From the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and the § Diabetes Unit, Laboratory of Clinical Investigation, NCCAM, National Institutes of Health, Bethesda, Maryland 20892-1755
Received for publication, November 7, 2002, and in revised form, December 30, 2002
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ABSTRACT |
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High density lipoprotein (HDL) activates
endothelial nitric-oxide synthase (eNOS), leading to increased
production of the antiatherogenic molecule NO. A variety of stimuli
regulate eNOS activity through signaling pathways involving Akt kinase
and/or mitogen-activated protein (MAP) kinase. In the present study, we
investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL
(10-50 µg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and
dominant negative Akt inhibited both HDL-mediated phosphorylation and
activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase)
inhibition or dominant negative PI3 kinase also blocked the
phosphorylation and activation of eNOS by HDL. Studies with genistein
and PP2 showed that the nonreceptor tyrosine kinase, Src, is an
upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase inhibition fully attenuated eNOS stimulation by HDL without
affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant
negative Akt did not alter HDL-induced MAP kinase activation. These
results indicate that HDL stimulates eNOS through common upstream,
Src-mediated signaling, which leads to parallel activation of Akt and
MAP kinases and their resultant independent modulation of the enzyme.
The risk for cardiovascular disease from atherosclerosis
is inversely proportional to serum levels of high density lipoprotein (HDL)1 (1, 2). HDL
classically serves to remove cholesterol from peripheral tissues in a
process known as reverse cholesterol transport. However, the mechanisms
by which HDL is atheroprotective are complex and not fully
understood, since circulating levels of HDL and the major HDL
apolipoprotein, apolipoprotein A-I, do not regulate reverse cholesterol
transport (3). We previously reported that HDL stimulates endothelial
nitric-oxide synthase (eNOS) activity in endothelial cells (EC) through
apolipoprotein A-I binding to scavenger receptor type I (SR-BI), the
high affinity HDL receptor (4). Similarly, HDL enhances endothelium-
and NO-dependent relaxation in aortas from wild-type but
not SR-BI knock-out mice. Recently, Li et al. (5) also
reported that HDL binding to SR-BI activates eNOS. The HDL-induced
increase in NO production may be critical to the atheroprotective
features of HDL, since diminished bioavailablity of endothelium-derived
NO has a key role in the early pathogenesis of
hypercholesterolemia-induced vascular disease and atherosclerosis
(6-8). However, the mechanisms by which HDL activates eNOS are yet to
be clarified.
eNOS is one of three isoenzymes that convert L-arginine to
L-citrulline plus NO. The activity of eNOS is regulated by
complex signal transduction pathways that involve various
phosphorylation events and protein-protein interactions. Many stimuli
modulate eNOS activity by activating kinases that alter the
phosphorylation of the enzyme (9-15). Akt kinase (also known as PKB)
activates eNOS by directly phosphorylating the enzyme at Ser-1179
(16-19). Akt itself is phosphorylated and activated by
phosphoinositide 3-kinase (PI3 kinase), which in turn is activated by a
tyrosine kinase (TK). Both receptor TK and nonreceptor TK are involved in PI3 kinase-Akt mediated eNOS activation by various agonists (19-22). In contrast to Ser-1179, phosphorylation of Thr-497 of eNOS
attenuates enzyme activity (12, 14, 15). eNOS is also modulated by MAP
kinases (23, 24), and unlike Akt, the effect of MAP kinases on eNOS
activity can be either positive or negative (9, 25-27). The role of
kinase cascades in signaling by HDL from SR-BI to eNOS is entirely unknown.
To better understand the basis of HDL action in endothelium, the
present investigation was designed to test the hypothesis that HDL
activation of eNOS entails the phosphorylation of the enzyme. We also
studied the potential roles of specific kinase cascades in HDL-mediated
eNOS stimulation. Using pharmacological inhibition or dominant negative
mutant forms of selective kinases in EC or COS M6 cells transfected
with eNOS and the HDL receptor, SR-BI, we investigated the involvement
of tyrosine kinases, PI3 kinase, Akt, and MAP kinases in HDL-mediated
eNOS activation. In addition to improving our specific understanding of
eNOS modulation, the elucidation of the signaling cascade(s) coupling
SR-BI to the enzyme provides important clues about multiple additional potential target of HDL action in EC.
Cell Culture and Transfection--
Primary ovine endothelial
cells were propagated and maintained as described previously (28) in
EGM-2 medium from BioWhittaker (Walkersville, MD). We have shown that
SR-BI expression is conserved in these cells up to at least passage 7 (4). For confirmation purposes, selected experiments were also done in
human aortic endothelial cells (purchased from BioWhittaker),
propagated in EGM-2 and used at passages 4-6, and in bovine aortic
endothelial cells, maintained in EGM-2 medium and studied at passages
5-8. COS M6 cells, which express a negligible amount of endogenous SR-BI and no eNOS, were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal calf serum (Invitrogen). COS M6
cells were transfected with various cDNAs using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instruction. The
transfected cells were used 48 h after transfection, and 70-80% transfection efficiency was typically achieved. cDNAs for wild-type eNOS, S1179A eNOS, AktAAA, AktMyr, and Sr Western Blot Analysis--
The methods used for Western blot
analysis generally followed those previously reported (34) using ECL
reagents (Amersham Biosciences) for chemiluminescence. Primary
antibodies were from the following sources. Anti-eNOS monoclonal
antibody was from BD Transduction Laboratories (San Diego, CA);
anti-phospho-Ser-1179 eNOS polyclonal antibody, anti-Akt polyclonal
antibody, and anti-phospho-Ser-473 polyclonal antibody were from Cell
Signaling Technology (Beverly, MA); anti-phospho-Thr-495 eNOS
polyclonal antibody and anti-ERK2 monoclonal antibody were from Upstate
Biotechnology, Inc. (Lake Placid, NY); anti-pMAPK polyclonal antibody
was from Promega (Madison, WI); and anti-PI3 kinase p85 subunit was
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish
peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies
were from Santa Cruz Biotechnology.
eNOS Activity Assay--
NOS activation was assessed in whole
cells by measuring [3H]L-arginine (45-70
Ci/mmol, 1 mCi/ml; PerkinElmer Life Sciences) conversion to
[3H]L-citrulline using previously reported
methods (28). In selected experiments, the cells were preincubated with
kinase inhibitors (genistein, PP2, wortmaninn, LY294002, or PD98059)
(Calbiochem) for 30 min at 37 °C. After the addition of HDL (10-50
µg/ml) or vascular endothelial growth factor (100 ng/ml) (Calbiochem)
and [3H]L-arginine, the cells were further
incubated for the indicated times. All findings were confirmed in at
least three independent studies.
Statistical Analysis--
Comparison between two groups was
accomplished using Student's t test, and comparison between
more than two groups employed Student's t tests with
Bonferroni correction for multiple comparisons. Significance was
accepted at the 0.05 level of probability.
HDL Stimulation of eNOS Requires Phosphorylation at
Ser-1179--
In order to determine whether HDL stimulation of eNOS
requires the phosphorylation of eNOS at Ser-1179, primary ECs were
incubated with HDL (10 or 50 µg/ml) for 20 min or with VEGF (100 ng/ml) for 5 min, serving as a positive control. The phosphorylation was detected by Western blotting using anti-phospho-specific eNOS antibody. As shown in Fig. 1a,
eNOS phosphorylation was observed with 10 or 50 µg/ml HDL treatment
as well as with 100 ng/ml VEGF treatment. Phosphorylation of eNOS by
HDL (30 µg/ml) was observed as early as 5 min and reached maximum at
10-20 min (data not shown). Comparable eNOS phosphorylation by HDL was
seen in the ovine, human, and bovine EC (data not shown).
We next determined whether the phosphorylation of eNOS at Ser-1179 is
required for the activation of eNOS by HDL. Either wild-type eNOS or
S1179A mutant eNOS was expressed in COS M6 cells along with SR-BI, and
the phosphorylation and activation of eNOS was assessed in response to
HDL (10 or 30 µg/ml). No phosphorylation of eNOS by HDL was detected
in S1179A-transfected cells, whereas wild-type eNOS phosphorylation was
apparent (Fig. 1b). In parallel, HDL did not stimulate eNOS
activation in cells transfected with S1179A eNOS, whereas HDL
stimulated eNOS activation in wild-type eNOS-expressing cells (Fig.
1c).
The dephosphorylation of eNOS at Thr-497 is involved in the activation
of eNOS by bradykinin and VEGF (9-11, 15). We investigated the effect
of HDL on the phosphorylation state of eNOS at Thr-497 using the
phospho-specific antibody against the site. As shown in Fig.
1d, in contrast to the observed change in Ser-1179
phosphorylation, HDL had no notable effect on the phosphorylation state
of Thr-497 of eNOS.
HDL Stimulation of eNOS Involves Akt Activation--
Since certain
stimuli such as estrogen and VEGF activate eNOS through Akt-mediated
phosphorylation of the enzyme, we investigated the involvement of Akt
in the phosphorylation and stimulation of eNOS by HDL. Akt is activated
by PI3 kinase through recruitment to the plasma membrane, where Akt
becomes phosphorylated at Ser-473 and Ser-308 (31, 35-38). ECs were
incubated with HDL (10 or 50 µg/ml) for 20 min. The phosphorylation
of Akt at Ser-473 was detected with HDL (Fig.
2a), and it occurred as early
as 5 min and reached a maximum at 30 min (Fig. 2b). We also
found that HDL (30 µg/ml, 20 min) caused the recruitment of Akt to
the plasma membrane (Fig. 2c), further indicating the
activation of Akt by the lipoprotein.
To determine whether Akt is responsible for the phosphorylation of eNOS
by HDL, either sham plasmid, a dominant negative Akt mutant (AktAAA,
designated DN), or a constitutively active Akt mutant (AktMyr,
designated CA) was expressed in COS M6 cells along with wild type eNOS
and SR-BI. The transfected cells were incubated with HDL (30 µg/ml)
for 0-30 min, and the phosphorylation of eNOS at Ser-1179 was
assessed. As shown in Fig. 2d, HDL caused phosphorylation of
eNOS in cells expressing endogenous wild-type Akt (sham), but no eNOS
phosphorylation was observed in cells expressing dominant negative Akt
(DN). Constitutively active Akt caused phosphorylation of eNOS in the
absence of HDL (CA, 0 min). These results suggest that HDL stimulates
Akt, which in turn phosphorylates eNOS.
PI3 Kinase Is Upstream of Akt Activation and eNOS
Activation--
Since PI3 kinase is a known upstream activator of Akt,
we next examined the effect of PI3 kinase inhibitors on both Akt and eNOS phosphorylation and activation. Primary ECs were preincubated with
the PI3 kinase inhibitor wortmannin (50 µM) for 20 min
before HDL stimulation (30 µg/ml), and the phosphorylation of Akt at Ser-473 was assessed. In parallel experiments, eNOS activity was measured. Wortmannin inhibited both Akt phosphorylation (Fig. 3a) and eNOS activation (Fig.
3b). In order to examine the involvement of PI3 kinase in
eNOS phosphorylation by HDL, the effect of another PI3 kinase
inhibitor, LY294002, was assessed. ECs were preincubated with LYL294002
(50 µM) for 20 min before HDL (30 µg/ml) stimulation for 0-20 min. As shown in Fig. 3c, LY294002 inhibited
HDL-mediated eNOS phosphorylation. To further confirm the involvement
of PI3 kinase in eNOS phosphorylation, sham vector or dominant negative PI3 kinase (Sr A Tyrosine Kinase Is Upstream of eNOS Activation by
HDL--
Tyrosine kinases frequently serve as upstream stimulators of
PI3 kinase. The involvement of nonreceptor tyrosine kinase (Src family)
has been demonstrated for eNOS stimulation by a variety of factors (21,
22). We determined the effect of the tyrosine kinase inhibitor,
genistein, on eNOS activation and phosphorylation by HDL. Primary
cultured endothelial cells were preincubated with or without Genistein
(50 µM) for 20 min before the addition of HDL (30 µg/ml) for 0-20 min. Phosphorylation states and activation of eNOS
were assessed. As shown in Fig. 4,
a and b, Genistein abrogated both eNOS
phosphorylation and activation. We next examined the involvement of the
Src kinase family. Endothelial cells were incubated with or without the
Src kinase-specific inhibitor, PP2 (0.1 µM), before HDL
stimulation (10 µg/ml, 20 min). PP2 inhibited both eNOS activation
and phosphorylation by HDL (Fig. 4, c and d).
These results suggest that HDL stimulates tyrosine kinases that
are most likely Src family kinases, which in turn activate the PI3 kinase-Akt pathway to ultimately lead to eNOS
phosphorylation.
HDL Activation of MAP Kinase--
The role of MAP kinases in HDL
stimulation of eNOS is not known. As such, we studied the effect of HDL
on MAP kinase activation in primary ECs. Cells were stimulated with HDL
(10 or 50 µg/ml) for 20 min, using fetal calf serum (10%, 5 min) or
VEGF (100 ng/ml, 5 min) as a positive control. The activation state of
MAP kinase was assessed by phospho-MAP kinase-specific antibody. As
shown in Fig. 5a, HDL
stimulated MAP kinase phosphorylation. The phosphorylation was detected
as early as 2 min and reached maximal phosphorylation at 30 min (Fig.
5b). The activation of MAP kinase by HDL was suppressed by
the MEK inhibitor PD98059 (Fig. 5c). Both PI3 kinase
inhibition (wortmannin (Wort)) (Fig. 5d) and Src
kinase inhibition (PP2) (Fig. 5e) also attenuated MAP kinase
activation by HDL, indicating that PI3 kinase and Src kinase are the
upstream effectors of MAP kinase activation by HDL. To determine
whether MAP kinase phosphorylation is required for eNOS activation by
HDL, EC were pretreated with MEK inhibitor (PD98059), and eNOS
activation was assessed. As shown in Fig. 5f, PD98059 (50 µM) completely abrogated eNOS activation by HDL.
Comparable MAP kinase activation by HDL was observed in the ovine,
human, and bovine EC (data not shown).
To determine whether MAP kinase activates eNOS through the
phosphorylation of the enzyme at Ser-1179, we examined the effect of
MEK inhibition on eNOS phosphorylation. As shown in Fig.
6a, PD98059 prevented MAP
kinase phosphorylation, but it did not attenuate eNOS phosphorylation,
suggesting that MAP kinase contributes to eNOS activation through a
different mechanism.
To further assess possible cross-talk between Akt and MAP kinase
signaling pathways, we determined whether the MAP kinase cascade
activates Akt. EC were treated with the MEK inhibitor, PD98059, and the
phosphorylation of Akt was assessed. As shown in Fig. 6b,
MEK inhibition had no effect on Akt phosphorylation by HDL, suggesting
that MAP kinase is not an upstream effector of Akt. We also determined
the effect of an Akt dominant negative mutant on MAP kinase
phosphorylation by HDL. As shown in Fig. 6c, dominant
negative Akt had no effect on HDL stimulation of MAP kinase, but the
phosphorylation of eNOS was fully prevented. This suggests that Akt
activation is not required for MAP kinase phosphorylation induced by
HDL.
In the present study, we have demonstrated that HDL stimulation of
eNOS occurs through two kinase pathways (Fig.
7). By binding to SR-BI, HDL causes eNOS
phosphorylation at Ser-1179 via TK-PI3 kinase-mediated activation of
Akt kinase. However, concomitant TK-PI3 kinase-mediated stimulation of
MAP kinase is necessary in order to enhance eNOS enzymatic activity in
response to the lipoprotein.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
85 were prepared as described previously (20). A mutant of eNOS with Ser-1179 converted to
alanine (S1179A eNOS) is unable to be phosphorylated at that site. The
triple Akt mutant AktAAA (K179A,T308A,S403A) is enzymatically inactive (29, 30), whereas the Akt mutant generated by fusing a
myristoylation signal to its amino terminus (AktMyr) is membrane-bound and constitutively active (31, 32). The mutant form of PI3 kinase
lacking the domain in the p85 subunit that is required for interaction
with the catalytic subunit (Sr
85) works as a dominant negative
mutant (33).
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ABSTRACT
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Fig. 1.
HDL stimulates eNOS through Ser-1179
phosphorylation. a, endothelial cells were treated with
HDL (10 or 50 µg/ml) for 20 min or VEGF (100 ng/ml) for 5 min at
37 °C. Cell lysates were analyzed by immunoblot using polyclonal
anti-phospho-serine 1179 eNOS antibody or monoclonal eNOS antibody.
b, COS M6 cells were transfected with SR-BI cDNA and
either cDNA for vector (sham), S1179A eNOS mutant (S1179A), or
wild-type eNOS for 48 h. The cells were treated with HDL (10 µg/ml) for 20 min, and cell lysates were analyzed by immunoblot using
polyclonal anti-phospho-serine 1179 eNOS antibody or monoclonal eNOS
antibody. c, COS M6 cells were transfected with SR-BI
cDNA and either cDNA for wild-type eNOS or S1179A mutant eNOS
for 48 h. eNOS activity was then assessed by measuring
[3H]L-arginine to
[3H]L-citrulline conversion over 20 min in
cells exposed to vehicle alone (basal (B)) or HDL (10 µg/ml). *, p < 0.05 versus basal.
d, endothelial cells were treated with HDL (30 µg/ml) for
20 min at 37 °C, and cell lysates were analyzed by immunoblot using
polyclonal anti-phospho-Thr-495 eNOS, anti-phospho-Ser-1179 eNOS, or
monoclonal eNOS antibodies.
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Fig. 2.
HDL stimulates eNOS phosphorylation through
Akt/PKB activation. a, endothelial cells were treated
with HDL (10, 50 µg/ml) for 20 min at 37 °C. Cell lysates were
analyzed by immunoblot using polyclonal anti-phospho-Ser-473 Akt/PKB
antibody (pAkt) or polyclonal Akt/PKB (Akt)
antibody. b, endothelial cells were treated with HDL (30 µg/ml) for 0-60 min, at 37 °C. Cell lysates were analyzed by
immunoblot using polyclonal anti-phospho-serine 473 Akt/PKB
(pAkt) antibody or polyclonal Akt/PKB (Akt)
antibody. c, endothelial cells were treated with or without
HDL (30 µg/ml) for 20 min, and cytosol (Cyt) and plasma
membrane (PM) fractions were isolated. The fractions were
analyzed by immunoblot using Akt/PKB polyclonal antibody or monoclonal
eNOS antibody. d, COS M6 cells were transfected with SR-BI
and eNOS cDNAs and with cDNA for either vector
(sham), dominant-negative AktAAA mutant (DN), or
constitutively active AktMyr (CA) for 48 h. Transfected
cells were treated with HDL (30 µg/ml) for 0-30 min at 37 °C. The
cells lysates were analyzed by immunoblot using anti-phospho-Ser-1179
eNOS polyclonal antibody (peNOS) or eNOS monoclonal
antibody.
85) was transfected in COS M6 cells expressing eNOS
and SR-BI, and the phosphorylation state of eNOS was assessed. As shown
in Fig. 3d, overexpression of dominant negative PI3 kinase inhibited eNOS phosphorylation at Ser-1179. These results suggest that
HDL phosphorylates and stimulates eNOS through PI3 kinase-mediated Akt
activation.
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Fig. 3.
PI3 kinase is involved in HDL-induced,
Akt-mediated eNOS phosphorylation and activation. a,
endothelial cells were pretreated with vehicle alone, or the PI3 kinase
inhibitor wortmannin (Wort; 50 µM) for 30 min.
The cells were then incubated with HDL (30 µg/ml) for 20 min, and
cell lysates were analyzed by immunoblot using anti-phospho-Ser-473
Akt/PKB (pAkt) polyclonal antibody or anti-Akt polyclonal
antibody. b, after pretreatment with vehicle alone or
wortmannin (HDL + Wort), eNOS activity was
assessed by measuring [3H]L-arginine to
[3H]L-citrulline conversion over 20 min in
cells exposed to vehicle (basal (B)), HDL (10 µg/ml), or
HDL plus wortmannin (50 µM). *, p < 0.05 versus basal; , p < 0.05 versus HDL alone. c, endothelial cells were
pretreated with vehicle alone or the PI3 kinase inhibitor LY294002 (50 µM) for 30 min. The cells were then treated with HDL (30 µg/ml) for 0, 10, or 20 min at 37 °C. Cell lysates were analyzed
by immunoblot using anti-phospho-Ser-1179 eNOS polyclonal antibody
(peNOS) or anti-eNOS monoclonal antibody (eNOS).
d, COS M6 cells were transfected with SR-BI and eNOS
cDNAs and with cDNA for either vector (sham) or
dominant negative PI3 kinase (Sr
p85) for
48 h. The cells were then treated with HDL (30 µg/ml) for 20 min, and cell lysates were analyzed by immunoblot using
anti-phospho-Ser-1179 eNOS polyclonal antibody (peNOS),
anti-eNOS monoclonal antibody (eNOS), or the anti-p85
subunit of PI3 kinase polyclonal antibody.
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Fig. 4.
Tyrosine kinase is involved in eNOS
phosphorylation and stimulation by HDL. a, endothelial
cells were pretreated with vehicle or genistein (50 µM)
for 30 min and then stimulated with HDL (30 µg/ml) for 0, 10, or 20 min. Cell lysates were analyzed by immunoblot using
anti-phospho-Ser-1179 eNOS polyclonal antibody (peNOS) or
anti-eNOS monoclonal antibody (eNOS). b, after
pretreatment with vehicle or genistein, eNOS activity was assessed by
measuring [3H]L-arginine to
[3H]L-citrulline conversion over 20 min in
cells exposed to vehicle (basal (B)), HDL (10 µg/ml), or
HDL plus genistein (50 µM). *, p < 0.05 versus basal; , p < 0.05 versus HDL alone. c, endothelial cells were
pretreated with PP2 (0.1 µM) for 30 min and exposed to
vehicle (B) or HDL (10 µg/ml) for 20 min. eNOS activity
was assessed as described for b. *, p < 0.05 versus basal;
, p < 0.05 versus HDL alone. d, endothelial cells were
pretreated with PP2 (0.1 µM) for 30 min and exposed to
vehicle or HDL (30 µg/ml) for 20 min. Cell lysates were analyzed by
immunoblot using anti-phospho-eNOS polyclonal antibody
(peNOS) or anti-eNOS monoclonal antibody
(eNOS).
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Fig. 5.
HDL activation of eNOS requires MAP kinase
stimulation through Src family kinase and PI3 kinase.
a, endothelial cells were treated with HDL (10 or 50 µg/ml) for 20 min, VEGF (100 ng/ml) for 5 min, or medium
containing 10% fetal calf serum (FCS) for 5 min. Cell
lysates were analyzed by immunoblot using anti-phospho-MAP kinase
polyclonal antibody (pMAPK) or anti-ERK2 monoclonal antibody
(ERK2). b, endothelial cells were treated with
HDL (30 µg/ml) for 0-60 min. Cell lysates were analyzed by
immunoblot using anti-phospho-MAP kinase polyclonal antibody or
anti-ERK2 monoclonal antibody. c, endothelial cells were
pretreated with the MEK inhibitor PD98059 (0-50 µM) for
30 min and then treated with or without HDL (30 µg/ml) for 20 min.
Cell lysates were analyzed by immunoblot using anti-phospho-MAP kinase
polyclonal antibody or anti-ERK2 monoclonal antibody. d,
endothelial cells were pretreated with PD98059 (50 µM) or
wortmannin (Wort) (1, 10, or 50 µM) for 30 min
and then treated with or without HDL (30 µg/ml) for 20 min. Cell
lysates were analyzed by immunoblot using anti-phospho-MAP kinase
polyclonal antibody or anti-ERK2 monoclonal antibody. e,
endothelial cells were pretreated with PP2 (0-10 µM) for
30 min and then treated with or without HDL (30 µg/ml) for 20 min.
Cell lysates were analyzed by immunoblot using anti-phospho-MAP kinase
polyclonal antibody or anti-ERK2 monoclonal antibody. f,
endothelial cells were pretreated with vehicle alone (basal
(B)) or the MEK inhibitor PD98059 (50 µM) for
30 min, and then eNOS activity was assessed by measuring
[3H]L-arginine to
[3H]L-citrulline conversion in cells exposed
to vehicle (basal), HDL (10 µg/ml), or HDL plus PD98058 (50 µM) over 20 min. *, p < 0.05 versus basal; , p < 0.05 versus HDL alone.
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Fig. 6.
MAP kinase and Akt/PKB activate eNOS in an
independent manner. a, endothelial cells were
pretreated with vehicle or the MEK inhibitor PD98059 (50 µM) for 30 min and then treated with HDL (30 µg/ml) for
0, 10, or 20 min. Cell lysates were analyzed by immunoblot using
anti-phospho-Ser-1179 eNOS polyclonal antibody (peNOS),
anti-eNOS monoclonal antibody (eNOS), anti-phospho-MAP
kinase polyclonal antibody (pMAPK), or anti-ERK2 monoclonal
antibody (ERK2). b, endothelial cells were
pretreated with vehicle or the MEK inhibitor PD98059 (50 µM) for 30 min, incubated with vehicle or HDL (30 µg/ml) for 20 min, and cell lysates were analyzed by immunoblot using
anti-phospho-serine 473 Akt (pAkt) polyclonal antibody or
anti-Akt polyclonal antibody. c, COS M6 cells were
transfected with SR-BI and eNOS cDNAs and either vector
(sham), dominant negative Akt/PKB (DN), or
constitutively active Akt/PKB (CA) cDNA for 48 h.
Cells were treated with HDL (30 µg/ml) for 0, 5, or 10 min, and the
cell lysates were analyzed by immunoblot as described for
a.
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Fig. 7.
Signaling pathways mediating HDL stimulation
of eNOS. HDL binding to SR-BI leads to the activation of a
tyrosine kinase (Src), which causes the activation of PI3 kinase. The
process(es) coupling SR-BI to tyrosine kinase is yet to be determined.
PI3 kinase induces the independent activation of both Akt and MAP
kinase pathways. Akt phosphorylates Ser-1179 of eNOS, and the basis for
MAP kinase-mediated activation of eNOS is currently unknown.
Importantly, the concerted impacts of both the Akt and MAP kinase
cascades are required for HDL-induced stimulation of eNOS enzymatic
activity.
The phosphorylation of eNOS regulates the activation of the enzyme by various stimuli including VEGF, estrogen, and shear stress (16-19). We have found that HDL also causes the phosphorylation of eNOS at Ser-1179 and that the phosphorylation is required for the activation of enzymatic activity (Fig. 1, a-c). In contrast to Ser-1179, HDL had no effect on the phosphorylation state of Thr-497 (Fig. 1d), suggesting that the lipoprotein does not regulate eNOS activation through the dephosphorylation of that residue. We also identified Akt as the kinase responsible for HDL-induced eNOS phosphorylation at Ser-1179 (Fig. 2, a-d). HDL stimulated Akt phosphorylation at Ser-473, one of the major phosphorylation sites of Akt in the regulatory domain that is often targeted by PI3 kinase (Fig. 2, a and b). The kinase-inactive mutant of Akt (AktAAA) efficiently inhibited HDL-induced eNOS phosphorylation. Furthermore, we determined whether PI3 kinase is an upstream regulator of Akt in the pathway leading to eNOS activation (Fig. 3, a-d). PI3 kinase activates Akt by recruiting the latter to the plasma membrane, which allows the phosphorylation of Akt at two key regulatory sites (Thr-308 and Ser-403), and Akt was recruited to plasma membrane by HDL stimulation (Fig. 2c). The inhibition of PI3 kinase by wortmannin resulted in decreased Akt phosphorylation at Ser-473 (Fig. 3a). In addition, the selective inhibitor of PI3 kinase LY294002 or dominant negative PI3 kinase also led to decreased eNOS phosphorylation and activation by HDL (Fig. 3, b-d). These cumulative results indicate that PI3 kinase stimulation of Akt leading to eNOS phosphorylation at Ser-1179 is critically involved in the activation of the enzyme by HDL (Fig. 7).
Additional proximal signaling events have been elucidated. We have demonstrated that a protein TK, most likely an Src family kinase, is a further upstream stimulator of the PI3 kinase/Akt pathway (Fig. 4, a-d). Typical PI3 kinase has regulatory subunits with two Src homology 2 domains that allow the enzyme to be activated by phosphotyrosine residues of a TK (39, 40). We speculate that HDL binding to SR-BI directly or indirectly causes tyrosine phosphorylation of Src kinase. Both the C-terminal and N-terminal domains of SR-BI are facing the cytoplasm and thus available for interaction with other proteins. At present, there is no evidence that SR-BI binds to a receptor TK or a nonreceptor TK. Further, it is not known whether HDL causes the phosphorylation of a tyrosine residue (tyrosine 489) within the C-terminal cytoplasmic domain of SR-BI, which could potentially bind to Src homology 2-containing adaptor molecules. Detailed studies of possible SR-BI-tyrosine kinase interactions are now warranted.
In addition to modulation by PI3 kinase-Akt, MAP kinases are also known to play a role in eNOS regulation by certain agonists (9, 25-27). In the present study, we have shown that HDL stimulates MAP kinase phosphorylation (Fig. 5, a-c) and that both PI3 kinase and Src kinase are upstream activators of MAP kinase activation by HDL (Fig. 5, d and e). It has been previously observed that HDL stimulates MAP kinase in EC as well as other cell types (41-43). However, the mechanisms mediating that process were not known prior to the current studies. Furthermore, the activation of MAP kinase is absolutely required for eNOS activation (Fig. 5f). However, MAP kinase activation does not play a role in HDL-induced Akt phosphorylation (Fig. 6b) or in eNOS phosphorylation (Fig. 6a). Moreover, dominant negative Akt had no effect on MAP kinase activation by HDL (Fig. 6c). Thus, there is no cross-talk between the Akt kinase and MAP kinase pathways, and the activation of both pathways is necessary for enhanced enzymatic activity in response to HDL (Fig. 7). Further experiments will be needed to elucidate the mechanism(s) by which MAP kinase contributes to eNOS stimulation by HDL, including the potential modulation of intracellular Ca2+ homeostasis.
Recently, Li et al. (5) reported that HDL stimulates eNOS in a ceramide-dependent manner. C2-ceramide stimulates MAP kinase via tyrosine kinase and PI3 kinase-mediated mechanisms in cultured airway smooth muscle cells (44, 45). It is possible that the HDL stimulation of MAP kinase observed in the present work occurred through ceramide production. Li et al. (5) also showed that HDL does not induce Akt kinase activation in CHO cells expressing eNOS and SR-BI. However, their studies were limited to assessments of Akt phosphorylation in the transfected CHO cells. Discrepancies between the observations made in the two studies may be related to the use of different cell paradigms. In the present work, we used ECs for endogenous Akt phosphorylation and COS M6 cells for cell transfection in which dominant negative mutants were employed to show that Akt is responsible for eNOS phosphorylation and activation (Fig. 2). We also used a combination of PI3 kinase inhibitors (Fig. 3) and dominant negative mutants (Figs. 2 and 3) to confirm the involvement of the PI3 kinase/Akt pathway in HDL-induced eNOS phosphorylation and activation. The eNOS phosphorylation observed in the ovine EC was also confirmed in the human and bovine endothelium. As such, multiple approaches have been employed in the current studies to implicate a key role for Akt kinase and phosphorylation in eNOS activation by HDL.
SR-BI is the high affinity HDL receptor, and HDL binding to SR-BI is
required for HDL-mediated cholesterol flux (46, 47). We showed
previously that HDL binding to SR-BI is also required for eNOS
activation by HDL (4). However, the initiating event that occurs upon
HDL binding to SR-BI to cause the proximal processes in signal
transduction by the lipoprotein is not known. We speculate that SR-BI
mediates the effect of HDL by two possible mechanisms. First,
alterations in membrane cholesterol pools may be involved. SR-BI
mediates changes in the cholesterol content of the plasma membrane, and
SR-BI and eNOS both reside in cholesterol-rich microdomains known as
caveolae, which are most likely a subset of lipid rafts, which contain
various signaling molecules including MAP kinases. It has been
demonstrated that cholesterol alterations induced by -cyclodextrin
activate MAP kinases (48). Along with potential cholesterol-related
mechanisms, there may be involvement of SR-BI C-terminal binding
protein(s). Using antibody blockade in isolated EC plasma membranes, we
previously showed that the C-terminal domain of SR-BI plays a role in
HDL-mediated eNOS stimulation (4). It is possible that a protein
binding to the C terminus of SR-BI mediates signal transduction from
SR-BI to a downstream effector such as a G protein and/or Src kinase. A
PDZ-containing protein, PDZK1 (also known as CLAMP, Diphor-1, CAP70, or
NaPi-Cap1) has been shown to bind to the C-terminal domain of SR-BI and
to be involved in cholesterol regulation (49). Now knowing that a Src
kinase is critically involved in the proximal signaling events induced
by HDL binding to SR-BI, in depth experimentation focused on the
possible roles of both cholesterol regulation and SR-BI adaptor
proteins in the upstream process can be pursued.
The intricate regulation of kinase cascades by HDL shown in this study
may help explain the impact of HDL on various other functions in EC.
For example, HDL stimulates the migration and proliferation of EC
(50-52). Since Akt is known to have a role in apoptosis, and MAP
kinase is involved in the proliferation and migration of EC during
reendothelialization after injury to the arterial wall (53, 54), it is
possible that the modulation of these pathways by HDL may be critical
to the regulation of EC turnover and movement. As such, our finding
that HDL is a potent stimulus of various kinases in EC enhances
both our specific understanding of the capacity of the
lipoprotein to modulate NO production and our overall knowledge of
other mechanisms by which HDL may be atheroprotective.
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ACKNOWLEDGEMENTS |
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We thank Helen Hobbs (McDermott Center for Human Growth and Development and Departments of Molecular Genetics and Biochemistry) for providing HDL and for insightful discussions and Divya Seetharam for valuable help with experiments of bovine aortic endothelial cells. We are indebted to Marilyn Dixon for preparing this manuscript.
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FOOTNOTES |
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* This work was supported by the Scientist Development Program of the American Heart Association (to C. M.) and National Institute of Health Grants HL 58888, HL 53546, and HD 30276 (to P. W. S.).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: Dept. of Pediatrics,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., Dallas, TX 75390. Tel.: 214-648-4574; Fax: 214-648-2481; E-mail:
chieko.mineo@utsouthwestern.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M211394200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; eNOS, endothelial nitric-oxide synthase; SR-BI, scavenger receptor B-I; PKB, protein kinase B; PI3 kinase, phosphoinositide 3-kinase; EC, endothelial cell; TK, tyrosine kinase; VEGF, vascular endothelial growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CHO, Chinese hamster ovary.
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