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
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ABSTRACT |
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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.
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
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 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 C 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.
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-PGF1 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 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
[ 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.
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.
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.
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 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.
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 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.
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.
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 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 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-PGF1 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
-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.
(13, 15, 16). PLD activation by angiotensin II in
rat VSMC and by
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The content
of 6-keto-PGF1
(the stable hydrolysis product of
prostacyclin) was measured as described previously (4).
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).
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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;
, value significantly different from that obtained in the presence of
vehicle of inhibitors, p < 0.05.
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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.
-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.
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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.
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
-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
-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-PGF1 production. VSMC
were treated as described above, and 6-keto-PGF1
production was then measured as described under "Experimental
Procedures." Data are expressed as the percent increase in
6-keto-PGF1
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.
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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.
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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
CaMKII
cPLA2
AA
HETEs
Ras/ERK
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.
-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
-subunits of heterotrimeric G proteins
(43) and also to decrease the activity of GTPase-activating protein
(44).
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.
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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. -AR,
-adrenergic
receptor; PGs, prostaglandins; EETs,
eposcyeicosatrienoic acids; PA, phosphatidic acid.
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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.
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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.
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
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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-PGF1, 6-keto-prostaglandin
F1
;
FPT, farnesyl protein transferase.
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