Phospholipase D Activation by Norepinephrine Is Mediated by 12(S)-, 15(S)-, and 20-Hydroxyeicosatetraenoic Acids Generated by Stimulation of Cytosolic Phospholipase A2

TYROSINE PHOSPHORYLATION OF PHOSPHOLIPASE D2 IN RESPONSE TO NOREPINEPHRINE*

Jean-Hugues ParmentierDagger, Mubarack M. Muthalif, Abdelwahab E. Saeed§, and Kafait U. Malik

From the Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, December 20, 2000, and in revised form, February 1, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A2 (cPLA2). Arachidonic acid (AA) released by cPLA2-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA2-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA2 activity or protein depletion with selective cPLA2 antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA2-dependent PLD activation by NE in VSMC. In addition to cPLA2, PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD2 (but not PLD1) mutant inhibited NE-induced PLD activity, and PLD2 was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA2-dependent PLD2 through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD2 in rabbit VSMC.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Norepinephrine (NE),1 the principal neurotransmitter released from post-ganglionic sympathetic fibers, stimulates arachidonic acid (AA) release for the synthesis of prostaglandins, which, in turn, act as physiological modulators of neurotransmitter release in various tissues, including blood vessels (1). In vascular smooth muscle cells (VSMC), the synthesis of prostaglandins elicited by NE is mediated by cytosolic phospholipase A2 (cPLA2) activation through alpha -adrenergic receptors (2, 3). In addition, Ca2+, calmodulin (CaM), and CaM-dependent kinase II (CaMKII) are required for NE-induced cPLA2 activation (4, 5). AA, the product of cPLA2 activity, is metabolized by three distinct pathways: cyclooxygenase, lipoxygenase, and cytochrome P450. 12-Hydroxyeicosatetraenoic acid (HETE) and 15-HETE (lipoxygenase metabolites) as well as 20-HETE (a cytochrome P450 4A (CYP4A) metabolite) are produced in VSMC (5).

NE also stimulates phospholipase D (PLD) activity in blood vessels (6). PLD catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and choline. Phosphatidic acid can be hydrolyzed by cPLA2 or sequentially metabolized by phosphatidate phosphohydrolase (PPH) and diacylglycerol (DAG) lipase to release additional AA (7, 8). NE has been reported to increase PLD activity in rat tail artery, cerebral cortex, and cardiac myocytes through stimulation of alpha -adrenergic receptors (6, 9, 10). Whether PLD activation contributes to AA release and prostaglandin synthesis elicited by NE is not known. Moreover, the mechanism by which NE stimulates PLD activity has not been established.

Activation of PLD by hormones and growth factors has been implicated in a wide range of cellular responses, including cellular trafficking, inflammatory and immune responses, mitogenesis, cellular differentiation, and apoptosis (see Ref. 11 for a review). To date, two PLD isoforms, PLD1 and PLD2, have been cloned and characterized. PLD1 has a low basal activity and is up-regulated by small G proteins (Arf, Rho, and Ral), protein kinase C, and phosphatidylinositol 4,5-bisphosphate in vitro. In contrast, PLD2 has a high basal activity, requires phosphatidylinositol 4,5-bisphosphate, and is also up-regulated by ARF and protein kinase C (11). Phosphorylation of PLD1 (12-14) and PLD2 (15) has been reported, as well as colocalization with some growth factor receptors and phospholipase Cgamma (13, 15, 16). PLD activation by angiotensin II in rat VSMC and by alpha -adrenergic receptor stimulation in Madin-Darby canine kidney cells has been reported to be independent of protein kinase C (17, 18). In addition, it has recently been demonstrated that NE stimulates PLD activity in rabbit VSMC via activation of the Ras/MAPK pathway by a protein kinase C-independent mechanism (14).

In VSMC as well as in other cell types, NE is able to activate simultaneously both cPLA2 and PLD (3, 14). Two recent reports demonstrate that activation of PLD by the calcium ionophore A23187 is strictly dependent on PLA2 in leukocytes (19) and human embryonic kidney 293 cells (20). However, the nature of the mediator(s) downstream of PLA2 involved in PLD activation is not known. In rabbit VSMC, 20-HETE and, to a lesser extent, 12(S)- and 15(S)-HETEs, metabolites of AA generated by NE-induced CaMKII-dependent cPLA2 activation, are able to stimulate MAPK activity (5). We have previously shown that NE stimulates PLD activity through a Ras/MAPK pathway (14) and that inhibition of cPLA2 activity blocks AA release (3) and attenuates the increase in MAPK activity elicited by NE in VSMC (5). Therefore, activation of PLD by MAPK in response to NE may be mediated by metabolites of AA. Activation of PLD, in turn, may further release AA for prostaglandin synthesis via the PPH/DAG lipase pathway. To test this hypothesis, we have investigated the effect of interventions that interfere with CaM, CaMKII, cPLA2 activity, and AA metabolism on NE-induced increases in PLD activity in VSMC. We have also examined the effect of inhibitors of the PLD/PPH/DAG lipase pathway on NE-induced AA release and prostacyclin synthesis in these cells. For the first time in a primary cell culture, with an endogenous receptor and without overexpression of a phospholipase, we demonstrate a novel mechanism for PLD activation by NE involving CaMKII-dependent cPLA2 stimulation and generation of the AA metabolites 12(S), 15(S)-, and 20-HETEs, which, in turn, activate the Ras/ERK pathway. Finally, we show that PLD2 is activated and tyrosine-phosphorylated through an ERK-dependent pathway by NE in rabbit VSMC.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- [3H]AA was purchased from PerkinElmer Life Sciences. [3H]Oleic acid was obtained from American Radiolabeled Chemicals (St. Louis, MO). NE, timolol, 1-butanol, 2-butanol, AA sodium salt, and oleic acid sodium salt were from Sigma. Methylarachidonyl fluorophosphonate and 12(S)-, 15(S)-, and 20-HETEs were from Cayman Chemical Co., Inc. (Ann Arbor, MI). W-7, calmidazolium, phosphatidylethanol, RHC-80267, propranolol, baicalein, indomethacin, 17-ODYA, and PD98059 were from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). 5,8,11,14-Eicosatetraynoic acid (ETYA), KN-92, KN-93, arachidonyltrifluoromethyl ketone, FPT III, and BMS-191563 were from Calbiochem. U0126 was from Promega (Madison, WI).

Culture of VSMC-- Aortas were rapidly removed from male New Zealand White rabbits, and the VSMC were isolated and cultured as described (2). Cells between passages 4 and 8 were plated in 24-well or 100-mm plates and maintained under 5% CO2 in M199 medium containing penicillin, streptomycin, Fungizone, and 10% fetal bovine serum.

Transient Transfections-- Cells in 100-mm dishes were transfected with wild-type (HA-tagged pCGN-PLD plasmids) and catalytically inactive mutant (K898R PLD1 and K758R PLD2; gifts from Dr. Michael Frohman, State University of New York, Stony Brook, NY) (21, 22) human PLD1 and murine PLD2 using a calcium phosphate precipitation method. Briefly, 10 µg of DNA was combined with 250 µM CaCl2 and mixed with Hepes-buffered saline buffer. After 20 min of incubation, the solution mixture was slowly added to the cells in the presence of serum-free M199 medium and incubated for 6 h. Cells were then washed twice with Hanks' balanced saline solution and allowed to recover with M199 medium containing 10% fetal bovine serum for 24 h. Cells were arrested overnight in 0.5% fetal bovine serum before the assays. Transfection efficiencies were measured by Western blot analysis.

For experiments involving inhibition of protein synthesis, VSMC were transfected with antisense or sense oligonucleotides designed from cPLA2 or sPLA2 cDNA sequences (3). Phosphorothioate oligonucleotides (synthesized at the Molecular Resource Center, University of Tennessee, Memphis, TN) were complexed with LipofectAMINE Plus reagents (Life Technologies, Inc.) according to the manufacturer's instructions. The oligonucleotide mixture was added to the cells for 24 h, and the medium was replaced with fresh M199 medium for another 24 h before the PLD assay. Efficiency of transfection with oligonucleotides was measured by Western blot analysis.

Phospholipase D Assay-- PLD activity was assayed as described with slight modifications (14). Briefly, nearly confluent rabbit VSMC were incubated with [3H]oleic acid (1 µCi/ml) for 16 h. Cells were then incubated with inhibitors and exposed to NE, AA, or 12(S)-, 15(S)-, or 20-HETE for an additional 10 min. VSMC were scraped into 2 ml of ice-cold methanol and 2 M HCl, and lipids were extracted with chloroform. The aqueous and insoluble fractions were saved for determination of transfection efficiencies, and a 40-µl aliquot was removed from the chloroform phase to estimate the content of radioactivity in the total lipid fraction. 0.8 ml from the chloroform phase was evaporated under nitrogen and resuspended in 50 µl of chloroform/methanol (9:1) containing phosphatidylethanol standard. Samples were spotted onto a silica gel thin-layer chromatography plate, and lipids were separated using the solvent system chloroform/acetone/methanol/acetic acid/water (50:20:12.5:10:7.5). Phosphatidylethanol was identified by the mobility of authentic standard visualized with iodine vapor. Lanes containing phosphatidylethanol were scraped, and radioactivity was measured by scintillation spectroscopy. Data are expressed as the ratio of [3H]phosphatidylethanol to total 3H-labeled lipids.

Arachidonic Acid Release-- VSMC were labeled with [3H]AA (1 µCi/ml) in 24-well clusters for 18 h as previously described (3). Cells were incubated with inhibitors for 1-4 h, washed three times with Hanks' balanced saline solution, and exposed to NE or vehicle for 10 min. The amount of free 3H released into the medium and the total radioactivity in the cells were counted by liquid scintillation spectroscopy. 3H released into the medium was calculated as a percent of the total cellular radioactivity and is referred to as fractional release of [3H]AA.

Radioimmunoassay of 6-Keto-PGF1alpha -- The content of 6-keto-PGF1alpha (the stable hydrolysis product of prostacyclin) was measured as described previously (4).

Western Blot Analysis-- The efficiency of transient transfection with both oligonucleotides and plasmids was determined by Western blot analysis. Briefly, VSMC treated with oligonucleotides were washed twice and scraped into ice-cold phosphate-buffered saline containing protease inhibitors. Cells were pelleted and resuspended in boiling Laemmli sample buffer. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with anti-cPLA2 antibody (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. The immunoblots were subsequently washed, incubated with horseradish peroxidase-linked secondary antibodies, rinsed, and developed with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). For transfection efficiencies with HA-tagged pCGN-PLD plasmids, the top aqueous layer and insoluble fraction from the PLD assay were precipitated with 4 volumes of ice-cold acetone, incubated for 1 h at -80 °C, pelleted, and dried under nitrogen. The pellet was resuspended in boiling Laemmli sample buffer and treated as described above. HA-PLD expression was detected with an HA probe (Santa Cruz Biotechnology).

PLD2 Phosphorylation-- VSMC were incubated with 10 µM NE in the presence of U0126 (10 µM) or its vehicle for 5 or 10 min. VSMC were then washed twice with ice-cold phosphate-buffered saline and lysed with 1 ml of ice-cold lysis buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% Igepal, 0.25% sodium deoxycholate, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and 1 mg/ml p-nitrophenyl phosphate)/100-mm Petri dish for 10 min on ice. The lysates were sonicated twice for 10 s and incubated at 4 °C for 10 min before centrifugation at 3000 rpm. The supernatant was incubated for 3 h with 7 µl of anti-PLD2 antibody (antiserum 26, a generous gift of Dr. Bourgoin, Centre Hospitalier Universitaire de Quebec, Quebec, Canada) and for 1 h with protein A-agarose beads (Life Technologies, Inc.). The immunocomplex was centrifuged, washed once with lysis buffer and twice with phosphate-buffered saline containing protease and phosphatase inhibitors, and dissolved in 6× Laemmli buffer. Immunoprecipitated proteins were separated by 8% SDS-polyacrylamide gel electrophoresis; transferred to nitrocellulose membranes; and probed with phospho-specific antibodies: anti-Tyr(P) (Upstate Biotechnology, Inc., Lake Placid, NY), anti-Ser(P) (Calbiochem), or anti-Thr(P) (Santa Cruz Biotechnology). Immunoreactivity was detected with an ECL system (Amersham Pharmacia Biotech). Each blot was reprobed with anti-PLD2 antibody (antiserum 27).

For the in vitro phosphorylation experiment, PLD2 was immunoprecipitated as described above and incubated with 25 units of activated ERK2 in MAPK buffer (Cell Signaling Technology, Boston, MA) in the presence of 15 µCi of [gamma -32P]ATP and 100 µM ATP for 10 and 20 min. Reactions products were separated by SDS-polyacrylamide gel electrophoresis (8% gel), dried, and detected by autoradiography. A positive control with immunoprecipitated PLD1 (14) as substrate was also used.

Statistical Analysis-- Results are expressed as the means ± S.D. from different batches of cells for PLD activity or arachidonic acid release. Data were analyzed by one-way analysis of variance. Student's t test was applied to determine the difference between treatments and their respective control values. The Newman-Keuls multiple range test was applied for comparison of treatments among multiple groups. The null hypothesis was rejected at p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ca2+/CaM/CaMKII Mediates NE-induced PLD Activation-- Activation of PLD by NE is dependent on extracellular calcium in VSMC (14), and cPLA2 activation is mediated through a CaM/CaMKII pathway in these cells (5). Therefore, we studied the possible involvement of CaM and CaMKII in NE-induced PLD activation. The CaM inhibitors W-7 (10 µM) and calmidazolium (10 µM) reduced PLD activity elicited by NE in VSMC (Fig. 1). The CaMKII inhibitor KN-93 (Fig. 1), which blocks NE-induced CaMKII activity in VSMC (3), but not its inactive analog KN-92 (data not shown), also inhibited PLD activity, indicating that a Ca2+/CaM/CaMKII pathway is involved in the activation of PLD by NE in these cells.


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Fig. 1.   Effect of the calmodulin antagonists W-7 and calmidazolium and the CaMKII inhibitor KN-93 on NE-induced PLD activity. Cells were labeled with [3H]oleic acid for 16 h and preincubated with W-7, calmidazolium (CMZ), or KN-93 for 30 min before the addition of 10 µM NE. PLD activity was measured as described under "Experimental Procedures." Data are expressed as the ratio of [3H]phosphatidylethanol (PEt) to total 3H-labeled lipids. Values are the means ± S.E. of three independent experiments performed in duplicate in different batches of cells. *, value significantly different from vehicle alone; dagger , value significantly different from that obtained in the presence of vehicle of inhibitors, p < 0.05.

cPLA2 Is Required for NE-induced PLD Activation-- The concomitant activation of cPLA2 (3) and PLD (14) by a Ca2+/CaM/CaMKII pathway in NE-stimulated VSMC and recent reports of PLA2-dependent PLD activation in cardiac sarcolemma (23), leukocyte cell lines (19), and human embryonic kidney 293 cells (20) suggested the possibility of an involvement of cPLA2 in NE-induced PLD activation in VSMC. Treatment of VSMC with a generic PLA2 inhibitor, methylarachidonyl fluorophosphonate (24), and arachidonyltrifluoromethyl ketone, a selective cPLA2 inhibitor (25), blocked PLD activation by NE (Fig. 2A). In addition, the selective depletion of endogenous cPLA2 with 1 µM cPLA2 antisense oligonucleotide for 48 h inhibited NE-induced PLD activity (Fig. 2B). cPLA2 sense and sPLA2 sense and antisense oligonucleotides did not significantly alter NE-induced PLD activation. Therefore, it appears that cPLA2 (but not sPLA2) is involved in NE-induced PLD activation in VSMC. AA, the product of phospholipid hydrolysis formed by activation of cPLA2, stimulated PLD activity in a concentration-dependent manner, reaching a peak at 25 µM (Fig. 3). Oleic acid, another unsaturated fatty acid, at similar or higher concentrations did not stimulate PLD activity, indicating a specific role of cPLA2-formed AA and/or its metabolites in PLD activation. 25 µM AA also stimulated PLD activity after depletion of cPLA2 with antisense oligonucleotides (data not shown). ETYA (5 µM), a non-hydrolyzable analog of AA that acts as a competitive inhibitor of AA in vivo or in vitro (26), reduced PLD activity elicited by NE (Fig. 2A). ETYA also inhibited AA-induced increases in PLD by 66% (22% increase versus 65% with AA alone), indicating that AA metabolites (but not the enzyme cPLA2 per se or AA) are responsible for PLD activation.


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Fig. 2.   Effect of cPLA2 inhibition on NE-induced PLD activity. A, [3H]oleic acid-labeled VSMC were treated with methylarachidonyl fluorophosphonate (MAFP), arachidonyltrifluoromethyl ketone (AACOCF3), or ETYA for 30 min, and PLD activity was measured as described under "Experimental Procedures." B, shown is the effect of cPLA2 antisense oligonucleotides on NE-induced PLD activation. [3H]Oleic acid-labeled VSMC were incubated with cPLA2 or sPLA2 antisense (A) or sense (SE) oligonucleotides for 48 h, and PLD activity was measured as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels obtained in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in duplicate on different batches of cells. *, value significantly different from vehicle (M199 medium (A) and LipofectAMINE (B)), p < 0.05. The middle panel is a representative Western blot showing the effect of cPLA2 and sPLA2 antisense and sense oligonucleotide treatment on cPLA2 protein levels. Lane 1, vehicle; lane 2, 0.5 µM cPLA2 antisense oligonucleotide; lane 3, 1 µM cPLA2 antisense oligonucleotide; lane 4, 1 µM cPLA2 sense oligonucleotide; lane 5, 1 µM sPLA2 antisense oligonucleotide; lane 6, 1 µM sPLA2 sense oligonucleotide. PEt, phosphatidylethanol.


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Fig. 3.   Effect of AA and oleate on PLD activity. [3H]Oleic acid-labeled VSMC were treated with different concentrations of AA or 50 µM oleic acid for 10 min. PLD activity was measured as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels obtained in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in duplicate on different batches of cells. *, value significantly different from basal levels, p < 0.05. PEt, phosphatidylethanol.

Lipoxygenase and CYP4A Products (HETEs) Are Involved in NE-induced PLD Activity-- AA is metabolized by three distinct pathways in blood vessels: cyclooxygenase (prostaglandins and thromboxane A2), lipoxygenase (HETEs), and cytochrome P450 (HETEs and epoxyeicosatrienoic acids) (27). It has been reported that 12-, 15-, and 20-HETEs are produced in VSMC (5, 28) and that both CYP4A and lipoxygenases are expressed in VSMC (5, 29, 30). To further characterize the role of cPLA2 in the PLD activation pathway, cells were treated with inhibitors of AA-metabolizing enzymes (Fig. 4A). Inhibitors of lipoxygenases (baicalein, 5 µM) (31) and CYP4A-dependent omega -hydroxylase/epoxygenase (17-ODYA, 5 µM) (32), but not cyclooxygenase (indomethacin, 10 µM), decreased NE- as well as AA-induced PLD activities, suggesting a significant contribution of the lipoxygenase and CYP4A pathway to NE-induced PLD activation. To gain more insight into the mechanism of cPLA2-mediated PLD activation, we tested the effect of metabolites of AA generated via lipoxygenase (12(S)- and 15(S)-HETEs) and CYP4A (20-HETE) in VSMC on PLD activity. As shown in Fig. 4B, 12(S)-, 15(S)-, and 20-HETEs (0.5 µM each) stimulated PLD activity approximately to the same extent as NE and AA. A stereoisomer of 12(S)-HETE (12(R)-HETE) had no effect on PLD activity (data not shown). In addition, AA- and HETE-induced PLD activity was not altered in antisense-directed cPLA2-depleted cells, and AA-induced PLD activity was inhibited by baicalein and 17-ODYA (data not shown). Based on these observations, we suggest that NE-induced PLD activation is cPLA2-dependent and is mediated by 12(S)-HETE and by 15(S)- and 20-HETEs, metabolites of lipoxygenase and CYP4A, respectively.


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Fig. 4.   Effect of inhibitors of AA metabolism on NE-induced PLD activity. A, cells were incubated with inhibitors of cyclooxygenase (indomethacin, 10 µM), lipoxygenase (baicalein, 5 µM), or CYP4A (17-ODYA, 5 µM) for 30 min and then exposed to 10 µM NE for 10 min. PLD activity was measured as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in duplicate on different batches of cells. *, value significantly different from vehicle, p < 0.05. B, shown are the effects of AA and HETEs on PLD activity. Cells were treated with 25 µM AA or 0.5 µM 12(S)-, 15(S)-, or 20-HETE for 10 min. PLD activity was measured as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in duplicate on different batches of cells. PEt, phosphatidylethanol.

HETEs Stimulate PLD through the Ras/MAPK Pathway-- We have previously reported that HETEs mediate NE-induced activation of Ras and ERK in rabbit VSMC (5). In addition, we have also shown that Ras/MAPK mediates NE-induced PLD activation in VSMC (14). It therefore seemed possible that HETEs mediate NE-induced PLD stimulation through activation of the Ras/MAPK pathway. To test this hypothesis, we selectively blocked the Ras/MAPK pathway and measured PLD activity stimulated by 12(S)-, 15(S)-, or 20-HETE (Fig. 5). Ras is post-translationally modified by farnesylation, and farnesyltransferase inhibitors such as FPT III and BMS-191563 produce aberrant Ras localization and loss of function (5, 33). FPT III (25 µM) and BMS-191563 (10 µM) preincubated for 18 h with VSMC did not show any cytotoxicity or modification of the basal PLD activity (data not shown). Consistent with the inhibitory effect of FPT III and BMS-191563 on NE-stimulated PLD activity (14), 12(S)-, 15(S)-, and 20-HETE-induced PLD activity was inhibited in cells treated with the Ras farnesyltransferase inhibitors. To further establish the importance of the Ras/MAPK pathway in HETE-mediated NE-induced PLD activation, we treated the cells with inhibitors of MEK: U0126 and PD98059. U0126 substantially reduced 12(S)-, 15(S)-, and 20-HETE-induced PLD activation (Fig. 5). PD98059, which reduces NE-induced PLD activation (14), also reduced the effect of HETEs to increase PLD activity (data not shown). U0126 required a shorter time of incubation (1 h) and a lower concentration (10 µM) compared with PD98059 (20-50 µM) to produce equivalent inhibition. These findings support the conclusion that 12(S)-, 15(S)-, and 20-HETE-induced PLD activation in VSMC is mediated by the Ras/ERK pathway.


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Fig. 5.   Effect of Ras/MAPK pathway inhibitors on AA-, 12(S)-, 15(S)-, and 20-HETE-induced PLD activation. Cells were incubated with inhibitors of Ras farnesyltransferase (FPT III (25 µM) or BMS-191563 (10 µM) for 18 h) or MEK (U0126, 10 µM, 1 h) and then exposed to 25 µM AA or 0.5 µM each HETE. PLD activity was measured as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in duplicate on different batches of cells. *, value significantly different from the vehicle in each group, p < 0.05.

Effect of PLD/Diacylglycerol Lipase Inhibition on NE-induced AA Release and Prostacyclin Production-- We have previously reported that NE stimulates AA release and prostacyclin synthesis in rabbit aortic VSMC via activation of cPLA2 (2). However, in Rat1 fibroblasts expressing alpha 1A-adrenergic receptors, the release of AA elicited by phenylephrine was shown to be mediated primarily by PLD activation and the PPH/DAG lipase pathway (8). To determine the contribution of PLD to NE-induced AA release and prostacyclin production in rabbit VSMC, we examined the effects of 1-butanol (a PLD inhibitor), 2-butanol (an inactive analog), propranolol (a beta -adrenergic receptor blocker that inhibits phosphatidate phosphohydrolase), and RHC-80267 (a DAG lipase inhibitor) (8) on AA release and prostacyclin synthesis. 1-Butanol (0.4% (v/v), 30 min), but not 2-butanol, reduced AA release, prostacyclin production, and PLD activity (Fig. 6). In addition, treatment with propranolol (10 µM, 30 min) or RHC-80267 (10 µM, 30 min) also inhibited NE-induced AA release and prostacyclin formation without any effect on PLD activity, indicating that arachidonic acid metabolites generated through this pathway do not participate in PLD regulation. Timolol, another beta -blocker, did not alter PLD activity (data not shown). Together, these results suggest an important contribution of PLD to AA release for prostaglandin production by NE via the PPH/DAG lipase pathway in rabbit VSMC.


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Fig. 6.   Effect of inhibitors of the PLD/DAG/AA pathway on NE-induced AA release in VSMC. A, [3H]AA-labeled VSMC pretreated with inhibitors of PLD (1-butanol, 0.4% (v/v), 30 min), phosphatidate phosphohydrolase (propranolol, 10 µM, 30 min), or DAG lipase (RHC-80267, 10 µM, 30 min) were stimulated with 10 µM NE for 10 min. AA release was then measured as described under "Experimental Procedures." Data are expressed as the percent increase in AA release over the basal fractional release in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in sextuplicate on different batches of cells. B, shown is the effect of PLD/DAG/AA pathway inhibitors on NE-induced 6-keto-PGF1alpha production. VSMC were treated as described above, and 6-keto-PGF1alpha production was then measured as described under "Experimental Procedures." Data are expressed as the percent increase in 6-keto-PGF1alpha production above basal levels in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in sextuplicate on different batches of cells. C, shown is the effect of PLD/DAG/AA pathway inhibitors on NE-induced PLD activation. [3H]Oleic acid-labeled VSMC were treated as described above, and PLD activity was then measured as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels obtained in unstimulated cells. Values are the means ± S.E. of three independent experiments performed in duplicate on different batches of cells. *, value significantly different from vehicle, p < 0.05 (A-C). PEt, phosphatidylethanol.

NE Selectively Activates PLD2-- Two isoforms of PLD, PLD1 and PLD2, have been identified (for review, see Ref. 11). The activity of PLD1 is increased by several stimuli, whereas PLD2 is thought to be constitutively active. However, recent findings indicate that PLD2 is also up-regulated by several agonists, primarily through an ARF-dependent pathway (34). To determine the PLD isoform activated by NE in VSMC, we overexpressed HA-tagged wild-type and catalytically inactive PLD1 and PLD2 in the expression vector pCGN (21, 22) in rabbit VSMC. Basal PLD activity was increased by 20% in cells overexpressing wild-type PLD2, consistent with a constitutive basal expression of PLD2, whereas the overexpression of wild-type PLD1 or the corresponding PLD1 and PLD2 mutants had no significant effect on basal PLD activity (Fig. 7A). We next examined the effects of these variants on NE-induced PLD activity. As illustrated in Fig. 7B, overexpression of catalytically inactive K758R PLD2 dramatically reduced NE-induced increases in PLD activity, whereas it was not altered in cells expressing K898R PLD1. These results suggest that PLD2 is the main isoform activated by NE in VSMC.


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Fig. 7.   Effect of PLD isoform mutants on PLD activity. A, cells were transiently transfected with wild-type (wt) or catalytically inactive mutant PLD1 or PLD2 for 48 h, and PLD activity was determined as described under "Experimental Procedures." Data are expressed as the percent increase in PLD activity above basal levels obtained in LipofectAMINE-treated cells. Values are the mean ± S.E. of three independent experiments performed in duplicate on different batches of cells. *, value significantly different from basal (LipofectAMINE), p < 0.05. B, effect of PLD isoform mutants on NE-induced PLD activation. PLD activity was determined during stimulation with NE. *, value significantly different from vehicle (LipofectAMINE), p < 0.05. The gel shown at the top is a representative Western blot showing the efficiency of transfection of pCGN-HA-PLD constructs using an anti-HA antibody. Lane 1, untransfected; lane 2, HA-PLD1; lane 3, HA-K898R PLD1; lane 4, HA-PLD2; lane 5, HA-K758R PLD2. PEt, phosphatidylethanol.

PLD2 Is Tyrosine-phosphorylated in Response to NE-- PLD1 and PLD2 phosphorylation has been described (12-15). We tested whether the activation of PLD2 by NE involves phosphorylation. VSMC lysates were immunoprecipitated with an anti-PLD2 antibody (antiserum 26) and immunoblotted with anti-PLD2 antiserum 27. This anti-PLD2 antiserum recognizes recombinant PLD2 protein expressed in Sf9 cells. As shown in Fig. 8, a band at ~100 kDa (the approximate PLD2 molecular mass) was detected. This band was not recognized by the antiserum neutralized with the peptide used for the preparation of this antibody (data not shown). When the blots were probed with anti-phosphotyrosine antibody, a strong tyrosine phosphorylation of endogenous PLD2 immunoprecipitated from VSMC exposed to NE for 5 and 10 min was observed. U0126, the selective MEK inhibitor, blocked this increase in NE-induced tyrosine phosphorylation of PLD2. In VSMC treated with NE, PLD2 phosphorylation on serine and threonine residues was also detected when the blots were probed with anti-phosphoserine and anti-phosphothreonine antibodies. However, this level of PLD2 serine and threonine phosphorylation was not altered in VSMC treated with NE or U0126 (data not shown). Since the MEK/ERK pathway is involved in PLD2 activation and phosphorylation elicited by NE, we studied the phosphorylation of PLD2, immunoprecipitated from arrested VSMC, by purified activated ERK2 (Cell Signaling Technology) in vitro. However, we failed to observe any increase in PLD2 phosphorylation elicited by ERK2 (data not shown). Therefore, it appears that NE selectively promotes tyrosine phosphorylation of PLD2, which is mediated by ERK through an as yet unknown tyrosine kinase.


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Fig. 8.   PLD2 is tyrosine-phosphorylated in response to NE. VSMC were pretreated with U0126 and/or stimulated with 10 µM NE (5 or 10 min). The cells were lysed, and PLD2 was immunoprecipitated (IP), separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with anti-phosphotyrosine (P-Tyr) antibody (upper panel). Anti-Ser(P) and anti-Thr(P) antibodies did not show any alteration in the basal PLD2 phosphorylation (data not shown). Expression of PLD2 was confirmed using anti-PLD2 antibody (lower panel). WB, Western blot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study demonstrates a novel mechanism of agonist-induced PLD activation whereby NE increases PLD2 activity in rabbit VSMC via CaMKII-dependent activation of cPLA2; generation of the AA metabolites 12(S)-HETE and 15(S)- and 20-HETEs through lipoxygenase and CYP4A, respectively; and stimulation of the Ras/MAPK pathway. This novel signaling pathway follows the sequence NE right-arrow CaMKII right-arrow cPLA2 right-arrow AA right-arrow HETEs right-arrow Ras/ERK right-arrow PLD2. Surprisingly, the increase in PLD2 activity is associated with its tyrosine phosphorylation through an ERK-dependent pathway. Moreover, activation of PLD by NE further releases AA for prostanoid synthesis through the PPH/DAG lipase pathway.

In rabbit VSMC, the increase in PLD activity caused by NE is dependent upon extracellular Ca2+ (14). On the other hand, in rat brain slices, NE does not require extracellular Ca2+ to produce this effect, but a Ca2+ concentration increase in the medium enhances NE-induced increases in PLD activity (6). Ca2+ and CaM have been shown to be involved in the regulation of PLD in osteoblast-like cells (35) and rat basophilic leukemia cells (36). In our study, the increase in PLD activity elicited by NE was also attenuated by two structurally distinct CaM antagonists, W-7 and calmidazolium. However, the mechanism by which Ca2+ and CaM stimulate PLD activity is not known. Our demonstration that the CaMKII inhibitor KN-93 attenuated NE-induced increases in PLD activity suggests that activation of PLD, similar to that of cPLA2 (5), is caused by CaMKII. We have previously shown that the NE-induced increase in PLD activity is mediated via the Ras/MAPK pathway by a protein kinase C-independent mechanism in rabbit VSMC (14). Moreover, the Ras/MAPK pathway in VSMC is activated by metabolites of AA generated through CYP4A and lipoxygenase, consequent to activation of cPLA2 by CaMKII (5). These observations and the demonstration that, in cardiac sarcolemma (23), leukocytes (19), and human embryonic kidney 293 cells (20), PLD stimulation is dependent upon PLA2 activity raised the possibility that NE-induced PLD activation in rabbit VSMC might be mediated by one or more products of phospholipid hydrolysis generated by PLA2, most likely AA and/or its metabolites. Supporting this view was our finding that inhibitors of PLA2, methylarachidonyl fluorophosphonate (24) and arachidonyltrifluoromethyl ketone (25), attenuated NE-induced increases in PLD activity. The effect of the latter agent in selectively inhibiting the activity of cPLA2 (25) and our findings that cPLA2 (but not sPLA2) antisense oligonucleotides attenuated NE-induced increases in PLD activity suggest that cPLA2 regulates PLD activity in VSMC. Inhibitors of PLA2 have also been shown to attenuate PLD activity caused by the ionophore A23187 in mouse lymphocytic leukemia L1210 cells (19). Since, in these cells, application of exogenous lysophosphatidylcholine reversed the effect of inhibitors of cPLA2 on PLD activation, it was implicated as the mediator of cPLA2 responsible for activating PLD (19). However, the role of endogenous lysophosphatidylcholine in PLD activation was not determined.

In this study, exogenous AA (but not oleic acid) increased PLD activity in rabbit VSMC, and this effect of AA was not inhibited by cPLA2 depletion. Moreover, the effect of both exogenous AA and NE on PLD activation in VSMC was attenuated by a non-hydrolyzable competitive inhibitor of AA metabolism, ETYA (26), indicating that one or more metabolites of AA (but not the fatty acid itself) mediate the effect of NE on PLD activation. Since inhibitors of lipoxygenases and CYP4A (but not cyclooxygenase) attenuated the effect of AA and NE on PLD activation, it appears that the metabolites of AA generated via lipoxygenases and CYP4A mediate the effect of NE on PLD activation in VSMC. In addition to its metabolism by cyclooxygenase to prostanoids, AA has also been shown to be metabolized by lipoxygenases and CYP4A in VSMC, mainly into HETEs, including 12- and 15-HETEs and 20-HETE, respectively (5). Our finding that 12(S)-, 15(S)-, and 20-HETEs increased PLD activity in VSMC supports our contention that they mediate the effect of NE and exogenous AA to increase PLD activity. A similar increase in the magnitude of PLD stimulation by 12(S)-, 15(S)-, and 20-HETEs indicates that hydroxylation of the AA backbone, at least at the 12-, 15-, or 20-position, promotes PLD activation. In addition, the stereoselectivity of the AA hydroxylation seems to be important for PLD activation because 12(R)-HETE failed to stimulate PLD activity in VSMC. Although some CYP4A isoforms can also metabolize AA into epoxyeicosatrienoic acids (37), these agents are not formed in detectable levels in rabbit aorta (27). However, we do not exclude a role for cytochrome P450-derived products of 12- or 15-lipoxygenase metabolites such as trihydroxyeicosatrienoic acids since these tri-HETEs are formed in rabbit aorta (38).

Increases in PLD activity brought about by NE via HETEs could be due to stimulation of the Ras/MAPK pathway for the following reasons. First, dominant-negative mutants of Ras and inhibitors of Ras and MEK have been shown to attenuate PLD activation caused by NE in VSMC (14). Second, 12(S)-, 15(S)-, and 20-HETEs have been reported to activate ERK via the Ras/Raf/MEK pathway (5). Our demonstration that agents that block farnesylation of Ras, which is required for its association with the plasma membrane and activation (5, 39) also attenuated 12(S)-, 15(S)-, and 20-HETE-induced PLD activation suggests that the effect of HETEs on PLD activation is mediated by Ras. Whether HETEs activate Ras by interacting with specific receptor sites is not known. There is some evidence that 12(S)- and 20-HETEs may act via specific receptors (40). 12(S)-HETE has been shown to bind to a 650-kDa multimeric complex that includes hsp70 in different cell types (41). Activation of Ras by HETEs could be due to a decrease in the activity of GTPase-activating proteins or post-translational modification of Ras by acylation, resulting in increases in membrane association. The lipidation of alpha -subunits of heterotrimeric G proteins by palmitoylation and of Ras by farnesylation is required for association with membranes or other proteins (42). AA has been reported to covalently bind to the alpha -subunits of heterotrimeric G proteins (43) and also to decrease the activity of GTPase-activating protein (44).

In this study, the NE-induced increase in PLD activity was also associated with an increase in AA release and prostacyclin production through sequential hydrolysis of phosphatidic acid by PPH and of DAG by DAG lipase (7, 8). Supporting this view is our finding that inhibition of PPH and DAG lipase attenuated NE-induced AA release and 6-keto-PGF1alpha production, but did not alter PLD activity. These data suggest that AA derived from the PLD/PPH/DAG lipase pathway does not contribute to PLD activation and is mainly metabolized into prostacyclin and probably other secreted eicosanoids and also lysophosphatidates (45). Therefore, AA derived from cPLA2 (but not from PLD) appears to be metabolized into PLD-stimulating HETEs.

PLD has been reported to occur in at least two isoforms, PLD1 and PLD2 (11). In A10 cells, a dedifferentiated cell line exhibiting characteristics of neointimal cells, angiotensin II activates mainly ARF-dependent PLD2, but not PLD1 (34). In our study, NE increased PLD activity through the PLD2 isoform. This suggests that the NE-induced increase in PLD activity and the associated release of AA and prostacyclin synthesis are due to selective activation of the PLD2 isoform in rabbit VSMC. Whether HETEs selectively activate PLD2 over PLD1 is not known and is under investigation. The increase in PLD2 activity in response to NE is most likely mediated through tyrosine phosphorylation of PLD2 by an ERK-dependent mechanism for the following reasons. First, NE increased tyrosine (but not serine and threonine) phosphorylation of PLD2. Second, inhibition of the ERK pathway by U0126 blocked NE-induced tyrosine phosphorylation of PLD2. Third, activated ERK2 was unable to phosphorylate PLD2 in vitro. Since ERKs are serine/threonine kinases, it appears that an ERK-dependent tyrosine kinase mediates the effect of NE on tyrosine phosphorylation of PLD2. We have previously reported that PLD1 is directly phosphorylated by ERK in response to NE in VSMC (14). However, the biological significance of this phosphorylation remains to be determined since the correlation between PLD1 phosphorylation and activation is not clear (13). In addition, care should be taken with the use of catalytically inactive K898R PLD1 since it does not act as dominant-negative in some cell lines (16).

In conclusion, this study provides evidence for a novel mechanism by which NE selectively increases PLD2 activity (Fig. 9) through stimulation of cPLA2 by CaMKII and generation of the AA metabolites 12(S)- and 15(S)-HETEs and 20-HETE via lipoxygenase and CYP4A, respectively, and activation of the Ras/ERK pathway by these HETEs. Moreover, the increase in PLD activity was associated with PLD2 tyrosine phosphorylation in response to NE, suggesting the involvement of a tyrosine kinase in an ERK-dependent pathway. Furthermore, activation of PLD by NE releases additional AA for eicosanoid/prostaglandin synthesis through the PPH/DAG lipase pathway.


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Fig. 9.   Schematic model of NE-induced PLD activation in VSMC. NE via stimulation of CaMKII activates cPLA2 and releases AA. AA is metabolized by cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LO). Eicosanoids generated via the cytochrome P450 and lipoxygenase pathways activate the Ras/MAPK pathway, which, in turn, promotes tyrosine phosphorylation and activation of PLD. Activation of the PLD/PPH/DAG pathway leads to further AA release from phosphatidylcholine. alpha -AR, alpha -adrenergic receptor; PGs, prostaglandins; EETs, eposcyeicosatrienoic acids; PA, phosphatidic acid.


    ACKNOWLEDGEMENTS

We thank Anne Estes for technical assistance and Dr. Lauren Cagen for editorial comments. We gratefully acknowledge Dr. Michael Frohman for generously supplying the PLD constructs and Dr. Sylvain Bourgoin for providing anti-PLD2 antisera.

    FOOTNOTES

* This work was supported in part by NHLBI Grant 19134-26 from the National Institutes of Health.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.

Dagger Recipient of a postdoctoral fellowship from the American Heart Association, Southeast Affiliate.

§ Supported by a National Institutes of Health minority postdoctoral fellowship.

To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Tennessee Health Science Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-6075; Fax: 901-448-7206; E-mail: kmalik@utmem.edu.

Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M011473200

    ABBREVIATIONS

The abbreviations used are: NE, norepinephrine; AA, arachidonic acid; VSMC, vascular smooth muscle cell(s); cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; CaM, calmodulin; CaMKII, CaM-dependent protein kinase II; 17-ODYA, 17-octadecynoic acid; HETE, hydroxyeicosatetraenoic acid; CYP4A, cytochrome P450 4A; PLD, phospholipase D; PPH, phosphatidate phosphohydrolase; DAG, diacylglycerol; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ETYA, 5,8,11,14-eicosatetraynoic acid; HA, hemagglutinin; 6-keto-PGF1alpha , 6-keto-prostaglandin F1alpha ; FPT, farnesyl protein transferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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