450 to hydroxy acid(s), one of which has been tentatively identified as 20-HETE (Fig. 2). The calculated amount of this product was approximately 0.5 mgymg of protein. Further studies are needed to confirm the identity of this peak. Moreover, we demonstrated that CYP4A protein, which may be involved in the formation of 20-HETE (37), is expressed in these cells (Fig. 2). 12(S), 15-, and 20-HETEs Stimulate MAPK and cPLA2 Activities in Rabbit VSMCs. AA has been shown to increase cPLA2 activity in human neutrophils through formation of 5-HETE (23)

12704


and to increase MAPK activity in rat aortic VSMCs through formation of 15-HETE (21, 22). In rabbit VSMCs, addition of 12(S)-, 15-, or 20-HETE, but not 12(R)- or 5-HETE (data not shown), increased MAPK and cPLA2 activities and AA release (Fig. 3). 20-HETE also caused activation of MAPK, measured as ERK1 and ERK2, in the presence of inhibitors of CYP450 and lipoxygenase (Fig. 3D). 20-HETE increased MAPK activity at concentrations as low as 0.1 mM; the maximal effect was produced at 1 mM. The effect of 12(S)-, 15-, and 20-HETE of stimulating MAPK and cPLA2 was attenuated by the MEK inhibitor PD-98059 (Fig. 3A), suggesting that these effects could be mediated by MEK or other signaling elements upstream of MEK. 20-HETE Mediates NE-Induced Activation of RasyRaf Pathways in Rabbit VSMCs. Many agonists stimulate MEK activity by promoting phosphorylation of proximal kinases, including c-Raf; Raf is known to be activated by Ras and recruited to the plasma membrane (38). In the present study, NE and 20-HETE increased Raf phosphorylation in VSMCs and increased the translocation of Ras to the plasma membrane. 17-ODYA and BACL blocked this effect of NE but not of 20-HETE (Fig. 4). Farnesylation of Ras by farnesyltransferase is required for proper membrane localization and activity of Ras (39, 40). FPT III, which inhibits farnesyltransferase activity (31), also blocked the NE- and 20HETE-induced translocation of Ras to the plasma membrane (Fig. 4B). Moreover, FPT III attenuated MAPK and cPLA2 activities and the AA release elicited by NE (Fig. 4C). Thus, 20-HETE may activate Raf in VSMCs by promoting the translocation of Ras to the plasma membrane. 20-HETE Mediates Ang II- and EGF-Induced RasyMAPK Activation. Ang II and EGF may also stimulate MAPK through CYP450 and lipoxygenase products of AA released by initial activation of cPLA2 by CaMKII. Ang II- and EGF-induced activation of MAPK was attenuated by inhibitors of CYP450 and lipoxygenase (Fig. 5A). Moreover, Ang II and EGF caused translocation of Ras to the plasma membrane, and this was inhibited by a combination of 17-ODYA and BACL and by FPT III (Fig. 5B). FPT III also attenuated MAPK activity and AA release elicited by Ang II and EGF (Fig. 5C). The inhibitory effect of 17-ODYA and BACL on the Ang II- and EGF-induced increase in MAPK activity and Ras translocation was reversed by 20-HETE (data not shown).

Proc. Natl. Acad. Sci. USA 95 (1998) CaMKII activates cPLA2 and releases AA and that products of AA generated via CYP450 and lipoxygenase stimulate MAPK, which then amplifies cPLA2 activity and releases additional AA. Although CaMKII activates cPLA2 in VSMCs, it inhibits PLA2 activity in rat brain synaptosomes (42). This could be due to differences in the species of PLA2; e.g., forskolin enhances cPLA2

DISCUSSION NE, through a1- and a2-adrenergic receptors, stimulates influx of extracellular Ca21 (41) and activates CaMKII in rabbit VSMCs (15). The CaMKII in turn stimulates MAPK and increases cPLA2 activity, resulting in the release of AA for prostaglandin synthesis (15). The present study demonstrates a mechanism by which a CYP450 metabolite of AA, 20-HETE, mediates NE-, Ang II-, and EGF-stimulated CaMKII-induced activation of the Rasy MAPK pathway. MAPK then amplifies cPLA2 activity and releases further AA for prostaglandin synthesis. In the present study, the cPLA2 inhibitor MAFP and the inhibitor of AA metabolism 5,8,11,14-eicosatetraynoic acid attenuated NE-induced MAPK activity. These results suggest that a metabolite of AA and not this fatty acid itself is involved in CaMKII-induced MAPK activation. An important finding is that inhibitors of CYP450 (17-ODYA) and lipoxygenase (BACL), but not of cyclooxygenase (indomethacin), attenuated the NE- and AA-induced increase in MAPK and cPLA2 and not the CaMKII activities in VSMCs. From these observations, it follows that products of AA generated by CYP450 and lipoxygenase but not by cyclooxygenase contribute to the activation of MAPK and cPLA2 elicited by NE. Although the combination of 17-ODYA, BACL, and the MEK inhibitor PD-98059 abolished the NEinduced increase in MAPK activity, measured as ERK1 and ERK2, it only partially reduced cPLA2 activity. We have reported that CaMKII mediates activation of cPLA2 and MAPK in response to NE (15). Therefore, it appears that NE-stimulated

FIG. 4. Involvement of RafyRas in NE-stimulated MAPK pathway. (A) Effect of NE and 20-HETE on Raf-1 kinase threonine phosphorylation in VSMCs. VSMCs were pretreated with 17-ODYA and BACL or vehicle and then stimulated with NE for 5 min, lysed, and immunoprecipitated with Sepharose-coupled anti-phosphothreonine antibody. The immunoprecipitates were subjected to SDSyPAGE and blotted with anti Raf-1 kinase monoclonal antibody and detected by the enhanced chemiluminescence method. (B) Translocation of Ras in response to NE (10 mM) and 20 HETE (0.25 mM) in the presence of 5 mM 17-ODYA and 5 mM BACL (30 min), farnesyltransferase inhibitor (FPT III, 25 mM, 24 h), or their vehicle. Cells were visualized with anti-Ras and Texas red-conjugated horse anti-mouse IgG by confocal microscopy. This figure is a representation of three experiments. (C) Effect of farnesyltransferase inhibitor (FPT III) on NEstimulated MAPK and cPLA2 activities and AA release. Basal values of MAPK and cPLA2 activities and AA release are given in text. The data shown represent the mean 6 SEM of three to six experiments on MAPK and cPLA2 activities and AA release in three batches of cells. p, Value significantly different from basal; †, value significantly different from that obtained with VEH of inhibitors.


Proc. Natl. Acad. Sci. USA 95 (1998)

12705

activities. These results raise the possibility that a product(s) of AA generated via CYP450, most likely 20-HETE, plays a major role in regulating NE-induced activation of MAPK and, consequently, amplification of cPLA2 activity. Our studies also indicate that MEK mediates NE- and 12(S)-, 15-, and 20-HETE-induced MAPK activation because the MEK inhibitor PD-98059 attenuated these agents’ effect of increasing MAPK activity. Since PD-98059 selectively inhibits MEK1 (26), it appears that MAPK stimulation is mediated primarily by MEK1. However, we cannot exclude the participation of other MEK family members that stimulate MAPKs, including c-jun N-terminal kinase and p38 MAPK. It is well established that Raf activates MEK (43). Our demonstration that the NE-increased phosphorylation of c-Raf was inhibited by ODYA and BACL is consistent with a role for Raf in activation of MEK by NE. Also supporting this view is the finding that 20-HETE increased Raf phosphorylation in VSMCs. NE stimulates Ras in human VSMCs (44), and Ras stimulates Raf by promoting its association with the plasma membrane (45). The finding that translocation of Ras by NE was blocked by inhibitors of CYP450 and lipoxygenase suggests that metabolites of AA activate Raf in VSMCs by promoting the translocation of Ras to the plasma membrane. The findings that 20-HETE translocated Ras to the plasma membrane and that the farnesyltransferase inhibitor FPT III blocked the Ras translocation elicited by NE and by 20-HETE supports this contention. The positive feedback mechanism of RasyMAPK activation by CYP450 and lipoxygenase products of AA may be a general mechanism in the actions of hormones like Ang II and growth factors such as EGF. Ang II and EGF are known to stimulate AA release, MAPK activity, and cell growth (12, 16, 46, 47). EGF increased CaMKII activity and AA release in rabbit VSMCs; this effect was inhibited by removal of calcium from the medium or addition of the CaMKII inhibitor KN-93 (data not shown). In the present study, both Ang II and EGF increased MAPK activity and the translocation of Ras to the plasma membrane, and these effects were inhibited by 17-ODYA and BACL and by the farnesyltransferase inhibitor FPT III. It would appear that these agents, like NE, generate AA metabolites through initial activation of cPLA2 by CaMKII, which stimulates Ras and activates MAPK, amplifies cPLA2 activity, and releases additional AA for prostanoid synthesis.

FIG. 5. Ang II and EGF stimulate RasyMAPK pathways through AA metabolites of CYP450 and lipoxygenase. (A) Effects of 17ODYA and BACL on Ang II- and EGF-stimulated MAPK activity. MAPK activity was measured by in-gel MAPK assay. (B) Translocation of Ras in response to Ang II and EGF in VSMCs. Arrested VSMCs were exposed to 5 mM 17-ODYA and 5 mM BACL (30 min), FPT III (24 h, 25 mM), or their vehicle. Cells were visualized by confocal microscopy. (C) Effects of FPT III on Ang II- and EGFstimulated MAPK activity. Basal values of MAPK are given in text. p, Value significantly different from basal; †, value significantly different from that obtained with VEH of FPT III. The data represent the mean 6 SEM of three to six experiments on three batches of cells.

activity in intact synaptosomes but does not alter PLA2 activity in rabbit VSMCs (41). In VSMCs, AA is metabolized by lipoxygenase into 5-, 12-, and 15-HETE (36). The present study indicates that rabbit VSMCs also express CYP450 4A enzyme, which may be involved in the formation of 20-HETE. 12(S)-, 15-, and 20-HETE increased MAPK and cPLA2 activities and AA release in rabbit VSMCs. However, 20-HETE (a CYP450 product) was more potent than 12(S)- or 15-HETE (lipoxygenase products) in stimulating MAPK and cPLA2 activity. Moreover, the CYP450 inhibitor (17-ODYA) produced a much greater reduction than the lipoxygenase inhibitor (BACL) in NE-induced MAPK and cPLA2

FIG. 6. Schematic model illustrating 20, 12(S)-, and 15-HETE as mediators of MAPK and cPLA2 activation in response to NE, Ang II, and EGF. In this model, activation of receptors with NE, Ang II, or EGF leads to an influx of Ca21 ions that bind to CaM and activate CaMKII. CaMKII activates cPLA2 and releases AA. CYP450 and, to a lesser, extent lipoxygenase metabolites of AA activate MAPK by the RasyRafyMEK pathway by a positive feedback mechanism. Activation of MAPK amplifies cPLA2 activity and further releases AA. R, receptor; RTK, receptor tyrosine kinase; G, G protein.

12706


In rat VSMCs, Ang II-induced MAPK activation has been reported to be Ras-independent (47, 48). However, another study conducted in the same cell system proposed that Ang II-induced MAPK activation was Ras-dependent and is mediated by an unidentified Ca21yCaM-dependent tyrosine kinase through transactivation of the EGF receptor (14, 49). The mechanism by which 20-HETE activates Ras is unknown and is currently being investigated. HETEs may activate Ras by hydroxy arachidonylation of Ras, which could promote its binding to the plasma membrane and subsequent activation. The modification of a subunits of G proteins by myristoylation, arachidonylation, andyor palmitoylation (50) and of Ras by farnesylation (39, 40) is known to be required for anchorage to membrane or interaction with other proteins. In human platelets, a subunits of G proteins (ai, aq, az, and a13) have been shown to covalently bind arachidonate and palmitate but not myristate (50). The present study proposes a signaling mechanism by which NE, Ang II, and EGF activate the RasyMAPK pathway through generation of an AA metabolite of CYP450, 20-HETE (Fig. 6). The activation of MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism (Fig. 6). This mechanism of RasyMAPK activation by 20-HETE might play a central role in other signaling processes involved in inflammation and in cell growth, proliferation, and differentiation. We thank Drs. Alan H. Stephenson and Andrew J. Lonigro (St. Louis University) for their generous help in HPLC separation of AA metabolites, Anne Estes for technical assistance, Dr. Lauren Cagen for scientific discussions, and Ms. Jin Emerson-Cobb for editing the manuscript. This work was supported by National Institutes of Health Grant 19134 from the National Heart, Lung and Blood Institute (to K.U.M.), an American Heart Association Tennessee Affiliate Award (I.F.B. and M.M.M.), and a Center for Neuroscience Fellowship (to S.F.). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Makita, K., Falck, J. R. & Capdevila, J. H. (1996) FASEB J. 10, 1456–1463. Vane, J. R. & Botting, R. M. (1997) Semin. Arthritis Rheum. 26, 2–10. Needleman, P. & Isakson, P. C. (1997) J. Rheumatol. 24, 6–9. McGiff, J. C., Steinberg, M. & Quilley, J. (1996) Trends Cardiovasc. Med. 6, 4–9. Brady, H. R. & Serhan, C. N. (1996) Curr. Opin. Nephrol. Hyperten. 5, 20–27. Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro, F., Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y., et al. (1997) Nature (London) 390, 618–622. Bonventre, J. V., Huang, Z., Taheri, M. R., O’Leary, E., Li, E., Moskowitz, M. A. & Sapirstein, A. (1997) Nature (London) 390, 622–625. Anderson, K. M., Roshak, A., Winkler, J. D., McCord, M. & Marshall, L. A. (1997) J. Biol. Chem. 272, 30504–30511. Heasley, L. E., Thaler, S., Nicks, M., Price, B., Skorecki, K. & Nemenoff, R. A. (1997) J. Biol. Chem. 272, 14501–14504. Della Rocca, G. J., van Biesen, T., Daaka, Y., Lutrell, D. K., Lutrell, L. M. & Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 19125–19132. Davis, R. J. (1993) J. Biol. Chem. 268, 14553–14556. Winitz, S., Gupta, S. K., Qian, N. X., Heasley, L. E., Nemenoff, R. A. & Johnson, G. L. (1994) J. Biol. Chem. 269, 1889–1895. Yang, H., Lu, D., Vinson, G. P. & Raizada, M. A. (1997) J. Neurosci. 17, 1660–1664. Eguchi, S., Matsumoto, T., Motley, E. D., Utsunomiya, H. & Inagami, T. (1996) J. Biol. Chem. 271, 14169–14175. Muthalif, M. M., Benter, I. F., Uddin, M. R. & Malik, K. U. (1996) J. Biol. Chem. 271, 30149–30157. Muthalif, M. M., Benter, I. F., Uddin, M. R., Harper, J. & Malik, K. U. (1998) J. Pharmacol. Exp. Ther. 284, 388–398. Enslen, H., Tokumitsu, H., Stork, P. J., Davis, R. J. & Soderling, T. R. (1996) Proc. Natl. Acad. Sci. USA 93, 10803–10808. Crespo, P., Xu, N., Simonds, W. F. & Gutkind, J. S. (1994) Nature (London) 369, 418–420.

Proc. Natl. Acad. Sci. USA 95 (1998) 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

50.

Jelsema, C. L. & Axelrod, J. (1987) Proc. Natl. Acad. Sci. USA 84, 3623–3627. Cui, X. & Douglas, J. G. (1997) Proc. Natl. Acad. Sci. USA 94, 3771–3776. Rao, G. N., Bass, A. S., Glasgow, W. C., Eling, T. E., Runge, M. S. & Alexander, R. W. (1994) J. Biol. Chem. 269, 32586–32591. Wen, Y., Nadler, J. L., Gonzales, N., Scott, S., Clauser, E. & Natarajan, R. (1996) Am. J. Physiol. 271, C1212–C1220. Wijkander, J., Flaherty, J. T. O., Nixon, A. B. & Wykle, R. L. (1995) J. Biol. Chem. 270, 26543–26549. Nebigil, C. & Malik, K. U. (1992) J. Pharmacol. Exp. Ther. 260, 849–858. Balsinde, J. & Dennis, E. A. (1996) J. Biol. Chem. 271, 6758–6765. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J. & Saltiel, A. (1995) Proc. Natl. Acad. Sci. USA 92, 7686–7689. Sumi, M., Kiuchi, K., Ishikawa, T., Ishii, A., Hagiwara, M., Nagatsu, T. & Hidaka, H. (1991) Biochem. Biophys. Res. Commun. 181, 968–975. Meade, E. A., Smith, W. L. & Dewitt, D. L. (1993) J. Biol. Chem. 268, 6610–6614. Sekiya, K. & Ohuda, H. (1982) Biochem. Biophys. Res. Commun. 105, 1090–1095. Zou, A. P., Ma, Y.-H., Sui, Z.-H., De Montellano, P. R. O., Clark, J. E., Masters, B. S. & Roman, R. J. (1994) J. Pharmacol. Exp. Ther. 268, 474–481. Leftheris, K., Kline, T., Vite, G. D., Cho, Y. H., Bhide, R. S., Patel, D. V., Patel, M. M., Schmidt, R. J., Weller, H. N., Andahazy, M. L., et al. (1995) J. Med. Chem. 39, 224–236. Huang, X.-C., Richards, E. M. & Sumners, C. (1996) J. Biol. Chem. 271, 15635–15641. Leslie, C. C. (1990) Methods Enzymol. 187, 216–225. Harder, D. R., Gebremedhin, D., Narayanan, J., Jefcoat, C., Falck, J. R., Campbell, W. B. & Roman, R. (1994) Am. J. Physiol. 266, H2098–H2107. Chi, E. Y., Henderson, W. A. & Klebanoff, S. J. (1982) Lab. Invest. 47, 579–585. Larrue, J., Rigaud, M., Razaka, G., Daret, D., Demond-Henri, J. & Bricaud, H. (1983) Biochem. Biophys. Res. Commun. 112, 242–249. Wang, M. H., Stec, D. E., Balazy, M., Mastyugin, V., Yang, C. S., Roman, R. J. & Schwartzman, M. L. (1996) Arch. Biochem. Biophys. 336, 240–250. Zhang, X.-F., Settleman, J., Kyriakis, J. M., Takeuchi-Suzuki, E., Elledge, S. J., Marshall, M., Bruder, J. T., Rapp, U. R. & Avruch, J. (1993) Nature (London) 364, 308–313. Zhang, F. L. & Casey, P. J. (1996) Annu. Rev. Biochem. 65, 241–269. Tschantz, W. R., Furfine, E. S. & Casey, P. J. (1997) J. Biol. Chem. 272, 9989–9993. Nebigil, C. & Malik, K. U. (1993) J. Pharmacol. Exp. Ther. 266, 1113–1124. Piomelli, D., Wang, J. K. T., Sihra, T. S., Nairn, A. C., Czernik, A. J. & Greengard, P. (1989) Proc. Natl. Acad. Sci. USA 86, 8550–8554. Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P. & Marshall, C. J. (1992) Cell 71, 335–342. Hu, A., Shi, X., Lin, R. Z. & Hoffmann, B. (1996) J. Biol. Chem. 271, 8977–8982. Zhang, X.-F., Settleman, J., Kyriakis, J. M., Takeuchi-Suzuki, E., Elledge, S. J., Marshall, M., Bruder, J. T., Rapp, U. R. & Avruch, J. (1993) Nature (London) 364, 308–313. Peppelenbasch, M. P., Qiu, R. G., de Vries-Smits, A. M., Tertoolen, L. G., de Laat, S. W., McCormick, F., Hall, A, Symons, M. H. & Bos, J. L. (1995) Cell 81, 849–856. Takahashi, T., Kawahare, Y., Okuda, M., Ueno, H., Takeshita, A. & Yokoyama, M. (1997) J. Biol. Chem. 272, 16018–16022. Liao, D.-F., Monia, B., Dean, N. & Berk, B. C. (1997) J. Biol. Chem. 272, 6146–6150. Eguchi, S., Namaguchi, K., Iwasaki, H., Matsumoto, T., Yamakawa, T., Utsunomiya, H., Motley, E. D., Kawakatsu, H., Owada, K. M., Hirata, Y., Marumo, F. & Inagami, T. (1998) J. Biol. Chem. 273, 8890–8896. Hallak, A., Muszbek, L., Laposata, M., Belmonte, E., Brass, L. F. & Manning, D. R. (1994) J. Biol. Chem. 269, 4713–4716.