From the Vascular Biology Research Center and
Division of Hematology, University of Texas Medical School,
Houston, Texas 77030 and § Department of Medicine,
University of Pennsylvania, School of Medicine,
Philadelphia, Pennsylvania 19104
Received for publication, June 19, 2000, and in revised form, October 18, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our recent study indicates that
lysophosphatidylcholine (LPC) enhances Sp1 binding and
Sp1-dependent endothelial nitric oxide synthase (eNOS)
promoter activity via the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK-1) signaling pathway (Cieslik, K., Lee, C.-M., Tang, J.-L., and Wu, K. K. (1999) J. Biol. Chem. 274, 34669-34675).
To identify upstream signaling molecules, we transfected human
endothelial cells with dominant negative and active mutants of Ras and
evaluated their effects on eNOS promoter activity. Neither mutant
altered the basal or LPC-induced eNOS promoter function. By contrast, a
dominant negative mutant of phosphatidylinositol 3-kinase Endothelial nitric oxide synthase
(eNOS),1 a member of the NOS
family, catalyzes the synthesis in blood vessels of nitric oxide, which
plays a key role in maintaining blood pressure homeostasis and vascular
integrity (1, 2). eNOS is constitutively expressed primarily in
endothelial cells, and its level of expression has been shown to be
up-regulated by exercise (3), shear stress (4), hypoxia (5), and
lysophosphatidylcholine (LPC; Ref. 6). That LPC is capable of
up-regulating the expression of vasoprotective eNOS is intriguing,
because LPC has emerged as an important mediator of vascular injury,
inflammation, and atherosclerosis (7). It has been postulated that
LPC-induced eNOS expression represents a crucial mechanism by which
arteries exert their plastic defense against vessel wall injury
(8).
LPC is generated from oxidized low density lipoprotein (9) or from
inflammatory cells as a result of phospholipase A2 action (10). It possesses a variety of proinflammatory and proatherogenic properties: 1) it increases chemotactic activities of monocytes and
T-lymphocytes (11, 12); 2) it has a mitogenic effect on macrophages
(13); 3) it activates expression of vascular adhesion molecule-1,
intercellular adhesion molecule-1, platelet-derived growth factor,
heparin binding epidermal growth factor, and cyclooxygenase-2 (14-17);
and 4) it increases thrombomodulin expression and reduces tissue factor
and tissue factor pathway inhibitor (18-20). The mechanisms by which
LPC exerts the myriad cellular and molecular actions are not entirely
clear. It has been suggested that LPC acts as a second messenger to
activate signaling molecules such as cAMP, mitogen-activated protein
kinase, protein kinase C (PKC), and phosphatidylinositol 3-kinase
(PI-3K; Refs. 13, 21-23). LPC has been shown to induce the DNA binding
activity of activated protein 1 (24), cAMP response element-binding
protein (25), and nuclear factor Cell Culture--
Two human endothelial cell lines, ECV304 and
EAhy926 cells, were used in our experiments, which yielded similar
results. ECV304 cells were cultured in Medium 199 (Life Technologies,
Inc.) containing 10% fetal bovine serum (FBS). EAhy926 cells were
cultured in Dulbecco's modified Eagle's medium (Life Technologies)
with high glucose and L-glutamine, containing 10% FBS and
HAT supplement (10 µM hypoxanthine and 1.6 µM thymidine) (Life Technologies). Unless otherwise
indicated, both types of cells were incubated in medium containing
0.5% FBS for 16 h before experiments.
Construction of Expression Vectors--
A 5'-flanking fragment
of the eNOS promoter at nucleotide positions from PI-3K Dominant Negative Mutant Constructs--
Two dominant
negative PI-3K constructs, Transfection Experiments--
To evaluate the effects of mutant
constructs on eNOS promoter activity, cells grown in 35-mm wells at
~50% confluence were incubated with 2 µg eNOS promoter construct
at 37 °C for 30 min followed by addition of 2 µg Ras or PI-3K
mutant constructs with 10 µl Lipofectin for 5 h. Medium was
removed and replaced with complete medium for 24 h. Cells were
washed, incubated in medium containing 0.5% FBS for 16 h, and
then incubated in fresh medium containing 5% FBS in the presence or
absence of 100 µM LPC for 5 h. Cells were harvested,
and the promoter activity was measured. For Sp1 binding experiments,
cells grown in 100-mm dishes to ~50% confluence were treated with 16 µg of mutant constructs and 80 µl Lipofectin for 5 h. Cells
were washed and incubated in complete medium for 24 h, and then
the medium was removed and replaced with fresh medium containing 0.5%
FBS for 15 h. Cells were again washed and incubated in fresh
medium containing 5% FBS with or without 100 µM LPC for
3 h. Cells were harvested, nuclear extracts were prepared, and the
gel shift assay was performed as described below.
eNOS Promoter Activity--
eNOS promoter activity was
determined by a procedure described previously (28). In brief, cells
were incubated in serum-free medium containing a mixture of 10 µl
Lipofectin and 2 µg eNOS promoter-luciferase construct at 37 °C
for 5 h. Medium was removed, and cells were washed and incubated
with fresh complete medium for 24 h. The medium was removed and
replaced with fresh medium containing 0.5% FBS. Cells were washed and
incubated in fresh medium containing 5% FBS with or without the
pharmacological inhibitor to be investigated for 1 h before
addition of 100 µM LPC for 5 h at 37 °C. For the
pertussis toxin (PTX) experiment, the medium was removed and replaced
with fresh medium containing 0.5% FBS with or without PTX for 16 h. The medium was replaced with fresh medium containing 5% FBS to
which 100 µM LPC was added. The cells were incubated for
an additional 5 h at 37 °C. The cells were harvested, and the
expressed luciferase activity was measured in a luminometer (Bioscan).
The results were expressed as a relative light units/µg protein.
Genistein, wortmannin, LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), AG-490 ( Electrophoretic Mobility Shift Assay--
The gel shift assay
was performed as described previously (29). Endothelial cells were
washed and incubated for 1 h in fresh medium containing 5% FBS in
the presence or absence of a pharmacological inhibitor followed by
addition of LPC for 3 h. 10 µg of nuclear extracts from these
treated cells were incubated in a binding buffer containing 25 mM Tris-HCl, pH 8.0, 50 mM KCl, 10% glycerol, 50 µg/ml bovine serum albumin, 6.0 mM MgCl2,
0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mM ZnCl2, 1.5 µg poly(dI-dC), and 0.03% Nonidet P-40 at room temperature for 15 min. A [ Jak2 Assay--
The Jak2 activity was assayed by its
autophosphorylation (33). Cells were incubated in fresh medium
containing 5% FBS in the presence or absence of an inhibitor for
1 h before addition of LPC for 15 min. Cells were then washed
twice with ice-cold buffered saline containing 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM
Na3VO4, and 0.05 M NaF and then
lysed in a Triton X-100 lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10%
glycerol, 2 mM EDTA, 1.5 mM MgCl2,
1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, and 0.05 M NaF for 1 h
at 4 °C with mild agitation. Detergent-insoluble material was
removed by centrifugation at 4 °C for 15 min at 12,000 rpm. Protein
concentration was determined using a bicinchoninic acid protein assay
(Pierce). 500 µg of the cell lysate proteins were immunoprecipitated
with an anti-Jak2 antibody (Santa Cruz) overnight in
immunoprecipitation buffer containing PBS, pH 7.4, 0.5 mM
EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml
leupeptin. Protein G Plus agarose (Santa Cruz) was added to absorb
immune complexes, and after washing twice with immunoprecipitation
buffer and once with Jak2 kinase buffer (0.02 M
1,4-piperazinediethanesulfonic acid, 3 mM
MnCl2, and 1 µg/ml leupeptin), the immune complexes were
resuspended in 50 µl of Jak2 kinase buffer containing 10 µCi
[ Western Blot Analysis--
The Western blot analysis was
performed by a procedure described previously (17). Cells were
incubated for 1 h in medium containing 5% FBS with or without an
inhibitor followed by incubation with LPC for 15 min. 20 µg of cell
lysate proteins were loaded to each lane of a 10% SDS gel. Anti-MEK-1
antibodies were purchased from Upstate Biotechnology. An antibody to
phosphorylated Raf-1 was obtained from New England Biolabs. An
anti-Jak2 antibody was obtained from Santa Cruz, and an antibody to
phosphorylated MEK-1 was from Calbiochem. An antibody to human eNOS was
obtained from Transduction Laboratories. The secondary goat anti-rabbit
horseradish peroxidase-conjugated antibody was obtained from Pierce.
The blots were detected by an enhanced chemiluminescence system (Pierce).
Ras Is Not Required for Basal or LPC-induced eNOS Promoter
Activity--
Results from our previous report (30) indicate that LPC
selectively activates MEK-1 and ERK1/2 via which Sp1 binding activity was increased and eNOS promoter activity was augmented. To determine whether LPC-induced eNOS expression depends on activation of upstream Ras, we transfected ECV304 cells or EAhy926 cells with a Ras dominant negative mutant, a constitutively active Ras mutant, or an empty vector
and determined the basal and LPC-induced eNOS promoter activity by
cotransfecting the cells with the luciferase expression vectors. Basal
and LPC-induced eNOS promoter activities (Fig. 1A) in cells transfected with
the control vector pUSE were comparable with those in native cells as
previously reported (29). Neither the dominant active nor the dominant
negative mutants significantly altered the basal or LPC-induced
activities (Fig. 1A). In accord with this, neither mutant
had a significant effect on Sp1 binding activity (Fig. 1, B
and C). It has been previously reported that transforming
growth factor PI-3K Jak2 Acts Downstream of PI-3K MEK-1 Is the Downstream Target of PI-3K Effect of PTX, AG-490, and LY 294002 on eNOS Protein
Levels--
The results so far indicate that LPC increased
Sp1-mediated eNOS promoter activity via the Gi-coupled
PI-3K Several laboratories including ours have reported that LPC
activates MEK-1, which in turn activates ERK1/2 (21, 30). Results from
our previous studies further indicate that the MEK-1/ERK1/2 signaling
pathway is essential for Sp1-dependent eNOS promoter activity induced by LPC (30). In the present study, we provide information for the first time that activation of the MEK-1 signaling pathway leading to eNOS promoter up-regulation depends on activation of
upstream PI-3K PI-3K catalyzes the synthesis of phosphatidylinositol
3,4,5-trisphosphate, which is an important second messenger for diverse cellular responses (40). At least four PI-3K isoforms have been characterized (41). Heterodimeric PI-3K Jak2 is a member of the Janus kinase family of nonreceptor protein
tyrosine kinases, which consists of three additional members: Jak1,
Jak3, and Tyk2. Each member has a conserved C-terminal kinase domain. Jak2 was reported to activate PI-3K It has been reported in HeLa and 3T3 cells that LPC can also activate
the c-Jun N-terminal kinase pathway and thereby increase AP1-dependent gene transcription (24). We did not
detect c-Jun N-terminal kinase (JNK) activation by LPC in endothelial
cells (30). Our results are consistent with other reports, which show that in vascular endothelial cells or smooth muscle cells, LPC predominantly activates the ERK1/2 pathway (21, 30, 54). It is possible
that PI-3K Previous studies suggest that LPC activates G proteins. A more recent
study shows in platelets and megakaryocytic cell lines that LPC
stimulates Gs, thereby increasing adenylyl cyclase and cellular cAMP levels (18). Our results indicate that LPC stimulates PTX-sensitive Gi in endothelial cells. The mechanism by
which LPC activates G proteins is unclear. It has been assumed that LPC
activates G proteins by membrane perturbation. LPC is an amphipathic molecule and has been shown to transit through plasma membrane and to
enter into cells at a rapid rate (59). It is unlikely that it will
exert a specific effect on activation of G proteins. Alternatively, its
action may be mediated by Gi-coupled receptor activation.
In view of the important roles that LPC plays in diverse pathophysiological processes, it should be valuable to determine whether its action is mediated by a specific receptor. Identification of such a specific receptor should have important therapeutic implications.
(PI-3K
) blocked the promoter activity induced by LPC. Wortmannin and
LY 294002 had a similar effect. AG-490, a selective
inhibitor of Janus kinase 2 (Jak2), also reduced the LPC-induced
Sp1 binding and eNOS promoter activity to the basal level. LPC induced
Jak2 phosphorylation, which was abolished by LY 294002 and the dominant
negative mutant of PI-3K
. LY 294002 and AG-490 abrogated MEK-1
phosphorylation induced by LPC but had no effect on Raf-1. These
results indicate that PI-3K
and Jak2 are essential for LPC-induced
eNOS promoter activity. This signaling pathway was sensitive to
pertussis toxin, suggesting the involvement of a Gi protein
in PI-3K
activation. These results indicate that LPC enhances
Sp1-dependent eNOS promoter activity by a pertussis
toxin-sensitive, Ras-independent novel pathway,
PI-3K
/Jak2/MEK-1/ERK1/2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (26). Despite these reports, the
signaling pathways that lead to transcriptional up-regulation of these
proinflammatory and growth factor genes remain unclear. By contrast,
the transcriptional regulation of eNOS is more extensively
investigated. The basal eNOS promoter activity depends on binding of
Sp1 to an Sp1 cognate site (
90 to
104) on the human eNOS
5'-flanking promoter region (27, 28). In our previous study, we have
shown that LPC up-regulates eNOS promoter activity by augmenting
specifically the Sp1 binding activity (29). We have further shown that
LPC selectively activates extracellular signal-regulated kinase 1/2
(ERK1/2) via mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase 1 (MEK-1), and PD98059
(2'-amino-3'-methoxyflavaone), a selective inhibitor of MEK-1,
abrogated the Sp1-dependent eNOS promoter up-regulation,
consistent with an essential role of MEK-1 and ERK1/2 in LPC-induced
promoter activity (30). The signaling pathway upstream of MEK-1
has not been reported. In this study, we searched for upstream
signaling kinases by using dominant negative mutants or selective
pharmacological inhibitors, or both. Here, we report identification of
PI-3K
as an essential signaling molecule in MEK-1 activation with
subsequent Sp1-dependent eNOS promoter up-regulation by
LPC. Our results further show that PI-3K
activates a downstream
Janus kinase 2 (Jak2), which in turn activates MEK-1. Neither Ras nor
Raf-1 activation is required for eNOS promoter activation by LPC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1322 to +22 was
obtained by polymerase chain reaction, using genomic DNA as a template
and synthetic oligomers as primers: EN1322G
(5'-AAAGATCTTCCATCTCCCTCCTCCTG-3') and EN3H
(5'-GGGAAGCTTGTTACTGTGCGTCCACTCTG-3'). The polymerase chain reaction
product purified from an agarose gel was digested with
BglII-HindIII and cloned into a promoterless luciferase expression vector, pGL3.
p85 and
110
, were cloned into a
promoterless vector, pCMV5, as described previously (31, 32). The
p85 construct lacks amino acid residues 478-513 of the p85 subunit
and functions as a dominant negative mutant for PI-3K
, -
, and
-
. The
110
construct lacks amino acids 948-981 and
functions as a dominant negative mutant of PI-3K
. Dominant negative
Ras (S17N) and dominant active Ras (Q61L) mutants and the control
vector pUSEamp (+) were obtained from Upstate Biotechnology.
-cyano-(3,4-dihydroxy)-N-benzylcinnamide), and
PTX were purchased from Calbiochem, and LPC was purchased from Avanti.
32P]ATP-labeled Sp1 oligonucleotide probe (29)
was added and incubated at room temperature for 15 min. The mixture was
electrophoresed at 12.5 V/cm on a 5% polyacrylamide gel with a buffer
containing 0.5× Tris borate-EDTA and 0.5% Nonidet P-40. The gel was
vacuum dried and autoradiographed. We have previously demonstrated that nuclear extracts prepared from ECV304 or EAhy926 cells form two retarded bands with a labeled probe containing the canonical Sp1 motif
(29). These two bands were competed out by a 100-fold molar excess of
unlabeled probe but not by Sp1-mutated probe. Our previous experiments
also demonstrated that LPC treatment invariably enhanced Sp1 binding by
~2-fold (29), and the augmented Sp1 binding was responsible for a
concordant increase in the eNOS promoter activity. These results have
been highly reproducible. Only the two shifted bands of Sp1-DNA
complexes are shown in the figures. The denser band was subject to
densitometry in all experiments.
32P]ATP for 30 min at 30 °C. The reaction was
stopped by addition of 2× Laemmlli buffer. Proteins were boiled for 5 min and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (TGF
1) activates the
Ras
MEK-1 signaling pathway in several types of cells including
endothelial cells (34-37), which may mediate up-regulation of the
promoter function of diverse genes in various cells (36, 37). We
therefore evaluated the effect of the dominant negative mutant of Ras
on TGF
1-induced MEK-1 activation measured as
phosphorylated MEK-1 on immunoblots. Our results confirmed that
TGF
1 (5 ng/ml) activated MEK-1, which was abrogated by
the dominant negative mutant of Ras but not by the control vector (Fig.
1, D and E). Taken together, these results
indicate that the signaling pathway by which LPC induced eNOS promoter
activity bypasses Ras as contrasted to the requirement of Ras for the
TGF
1 signaling in endothelial cells.
View larger version (32K):
[in a new window]
Fig. 1.
Effect of constitutively active Ras mutant
(H-Ras(+)), dominant negative Ras mutant
(H-Ras( )), and control vector on eNOS
promoter activity and Sp1 binding. A, eNOS promoter
activity was measured as expressed luciferase activity in relative
light units (RLU)/mg protein. The white
bars denote basal and the black bars denote
LPC-stimulated values. Each bar is the mean ± S.D. of
three experiments. B, representative autoradiograph showing
the two Sp1-specific shifted bands determined by electrophoretic
mobility shift assay. C, control cells; L,
LPC-treated cells. C, densitometric analysis of Sp1
binding from three experiments. Each bar (white,
basal; black, LPC-treated) is the mean ± S.D. of three
experiments. D, as a positive control, we treated EAhy926
cells with TGF
1 (5 ng/ml) for 30 min, which was
previously shown to induce Ras-dependent MEK-1 activation.
MEK-1 activation was determined by the level of phosphorylated MEK-1,
using an antibody specific for phosphorylated MEK-1. A representative
graph shown here revealed that TGF
1 induced MEK-1
phosphorylation (lane 2 versus lane 1), which was
abrogated by the dominant negative mutant of Ras (lane 4).
E, densitometric analysis of D of three
experiments. Each bar (white, basal;
black, TGF
1-treated) is the mean ± S.D.
of three experiments.
Activation Is Essential for Sp1-dependent eNOS
Promoter Function--
To search for an upstream kinase that transmits
signals to augment the eNOS promoter activity via the MEK-1/ERK1/2
pathway, we evaluated the effects of selective PI-3K inhibitors on eNOS promoter activity and Sp1 binding activity. Neither wortmannin (50 nM) nor LY 294002 (50 µM) inhibited basal Sp1
binding activity (Fig. 2, A
and B) or eNOS promoter activity (Fig. 2C) but
reduced the LPC-induced Sp1 binding activity (Fig. 2, A and
B) and eNOS promoter activity (Fig. 2C) to the
basal level. Because neither wortmannin nor LY 294002 is a selective
inhibitor of PI-3K isoforms, we used
p110
and
p85 to identify
the isoform of PI-3K that is involved in LPC-induced eNOS
transcription. LPC-induced eNOS promoter activity was blocked by
p110
transfection but not by
p85 transfection (Fig.
3). Neither mutant reduced the basal
activity, and the control vector had no effect on LPC-induced or basal
promoter activity (Fig. 3). Thus, LPC-induced eNOS promoter function
depends on activation of PI-3K
. PI-3K
activation has been
reported to be linked to an upstream Gi activation, when
Gi
is dissociated from Gi
and interacts with PI-3K
(38). We therefore determined whether the
LPC-induced eNOS promoter function was PTX sensitive. The eNOS promoter
activity induced by LPC was abrogated by PTX treatment (Fig.
4A). This was confirmed by a
reduction of the Sp1 binding activity to the basal level by PTX (100 ng/ml; Fig. 4, B and C). Neither the basal eNOS
promoter activity nor the basal Sp1 binding activity was perturbed by
PTX. These results suggest that the action of LPC on eNOS promoter
activity is mediated by PTX-sensitive, Gi-coupled
activation of PI-3K
.
View larger version (28K):
[in a new window]
Fig. 2.
Suppression of LPC-induced Sp1 binding and
eNOS promoter activity by PI-3K-selective inhibitors wortmannin
(W) and LY 294002 (LY). A,
representative autoradiograph of Sp1 binding to labeled probe;
W (50 nM) and LY (50 µM) suppressed the LPC-induced Sp1 binding to the basal
level (lanes 3 versus 5 and
7 versus 9). C, control
cells; L, LPC-treated cells. B,
densitometric analysis of Sp1 binding of three experiments. Each
bar is the mean ± S.D. C, eNOS promoter
activity. White and black bars denote basal and
LPC-stimulated luciferase activities, respectively. Both W
(50 nM) and LY (50 µM) reduced the
LPC-stimulated activity to the basal level.
View larger version (22K):
[in a new window]
Fig. 3.
Dominant negative mutant of
PI-3K
(
p110
)
but not
p85 suppressed LPC-induced eNOS
promoter activity. Each bar is the mean ± S.D. of
three experiments.
View larger version (14K):
[in a new window]
Fig. 4.
Effect of PTX (100 ng/ml) on eNOS promoter
activity (A) and Sp1 binding (B and
C). Each bar is the mean ± S.D. of
three experiments.
--
Genistein at concentrations
of
50 µM inhibited Sp1 binding activity (Fig.
5, A and B) but had
no significant effect at 5 µM. The eNOS promoter activity
was also inhibited by genistein at 50 µM (Fig.
5C). Because receptor tyrosine kinase is reported to be
inhibited by genistein at a 5 µM concentration
(IC50 = 2.6 µM; Ref. 39), whereas cytosolic
tyrosine kinase is inhibited by higher genistein concentrations, our
results suggest the involvement of a cytosolic tyrosine kinase in eNOS
promoter activity. AG-490, a selective inhibitor of a cytosolic
tyrosine kinase, Jak2, reduced the Sp1 binding to the basal level (Fig.
6, A and B) with a
concordant suppression of eNOS promoter activity (Fig. 6C).
LPC-induced Jak2 phosphorylation was blocked by AG-490 (50 µM; Fig. 7A).
The Jak2 level was, on the other hand, unchanged (Fig. 7B).
Interestingly, Jak2 phosphorylation was abolished by treatment with LY
294002 (50 µM) or PTX (100 ng/ml; Fig. 7A),
suggesting that Jak2 activation is downstream of PI-3K
activation.
This was supported by the results demonstrating that Jak2
phosphorylation was abolished in cells transfected with
p110
but
not with
p85 (Fig. 7C).
View larger version (18K):
[in a new window]
Fig. 5.
Effect of genistein (GE) on Sp1
binding and eNOS promoter activity. A,
concentration-dependent inhibition of Sp1 binding to
labeled probe in control (C) or LPC-treated (L)
cells. Concentrations for GE are in µM. Sp1
competitor denotes a 100-fold molar excess of unlabeled probe.
B, densitometric analysis of results from three experiments.
C, inhibition of LPC-induced eNOS promoter activity by 50 µM GE.
View larger version (14K):
[in a new window]
Fig. 6.
Suppression of Sp1 binding and eNOS promoter
activity by a selective Jak2 inhibitor, AG-490 (AG; 50 µ CM;1M). A, representative figure of
Sp1 gel shift. , negative control; C, control cells;
L, LPC-treated cells. B, densitometric
analysis of results from three experiments. C, eNOS promoter
activity of three experiments. AG-490 (50 µM) suppressed
the LPC-induced eNOS promoter activity.
View larger version (16K):
[in a new window]
Fig. 7.
A, Western blot analysis of
phosphorylated Jak2 (Jak2-P) detected by using an antibody
specific for Jak2-P. LPC induced Jak2-P (lane 3), which was
suppressed by PTX (100 ng/ml), AG-490 (50 µM), and LY
294002 (50 µM). Lane 1, negative control.
B, Jak2 level detected by a Jak2 antibody. C,
influence of p85 and
p110
on Jak2-P. Only
p110
suppressed LPC-induced Jak2 phosphorylation. C, control
cells; L, LPC-treated cells; AG, AG-490;
LY, LY 294002.
and Jak2--
We then
determined whether Raf-1 or MEK-1 is the downstream target of Jak2. We
measured the levels of phosphorylated MEK-1 and Raf-1-P, because
phosphorylation of these two signaling molecules correlate with their
activation. Raf-1-P was detected in unstimulated cells, which was not
enhanced by LPC (Fig. 8A).
AG-490 (50 µM), LY 294002 (50 µM), and PTX
(100 ng/ml) had no effect on Raf-1-P levels (Fig. 8A). The
Raf-1 protein levels were also unaffected by any of the treatments
(data not shown). By contrast, the phosphorylated MEK-1 level was
increased by ~2-fold in response to LPC stimulation, and this
increase was blocked by AG-490 (50 µM), as well as by LY
294002 (50 µM) and PTX (100 ng/ml; Fig. 8, B
and C). The MEK-1 protein level was unaltered (Fig.
8D). These results are consistent with MEK-1 as the target
of the PI-3K
/Jak2 signaling pathway.
View larger version (17K):
[in a new window]
Fig. 8.
Western blot analysis of Raf-1 and
MEK-1. A, detection of phosphorylated Raf-1
(Raf-1-P) using an antibody specific for Raf-1-P.
B, detection of MEK1-P using an antibody specific for
phosphorylated MEK-1. C, Densitometric analysis of
B from three experiments. Each bar
(white, control; black, LPC-treated) is the
mean ± S.D. of three experiments. D, MEK-1
level detected by an antibody for MEK-1. C, control cells;
L, LPC-treated cells; AG, AG-490; LY,
LY 294002.
Jak2
MEK-1 pathway. To confirm that this pathway is
involved in eNOS protein expression, we evaluated the effects of
several inhibitors on eNOS protein levels. LPC increased eNOS protein
over the basal level by ~2-fold, which is in agreement with the
extent of increase in eNOS promoter activity. The eNOS protein increase
was reduced to the basal level by PTX, AG-490, and LY 294002 (Fig.
9, A and B).
View larger version (23K):
[in a new window]
Fig. 9.
Western blot analysis of eNOS protein levels
in endothelial cells in response to LPC stimulation in the presence and
absence of inhibitors of G1 protein (PTX, 100 ng/ml), Jak2
(AG-490, 50 µM), or PI-3K
(LY-294002, 50 µM).
A, representative Western blot showing that LPC increased
eNOS protein, which was abrogated by each of the inhibitors.
B, densitometric analysis of the Western blots from three
experiments. Each bar (white, basal;
black, LPC-treated) is the mean ± S.D. of three
experiments. C, control cells; L, LPC-treated
cells; AG, AG-490; LY, LY 294002.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Jak2. Several pieces of evidence from the present
study indicate that PI-3K
and Jak2 activations are essential for
eNOS promoter up-regulation by LPC: 1) wortmannin and LY 294002 block
LPC-induced MEK-1 phosphorylation (Fig. 8B), Sp1 binding (Fig. 2, A and B), and eNOS promoter activity
(Fig. 2C); 2) the dominant negative mutant of p110
selectively abrogated eNOS promoter activity increased by LPC (Fig. 3);
and 3) AG-490, a selective inhibitor of Jak2, reduced LPC-induced Sp1
binding (Fig. 6, A and B), eNOS promoter activity
(Fig. 6C), and MEK-1 phosphorylation (Fig. 8B) to
the basal level. Importantly, our results indicate that Jak2 acts
downstream of PI-3K
. Compelling evidence to support this conclusion
includes the abrogation of Jak2 autophosphorylation by
110
(Fig.
7C) as well as by LY 294002 (Fig. 7A). By
contrast, the conventional upstream signaling molecules of MEK-1,
i.e. Ras and Raf-1, do not play a significant role in
signaling LPC-induced eNOS promoter activity. Neither dominant negative
nor dominant active Ras mutants exerted any effect on basal or
LPC-induced Sp1 binding and eNOS promoter activity (Fig. 1).
Furthermore, LPC did not increase Raf-1 phosphorylation (Fig.
8A). Taken together, these findings indicate that LPC
induces eNOS promoter function in endothelial cells by a novel
signaling pathway involving PI-3K
Jak2
MEK-1
ERK1/2.
, PI-3K
, and PI-3K
constitute a p110 catalytic subunit and a p85 or p55 regulatory subunit. The heterodimeric PI-3K
constitutes a p110
catalytic subunit, which does not interact with p85 adaptors but forms a dimer
with a p101 regulatory subunit. The dominant negative deletion mutants
p110
and
p85 specifically inhibit PI-3K
and PI-3K
, -
,
or -
, respectively. Our results clearly show that
p110
but not
p85 blocked LPC-induced eNOS promoter activity signaled via the
MEK-1/ERK1/2 pathway. Both PI-3K
and PI-3K
have been shown to
transmit signals to activate MEK-1 in different cell types via a common
pathway involving Shc, Grb2, and SOS (son of sevenless) with subsequent activation of Ras and
Raf-1 (42-44). eNOS promoter activation by LPC does not signal through
this pathway, because neither Ras nor Raf-1 activation was required for
LPC-induced eNOS promoter activity. Alternatively,
Gi
-stimulated PI-3K
has recently been
reported to activate PKC
(45, 46), and PKC
activates MEK-1
independently of Ras and Raf-1 (44). The LPC-induced eNOS promoter
up-regulation may be signaled through this pathway. In our preliminary
work, we found that several PKC inhibitors blocked ERK1/2 activation
and eNOS promoter activity,2
supporting the involvement of a PKC in this pathway. Furthermore, Jak2
activation in thrombin-stimulated platelets (G protein-coupled receptor) is downstream of PKC activation (47). It is possible that a
PKC isoform, such as atypical PKC
, serves as an intermediate signal
between PI-3K
and Jak2 in the LPC-induced MEK-1 activation. Further
studies are required to evaluate this possibility. It was reported that
PI-3K
possesses a protein kinase activity that signals to ERK1/2 in
addition to the well characterized lipid kinase activity, which signals
to protein kinase B/Akt (48). It is unclear whether signaling of
PI-3K
to Jak2 and MEK-1 is mediated by its protein kinase activity
or via lipid kinase activity, nor is it clear whether the Akt pathway
is involved in ERK1/2-mediated promoter up-regulation by LPC.
Nevertheless, it should be noted that Akt activation leads to
phosphorylation of a serine residue located at the C-terminal region of
eNOS, and the phosphorylated eNOS exhibits enhanced catalytic activity
(49). Thus, the PI-3K pathway(s) occupies a central position in
regulating eNOS activity.
, but the exact mechanism by which these two molecules interact is unclear (50). This Jak2-dependent PI-3K
pathway is unlikely to signal
LPC-induced eNOS promoter function, because transfection of
p85,
which blocks PI-3K
activity, had no effect of eNOS promoter
activity. Jak2 has been reported to be directly associated with and
stimulated by the angiotensin II receptor (51, 52). The relevance of these reports to LPC-induced signaling Jak2 is unclear, because it is
unknown whether the action of LPC is receptor-mediated. However, on the
basis of our experimental results, it is unlikely that Jak2 is directly
activated by LPC receptor, even if such a receptor exists. Our results
clearly show that Jak2 activation is downstream of PI-3K
. A previous
study has shown that PI-3K
activates Bruton's tyrosine kinase in
lymphocytes (53). Further studies are likely to identify additional
nonreceptor protein-tyrosine kinases that are activated by PI-3K
.
The mechanism by which PI-3K
activates Jak2 is unclear. It is
possible that phosphatidylinositol 3,4,5-trisphosphate generated by
PI-3K
may bind to the JH motif (Jak
homology domain) located in the catalytic domain of Jak2, leading to Jak2 activation. The JH motif is structurally similar to the
Src homology 2 domain (SH2), and phosphatidylinositol
3,4,5-trisphosphate is known to bind the Src homology 2 domain for
activation of kinases. Alternatively, PI-3K
may activate Jak2
through its protein kinase activity (48).
Jak2
MEK-1
ERK1/2 may represent a common signaling
pathway through which LPC exerts its transcriptional responses in
vascular cells. LPC has been shown to increase mRNA or protein
expression, or both, of a myriad of endothelial genes, including
thrombomodulin, tissue plasminogen activator, plasminogen activator
inhibitor 1, intercellular adhesion molecule 1, vascular cell adhesion
molecule 1, platelet-derived growth factor, and heparin binding
epidermal growth factor-like growth factor (55, 14-16). How LPC
increases the expression of these biologically active genes is
generally unknown. It is unclear whether LPC augments the promoter
activities of these genes, nor is it known which transactivators are
stimulated to account for the increased expression of such a diverse
group of genes. Results from our study suggest that LPC may stimulate
the promoter activities of certain genes, such as thrombomodulin,
vascular cell adhesion molecule 1 and plasminogen activator inhibitor
1, that depend in part on Sp1 binding for their transcriptional
activation (56-58) by a similar PI-3k
Jak2 signaling pathway,
resulting in increased Sp1 binding and promoter activity. Further
studies are needed to test this hypothesis.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Susan Mitterling for editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants P50 NS23327 and RO1 HL50675.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.
¶ To whom correspondence should be addressed: Vascular Biology Research Center and Division of Hematology, University of Texas Medical School, 6431 Fannin, MSB 5.016, Houston, TX 77030. Tel.: 713-500-6801; Fax: 713-500-6812; E-mail: Kenneth.K.Wu@uth.tmc.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M005305200
2 C.-M. Lee, K. Cieslik, and K. K. Wu, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: eNOS, endothelial nitric oxide synthase; LPC, lysophosphatidylcholine; PI-3K, phosphatidylinositol 3-kinase; PTX, pertussis toxin; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Jak, Janus kinase; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; TGF, transforming growth factor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Moncada, S., Palmer, R. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109-142[Medline] [Order article via Infotrieve] |
2. | Wu, K. K. (1995) Adv. Pharmacol. 33, 179-207[Medline] [Order article via Infotrieve] |
3. | Sessa, W. C., Pritchard, K., Seyedi, N., Wang, J., and Hintze, T. H. (1994) Circ. Res. 74, 3349-3353 |
4. |
Nadaud, S.,
Philippe, M.,
Arnal, J. F.,
Michel, J. B.,
and Soubrier, F.
(1996)
Circ. Res.
79,
857-863 |
5. |
Arnet, U. A.,
McMillan, A.,
Dinerman, J. L.,
Ballermann, B.,
and Lowenstein, C. J.
(1996)
J. Biol. Chem.
271,
15069-15073 |
6. |
Zembowicz, A.,
Tang, J.-L.,
and Wu, K. K.
(1995)
J. Biol. Chem.
270,
17006-17010 |
7. | Witztum, J., and Steinberg, D. (1991) J. Clin. Invest. 88, 1785-1792[Medline] [Order article via Infotrieve] |
8. | Wu, K. K. (1998) Proc. Assoc. Am. Physicians 110, 163-170[Medline] [Order article via Infotrieve] |
9. | Parthasarathy, S., Streinbrecher, U. P., Barnett, J., Witztum, J. L., and Steinberg, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3000-3004[Abstract] |
10. | Asaoka, Y., Yoshida, K., Sasaki, Y., Nishizuka, Y., Murakami, M., Kudo, I., and Inoue, K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 716-719[Abstract] |
11. | Quinn, M. T., Parthasarathy, S., and Steinberg, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2805-2809[Abstract] |
12. | Asaoka, Y., Oka, M., Yoshida, K., Saski, Y., and Nishizuka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6447-6451[Abstract] |
13. |
Sakai, M.,
Miyazaki, A.,
Hakmata, H.,
Sasaki, T.,
Yui, S.,
Yamazaki, M.,
Shichiri, M.,
and Horiuchi, S.
(1994)
J. Biol. Chem.
269,
31430-31435 |
14. | Kume, N., and Gimbrone, M. A. (1994) J. Clin. Invest. 93, 907-911[Medline] [Order article via Infotrieve] |
15. | Kume, N., Cybulski, M. I., and Gimbrone, M. A. (1992) J. Clin. Invest. 90, 1138-1144[Medline] [Order article via Infotrieve] |
16. | Nakamo, T., Raines, E. W., Abaraham, J. A., Klagsbrun, M., and Ross, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1069-1073[Abstract] |
17. | Zembowicz, A., Jones, S. L., and Wu, K. K. (1995) J. Clin. Invest. 96, 1688-1692[Medline] [Order article via Infotrieve] |
18. |
Yuan, Y.,
Schoenwaelder, S. M.,
Salem, H. H.,
and Jackson, S. P.
(1996)
J. Biol. Chem.
271,
27090-27098 |
19. |
Engelman, B.,
Zieseniss, S.,
Brand, K.,
Page, S.,
Lentschat, A.,
Ulmer, A. J.,
and Gerlach, E.
(1999)
Atheroscler. Thromb. Vasc. Biol.
19,
47-53 |
20. | Sato, N., Kokame, K., Miyata, T., and Kato, H. (1998) Thromb. Haemost. 79, 217-221[Medline] [Order article via Infotrieve] |
21. |
Wong, J. T.,
Tran, K.,
Pierce, G. N.,
Chan, A. C., O, K.,
and Choy, P. C.
(1998)
J. Biol. Chem.
273,
6830-6836 |
22. |
Yamakawa, T.,
Eguchi, S.,
Yamakawa, Y.,
Motley, E. D.,
Numaguchi, K.,
Utsunomiya, H.,
and Inagami, T.
(1998)
Hypertension
31,
248-253 |
23. | Nishioka, H., Horiuchi, H., Arai, H., and Kita, T. (1998) FEBS Lett. 441, 63-66[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Fang, X.,
Gibson, S.,
Flowers, M.,
Furui, T.,
Bast, R. C.,
and Mills, G. B.
(1997)
J. Biol. Chem.
272,
13683-13689 |
25. | Ueno, Y., Kume, N., Miyamoto, S., Morimoto, M., Kataoka, H., Ochi, H., Nishi, E., Moriwaki, H., Minami, M., Hashimoto, N., and Kita, T. (1999) FEBS Lett. 457, 241-245[CrossRef][Medline] [Order article via Infotrieve] |
26. | Zhu, Y., Lin, J. H.-C., Liao, H.-L., Verna, L., and Stemerman, M. B. (1997) Biochim. Biophys. Acta 1345, 93-98[Medline] [Order article via Infotrieve] |
27. |
Zhang, R.,
Min, W.,
and Sessa, W. C.
(1995)
J. Biol. Chem.
270,
15320-15326 |
28. | Tang, J.-l., Zembowicz, A., Xu, X.-M., and Wu, K. K. (1995) Biochem. Biophys. Res. Commun. 213, 673-680[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Cieslik, K.,
Zembowicz, A.,
Tang, J.-L.,
and Wu, K. K.
(1998)
J. Biol. Chem.
273,
14885-14890 |
30. |
Cieslik, K.,
Lee, C.-M.,
Tang, J.-L.,
and Wu, K. K.
(1999)
J. Biol. Chem.
274,
34669-34675 |
31. |
Ma, A.,
and Abrams, C. S.
(1999)
J. Biol. Chem.
274,
28730-28735 |
32. |
Ma, A.,
Metjian, A.,
Bagrodia, S.,
Taylor, S. A.,
and Abrams, C. S.
(1998)
Mol. Cell. Biol.
18,
4744-4751 |
33. | Storz, P., Doppler, H., Pfizenmaier, K., and Muller, G. (1999) FEBS Lett. 464, 159-163[CrossRef][Medline] [Order article via Infotrieve] |
34. | Santibáñez, J. F., Iglesias, M., Frontelo, P., Martinez, J., and Quintanilla, M. (2000) Biochem. Biophys. Res. Commun. 273, 521-527[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Yamamoto, H.,
Atsuchi, N.,
Tanaka, H.,
Ogawa, W.,
Abe, M.,
Akira, T.,
and Ueno, H.
(1999)
Eur. J. Biochem.
264,
110-119 |
36. |
Hu, P. P.,
Shen, X.,
Huang, D.,
Liu, Y.,
Counter, C.,
and Wang, X.-F.
(1999)
J. Biol. Chem.
274,
35381-35387 |
37. |
Inoue, N.,
Venema, R. C.,
Sayegh, H. S.,
Ohara, Y.,
Murphy, T. J.,
and Harrison, D. G.
(1995)
Arterioscler. Thromb. Vasc. Biol.
15,
1255-1261 |
38. |
Lopez-Ilasaca, M.,
Crespo, P.,
Pellici, G.,
Gutkind, J. S.,
and Wetzker, R.
(1997)
Science
275,
394-397 |
39. |
Akiyama, T.,
Ishida, J.,
Nakagawa, S.,
Ogawara, H.,
Watanabe, S.,
Itoh, N.,
Shibuya, M.,
and Fukami, Y.
(1987)
J. Biol. Chem.
262,
5592-5597 |
40. | Toker, A., and Gantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve] |
41. | Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., and Waterfield, M. D. (1997) Trends Biochem. Sci. 22, 267-272[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Leopoldt, D.,
Hanck, T.,
Exner, T.,
Maier, U.,
Wetzker, R.,
and Nurnberg, B.
(1998)
J. Biol. Chem.
273,
7024-7029 |
43. | Van Biesen, T., Hawes, B. E., Luttrell, D. K., Krueger, K. M., Touhara, K., Porfiri, E., Sakaue, M., Luttrell, L. M., and Lefkowitz, R. J. (1995) Nature 376, 781-784[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Hawes, B. E.,
Luttrell, L. M.,
Van Biesen, T.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
12133-12136 |
45. |
Wang, Y.-X.,
Dhulipala, P. D. K.,
Li, L.,
Benovic, J. L.,
and Kotlikoff, M. I.
(1999)
J. Biol. Chem.
274,
13859-13864 |
46. |
Takeda, H.,
Matozaki, T.,
Takada, T.,
Noguchi, T.,
Yamao, T.,
Tsuda, M.,
Ochi, F.,
Fukunaga, K.,
Inagaki, K.,
and Kasuga, M.
(1999)
EMBO J.
18,
386-395 |
47. | Rodriguez, B., and Watson, S. P. (1994) FEBS Lett. 352, 335-338[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Bondeva, T.,
Pirola, L.,
Bulgarelli-Leva, G.,
Pubio, I.,
Reinhard, W.,
and Wymann, M. P.
(1998)
Science
282,
293-296 |
49. | Fulton, D., Gratton, J. P., McCabe, T. J., Fontana, J., Fujio, Y., Walsh, K., Franke, T. F., Papapetropoulos, A., and Sessa, W. C. (1999) Nature 399, 597-601[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Al-Shami, A.,
and Naccache, P. H.
(1999)
J. Biol. Chem.
274,
5333-5338 |
51. | Marrero, M. B., Schleffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., and Bernstein, K. E. (1995) Science 375, 247-250 |
52. |
Ali, M. S.,
Sayeski, P. P.,
Dirksen, L. B.,
Hayzer, D. J.,
Marrero, M. B.,
and Bernstein, K. E.
(1997)
J. Biol. Chem.
272,
23382-23388 |
53. |
Li, Z.,
Wahl, M. I.,
Equinoa, A.,
Stephens, L. R.,
Hawkins, P. T.,
and Witte, D. N.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13820-13825 |
54. |
Rikitake, Y.,
Kawashima, S.,
Yamashita, T.,
Ueyama, T.,
Ishido, S.,
Hotta, H.,
Hirata, K.,
and Yokoyama, M.
(2000)
Atheroscler. Thromb. Vasc. Biol.
20,
1006-1012 |
55. | Sato, N., Kokame, K., Shimokado, K., Kato, H., and Miyata, T. (1998) J. Biochem. (Tokyo) 123, 1119-1126[Abstract] |
56. |
Shingu, T.,
and Bornstein, P.
(1994)
J. Biol. Chem.
269,
32551-32557 |
57. |
Neish, A. S.,
Khachigian, L. M.,
Park, A.,
Baichwal, V. R.,
and Collins, T.
(1995)
J. Biol. Chem.
270,
28903-28909 |
58. |
Chen, Y. Q.,
Su, M.,
Walia, R. R.,
Hao, Q.,
Covington, J. W.,
and Vaughan, D. E.
(1998)
J. Biol. Chem.
273,
8225-8231 |
59. |
Mohandas, N.,
Wyatt, J.,
Mel, S. F.,
Rossi, M. E.,
and Shohet, S. B.
(1982)
J. Biol. Chem.
257,
6537-6543 |