Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1159-1164
doi: 10.1161/hq0701.092473
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1159.)
© 2001 American Heart Association, Inc.
LDL-Activated p38 in Endothelial Cells Is Mediated by Ras
Yi Zhu;
Hailing Liao;
Nanping Wang;
Kuo-Sheng Ma;
Lynne K. Verna;
John Y.-J. Shyy;
Shu Chien;
Michael B. Stemerman
From the Division of Biomedical Sciences, University of California,
Riverside, and the Department of Bioengineering and Whitaker Institute of
Biomedical Engineering, University of California, San Diego, La Jolla (S.C.),
Calif.
Correspondence to Yi Zhu, MD, Division of Biomedical Sciences, University of California, Riverside, CA 92521. E-mail yi.zhu{at}ucr.edu
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Abstract
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AbstractEndothelial
dysfunction is a major atherogenic
proinflammatory event. LDL causes
the activation and phenotypic
changes of cultured vascular
endothelial cells (ECs). We previously
reported that
LDL activates c-Jun and AP-1 in ECs. In this study,
we
demonstrated that p38ATF-2 is activated by LDL in
human ECs
and that this activation is mediated by Ras. When
ECs are incubated
with LDL in pathophysiological concentrations,
the
p38-mediated ATF-2 phosphorylation and ATF-2
transactivation
are increased in a time- and dose-dependent manner. To
elucidate
the upstream mechanism in LDL-activated p38 in ECs,
we demonstrate
that LDL increases Ras translocation from the cytoplasm
to the
cellular membrane, with concurrent increases in Ras binding
activity
to GSTRaf-1. Overexpression of RasN17, a dominant negative
mutant
of Ras, attenuates the LDL-induced increases in (1)
phosphorylation
of ATF-2, (2)
phosphorylation of c-Jun, (3) AP-1 binding, and
(4)
AP-1driven luciferase activity. To study the effect
of p38 in the
regulation of an LDL targeting gene, we show that
a specific p38
inhibitor attenuates LDL-induced E-selectin at
the mRNA
level. Thus, LDL activates both p38 and JNK signaling
pathways
through Ras activation, and furthermore, these events
may play an
important role in LDL-induced endothelial
activation.
Key Words: p38 ATF-2 Ras LDL ECs
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Introduction
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Endothelial
dysfunction is one of the earliest proinflammatory
vascular events
leading to
atherosclerosis.
1
Native LDL has
been implicated in initiating
endothelial cell (EC)
dysfunction.
2 When incubated
with LDL, ECs in culture are activated, and
several genes
involved in atherogenesis, including E-selectin,
intercellular adhesion
molecule (ICAM)-1, and vascular cell
adhesion molecule (VCAM)-1, are
upregulated.
3 4 5 6
EC activation
by LDL involves mobilization of calcium, activation of
protein
kinases, and an increase in the transactivation of AP-1
transcription
factor.
3 4 7 8 9
The effects of LDL on intracellular signal transduction
leading to EC
activation, however, have been studied only to
a limited
extent.
In response to stimulation by mitogens, cytokines,
UV irradiation, and other environmental stresses, membrane-associated
small GTPase molecules, eg, p21-Ras, are activated. Ras cycles
between an active GTP-bound and an inactive GDP-bound state,
functioning as a molecular switch in response to cell-activating
stimuli. Ras can trigger at least 3 diverging mitogen-activated
protein kinase
(MAPK) cascades. The first cascade is mediated by
Raf-1 activation and transmits signals through MAPK/extracellular
signal-regulated kinase (ERK) kinase
(MEK)1/2 to activate ERK. The second cascade
operates through MEKK1 and JNKK to activate c-Jun NH2-terminal
kinases (JNKs). The third cascade leads to p38 activation. Efficient
activation of p38 requires phosphorylation of Thr-180
and Tyr-182. At least 3 Thr/Thy kinases (MKK3, MKK4/SEK1, and MKK6)
phosphorylate and activate p38. This leads to the
activation of multiple transcription factors, such as ATF-2 and CHOP,
that induce the expression of proinflammatory genes, such as
E-selectin.10
ATF-2 can form heterodimers with c-Jun, which positively
regulate c-Jun promoter
activity.11 Recently, we
found that LDL increases AP-1 activity and activates the
JNKc-Jun pathway but not the ERK/c-Fos pathway in human
ECs.8 9 This report
was designed to study whether LDL activates the p38ATF-2
pathway in ECs and to elucidate the upstream signaling involved. Our
study demonstrates that LDL enhances Ras translocation to membrane and
Ras activation in human umbilical vein ECs (HUVECs) and that these
events activate both p38ATF-2 and JNKc-Jun signaling
pathways. Furthermore, we show that LDL-induced E-selectin is blocked
by a specific p38 inhibitor.
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Methods
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Cell Culture and LDL Isolation
HUVECs were isolated and maintained as described
previously.
8 All experiments
were performed with cells up to passage 3 in
EC medium and cultured to
confluence before LDL treatment. LDL
was isolated from nonfrozen human
plasma as
described.
8 9 The
LDL
preparations contained <0.0005 U endotoxin/mg
cholesterol
as determined by the chromogenic
Limulus test
(BioWhittaker).
For all studies, LDL was used at
a final cholesterol culture
concentration of 240 mg/dL
(6.24 mmol/L).
Western Blotting Analysis
Cellular membrane and cytosolic proteins from
whole-cell lysate were isolated as previously
described.12 Western
analyses with antibodies against H-Ras (Transduction
Laboratories) or Anti-ACTIVE MAPK
pAb antibody (Promega) were performed as
previously
described.9 12
Affinity Precipitation/Immunoblot
of Activated Ras
GST fusion protein, corresponding to the human
Ras-binding domain (RBD, residues 1 to 149) of Raf-1 bound to
glutathione agarose, was from Upstate
Biotechnology. The procedure used to measure Ras binding on
GSTRaf-1 RBD was described by company protocol (Upstate
Biotechnology).
ATF-2 and c-Jun Phosphorylation
Assay
The bacterial expression vector GSTATF-2 (residues
1 to 109)13 was provided by
R.J. Davis (University of Massachusetts Medical School). A GSTATF-2
fusion protein was isolated and purified. The assay for c-Jun
phosphorylation was performed as
described.9 The procedures
for the ATF-2 phosphorylation assay were similar to
those for c-Jun, except that the cell extracts were incubated with
GSTATF-2. In the study of the role of p38 on ATF-2
phosphorylation, p38 protein was immunoprecipitated
with an antibody against p38. Then, the immunoprecipitated p38 was
incubated with agarose-bound GSTATF-2 in a kinase
buffer.14
Recombinant Adenoviruses
The recombinant adenovirus Ad-RasN17 encoding for
RasN17 was constructed as described
previously.15 The
adenoviruses were plaque-purified, expanded and titrated in 293 cells,
and purified by cesium chloride
methods.16 For adenoviral
infection, confluent HUVECs were exposed to adenoviral vectors
(Ad-RasN17 or Ad-ß-gal as control) at a multiplicity of infection of
100 to 500 for 2 hours. After the viruses had been washed out, HUVECs
were continuously incubated for 18 to 24 hours before the
treatment.16
Plasmids and Transfection
For transactivation experiments, we used the
Targefect transfection method (Targeting Systems). The in vivo
trans-reporting system was purchased from
Stratagene. This system includes a pFAc-Jun,
pFCATF-2, pFA2-Elk, or pFA-CHOP (CHOP is a transcription factor
specific response to p38 activation) as an activator
plasmid, and a reporter plasmid (pFR-Luc). pFC-dbd plasmid and pFC-MEKK
plasmid were used as a negative and positive control, respectively.
pRSV-ß-gal was cotransfected as a transfection control. After 24
hours of LDL or phorbol 12-myristate 13-acetate (PMA) exposure,
samples were collected and assayed for luciferase activity. The results
were normalized against
ß-galactosidase.9
Electrophoretic Mobility Shift Assay
After infection by the adenoviral construct
Ad-RasN17, HUVEC monolayers on 100-mm dishes were exposed to 240 mg/dL
of LDL for 6 hours or to 50 ng/mL PMA for 2
hours.8 Nuclear extracts were
prepared, and an electrophoretic mobility shift assay was performed
with consensus sequences for AP-1 and nuclear factor (NF)-
B as
described.8
Northern Hybridization
Total RNA isolation and Northern analysis for
hABC1 and von Willebrand factor (vWF) expression were
performed.8 The probes of
E-selectin and vWF cDNA were labeled with
[
-32P]dCTP by DECApriming
(Ambion) as previously
described.6
Statistics
Quantitative data were expressed as mean±SEM.
Statistical significance of the data was evaluated by Students
t test. Probability values of
P<0.05 were considered
significant. For nonquantitative data, the results were expressed as
representative of
3 independent
experiments.
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Results
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LDL Increases p38 Activity in ECs
We have previously shown that LDL activates
JNK/c-Jun, but not
ERK/c-Fos, in ECs and proposed that Ras may play an
important
role in LDL-induced EC
activation.
8 9 As
another major MAPK
pathway induced by Ras, p38/ATF-2 activation by LDL
in ECs was
investigated in this study. Confluent HUVECs were exposed to
LDL
in concentrations up to 240 mg/dL for various times. Cell lysates
were
collected from various samples for ATF-2
phosphorylation assay.
As shown in
Figure 1A

, LDL increases ATF-2
phosphorylation 30
minutes after LDL exposure, reaching
a peak at 2 hours. To confirm
the involvement of p38 in ATF-2
phosphorylation, we immunoprecipitated
p38 proteins
with anti-p38 antibody and then used the precipitated
p38 to perform
the kinase activity assay. A similar pattern
of ATF-2
phosphorylation was obtained with the
p38-immunoprecipitated
samples compared with whole-cell lysates.
Exposure of ECs to
different concentrations of LDL caused increased p38
activity
in a dose-dependent fashion, with incremental increases over
the
concentration range of 160 to 240 mg/dL
(Figure 1B

). Therefore,
LDL at a concentration of 240 mg/dL
was used in the rest of
the experiments.

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Figure 1. Effect of LDL on ATF-2 phosphorylation. A, HUVECs were incubated with 240 mg/dL LDL for different periods of time as indicated. B, HUVECs were incubated with different concentrations of LDL for 2 hours. Cellular protein was extracted in a lysis buffer. The cell extracts were incubated with agarose-bound GSTATF-2 (A and B, top). The p38 proteins in the extracts were immunoprecipitated by an anti-p38 antibody, and then the precipitated p38 was incubated with agarose-bound GSTATF-2 (A and B, bottom). After being pelleted and washed, the GSTATF-2 beads were resuspended and incubated in a kinase buffer containing [32P]ATP. The samples were then separated by SDS-polyacrylamide gel electrophoresis and further analyzed by autoradiography. Data are representative of 3 independent experiments.
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LDL Increases ATF-2 and c-Jun Transactivation
in ECs
To further investigate whether LDL activates
ATF-2 transactivation in ECs as a consequence of p38 activation, ECs
were cotransfected with activator plasmids pFCATF-2,
pFA-CHOP, pFAc-Jun, or pFA2-Elk, and the reporter plasmid pFR-Luc.
pFC-MEKK plasmid was used as a positive control in the groups of
pFCATF-2, pFA-CHOP, and pFAc-Jun transfection. PMA was a positive
control for pFA2-Elk transfection. pFC-dbd plasmid containing the DNA
binding domain of the yeast GAL4 but lacking any activation domain
served as negative control. Eighteen hours after transfection, cells
were exposed to LDL for 24 hours, and samples were collected for
luciferase activity assay. LDL increased ATF-2 transactivation activity
by 2.8-fold, CHOP by 2.9-fold, and c-Jun by 3-fold. LDL failed to
activate pFA2-Elk, however, whereas PMA induced activity
>6-fold (see
Figure 2
). The cotransfection of pFC-dbd and pFC-luc had
only minimum luciferase activity, and LDL exposure caused no change
(data not shown).9 Thus, LDL
promotes both ATF-2 and c-Jun activation, but not Elk activation, in
human ECs. These data, together with our previous reports showing that
LDL activates
JNK/c-Jun,8 9
indicate that LDL activates ECs through both p38 and JNK
pathways.

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Figure 2. Effect of LDL on ATF-2 and c-Jun transactivation. HUVECs were cotransfected with pFCATF-2, pFA-CHOP, pFA-cJun or pFA2-Elk, and a reporter plasmid (pFR-Luc). pFC-MEKK plasmid was used as a positive control in each transfection except in the pFA2-Elk group, in which 50 ng/mL of PMA served as positive control. pRSV-ß-gal was cotransfected as a transfection control. After exposure to LDL or PMA for 24 hours as indicated, the samples were collected and assayed for luciferase expression. The results were normalized against ß-galactosidase. Data are presented as mean±SD of relative luciferase activities in 3 independent experiments, each performed in triplicate.
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LDL Increases Ras Membrane Translocation and
Activity in HUVECs
We studied whether Ras is an upstream molecule for the
activation of p38 and JNK cascades in ECs by LDL. We first examined the
membrane translocation of Ras by determining Ras abundance in
whole-cell lysates and the cell membrane. In whole-cell lysates, LDL
did not increase the amount of Ras up to 6 hours. Ras was increased by
LDL exposure in 1 to 2 hours, however, in the membrane fraction of the
cells
(Figure 3A
). Thus, LDL promotes Ras translocation to the
cytoplasmic membrane. To test Ras activation by LDL, we used GSTRaf-1
RBD conjugated with agarose to pull down the GTP-bound form of
Ras.17 The Raf-1bound Ras
was increased by LDL exposure (see
Figure 3B
). The LDL-increased Ras activation began at 30
minutes, reached a peak at 2 hours, and returned to basal level at 6
hours. These data demonstrate LDL activation of Ras in human
ECs.

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Figure 3. Effect of LDL on Ras GTP binding and translocation. Confluent HUVECs were exposed to 240 mg/dL of LDL for different times as labeled. Cell lysates were extracted and quantified. A, The total cellular proteins and membrane proteins were isolated separately. Equal amounts of proteins (15 µg/lane for cell lysate, 5 µg/lane for membrane proteins) were separated with SDSpolyacrylamide gel electrophoresis (PAGE). B, Cellular proteins (500 µg) were incubated with agarose-bound GSTRaf-1 RBD for 30 minutes at 4°C in Mg-containing lysis buffer. The agarose beads were collected and washed. The Ras proteins in the supernatant were separated on SDS-PAGE. The blots in A and B were analyzed by Western blotting with antibodies against Ras. C, HUVECs were transfected with a plasmid encoding wtRas or RasN17, respectively, and then exposed to LDL for 2 hours. Cell lysates were extracted and quantified. Western blotting for Ras and GSTRaf-1 Ras binding assay were performed as described above. Results are representative of 3 independent experiments.
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RasN17 is a dominant negative mutant of Ras in which the
Lys-17 in the wild type has been replaced by an Asn. RasN17 competes
with the endogenous Ras and thus inhibits the Ras-mediated
signaling pathway.15 We
overexpressed the wild type of Ras (wtRas) or RasN17 in HUVECs, which
were then treated with LDL for 2 hours. The cell lysates were collected
for the GSTRaf-1 binding assay. Raf-1bound Ras in cells with wtRas
overexpression was increased by LDL exposure. In contrast, RasN17
overexpression blocked this Ras activation in the LDL-treated samples
as well as untreated controls
(Figure 3C
, bottom). Furthermore, LDL caused Ras
translocation to the cell membrane, as detected by
immunoblotting, in wtRas-transfected cells (data not
shown). Therefore, LDL activates both endogenous
and overexpressed wtRas, but not the Ras mutant with low GTP affinity.
Dominant Negative Mutant of Ras Attenuates LDL
Activation of P38 and JNK
To elucidate the role of Ras in LDL activation of P38,
we infected HUVECs with an adenoviral construct, Ad-RasN17, to
overexpress RasN17. These cells, as well as control cells (infected
with adRSV-ß-gal), were then exposed to LDL. Cell lysates were
collected for ATF-2 and c-Jun phosphorylation assays.
In parallel experiments, cells were treated with PMA, thus serving as a
positive control. As shown in
Figure 4
, both LDL and PMA increased ATF-2 and c-Jun
phosphorylation in cells infected with Ad-ß-gal (top
2 panels). These effects were attenuated in cells infected with
Ad-RasN17. The results suggest that Ras plays an important role in the
signaling of LDL activation of p38 and JNK. Because PMA, but not LDL,
induced ERK1/2 phosphorylation in
ECs,9 the
inhibitory effect of RasN17 on PMA-induced ERK1/2
phosphorylation is shown in
Figure 4
, bottom.

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Figure 4. Effect of Ad-RasN17 on LDL-induced ATF-2 and c-Jun phosphorylation. HUVECs were infected with Ad-RasN17 (left) or Ad-ß-gal (right) as a control for 2 hours. Twenty-four hours after infection, the cells were exposed to LDL (240 mg/dL) for 2 hours or to PMA (50 ng/mL) for 30 minutes. Cell lysates were extracted and were incubated with agarose-bound GSTATF-2 (top) or GSTc-Jun (second panel). After pelleting and washing, the GST-fusion protein beads were resuspended and incubated in a kinase buffer containing [32P]ATP. The samples were then separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography. Immunoblotting with anti-Ras antibody shows positive results in cells infected with Ad-RasN17 but not with Ad-ß-gal (third panel). Bottom, phospho-ERK1/2 was detected by Western blotting with primary antibodies against anti-ACTIVE MAPK pAb. Data are representative of 3 independent experiments.
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Dominant Negative Mutant of Ras Attenuates the
LDL-Induced AP-1 Activation
Because mutant Ras blocks p38/ATF-2 and JNK/c-Jun
pathways and Jun/ATF-2 heterodimers bind to the AP-1 site in the
promoters of many genes (eg, the gene encoding
c-Jun),11 RasN17 should
attenuate LDL-induced AP-1 activation. To test this, we investigated
the modulation of AP-1 binding by LDL using electrophoretic mobility
shift assay in Ad-RasN17infected ECs. As shown in
Figure 5
, RasN17 abolished the LDL-induced increase in AP-1
binding and reduced the PMA-induced increase in AP-1 binding. Many
inducible molecules in ECs, eg, VCAM-1 and ICAM-1, contain a number of
AP-1like and NF-
Blike binding motifs within their 5' promoter
regions.4 18 19
Because LDL activated AP-1 but not
NF-
B,8 LDL-induced Ras-p38
and Ras-JNK pathways should have little effect on NF-
B activation.
PMA increased the binding activity of NF-
B, and this effect was not
blocked by Ad-RasN17 in ECs
(Figure 5
).

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Figure 5. Effect of Ad-RasN17 on LDL-induced AP-1 binding. HUVECs were infected with Ad-RasN17 or Ad-ß-gal as a control for 2 hours. Twenty-four hours after infection, the cells were exposed to LDL (240 mg/dL) for 6 hours or to PMA (50 ng/mL) for 2 hours. Nuclear extracts were prepared, a gel shift assay was performed using a consensus AP-1 or NF- B as a probe, and the results were analyzed by autoradiography. Data are representative of 3 independent experiments.
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To test whether the blockage of p38 and JNK by RasN17 leads
to a similar outcome in LDL-induced AP-1 activation, we cotransfected
pAP-1-luc and RasN17 plasmids into HUVECs. The transfected cells were
then exposed to LDL. As shown in
Figure 6
, after RasN17 transfection, LDL no longer induced
AP-1 activation (P<0.01). The
basal level of AP-1 activity was also decreased in RasN17 transfection
samples compared with controls. Consequently, a Ras-dependent pathway
appears to be involved in the AP-1 activation by
LDL.

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Figure 6. Effect of RasN17 on LDL-induced AP-1driven promoter activation. HUVECs were cotransfected with p3xAP-1-luc and pRSV-ß-gal with or without RasN17 plasmids. Posttransfected cells were incubated for 24 hours with 240 mg/dL of LDL. Promoter activities were measured by use of the reporter luciferase and normalized with ß-galactosidase. Inset, Representative Western blot of mutant Ras overexpression detected by an anti-Ras antibody. Data are mean±SD of the relative luciferase activities in 3 independent experiments, each performed in triplicate.
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A Specific p38 Kinase Inhibitor
Blocks LDL-Induced E-Selectin
We previously reported that LDL increased
endothelial cell adhesiveness by inducing adhesion
molecules, including
E-selectin.6 To elucidate the
role of p38 in the induction of LDL-induced targeting genes, we chose
E-selectin as an example in ECs, because it is an ATF-2regulatory
gene.10 We exposed HUVECs to
LDL with or without SB203580, a specific p38 kinase
inhibitor, for 48 to 72 hours. Total RNA was collected from
various samples for Northern analysis. As shown in
Figure 7
, SB203580 blocked LDL-increased E-selectin mRNA in
both LDL exposure times. vWF mRNA was also detected as an
endothelial internal
control.

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Figure 7. Effect of p38 inhibitor on LDL-induced E-selectin mRNA in ECs. Confluent HUVECs were exposed to 240 mg/dL of LDL with or without p38 inhibitor SB203580 (10 µmol/L) for 48 to 72 hours as labeled. RNA was isolated, and 15-µg samples of total RNA were resolved by gel electrophoresis and then hybridized with [ -32P]-labeled E-selectin or vWF cDNA as labeled. Results shown are representative of 3 independent experiments. Resulting hybridization bands were quantified by densitometry and normalized against vWF. Relative density is expressed as a percentage of basic control (lane 1).
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Discussion
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Hypercholesterolemia is a
well-known risk factor for the development
of
atherosclerosis. The elevated level of LDL is
associated
with endothelial dysfunction and lesion
formation.
20 21
LDL
activates JNK,
9
promotes AP-1 binding,
8 and
induces adhesion
molecules in cultured human vascular
ECs.
4 6 The
present study
was designed to determine whether LDL
activates ECs by promoting
p38, another MAPK family member, and
whether Ras mediates LDL
activation of the p38 pathway. Our findings
demonstrate that
(1) LDL activates the p38-mediated signaling
pathway, (2) LDL
increases Ras membrane translocation and activation,
(3) a dominant
negative mutant of Ras attenuates LDL-induced ATF-2 and
c-Jun
phosphorylation and AP-1 activation, and (4) a
specific p38
kinase inhibitor attenuates LDL-induced
induction of E-selectin
at the mRNA level.
Originally identified as a proto-oncogene, Ras has been
broadly studied for its function and regulation. There is little
information, however, on the physiological and
pathophysiological roles of Ras in
cardiovascular cells, especially in
endothelium exposed to lipoprotein. As a small GTPase
molecule, Ras moves to the plasma membrane after the synthesis in free
cytoplasmic ribosomes and the farnesylation of its posttranslational
C-terminal. Ras activation can trigger at least 3 diverging MAPK
cascades, ie, p38, JNK, and ERK. Distinct intracellular MAPK signaling
cascades are differentially activated to orchestrate
transcriptional activation in response to various extracellular
stimuli. LDL activates the membrane-associated Ras in human
ECs, which in turn preferentially activates p38 and JNK, the 2
MAPKs particularly involved in stress responses. Both native LDL and
oxidized LDL have been found to stimulate p44/42 and p38 MAPKs in
smooth muscle cells and
microphages.22 23 24 25
In contrast, neither native LDL nor
Cu2+-oxidized LDL stimulates ERK activity in
bovine aortic ECs.25 It is
not clear why LDL causes this differential activation of MAPKs in ECs.
Fluid shearing of vascular ECs increases the JNK activity by >10-fold
but activates ERK to a much lesser
degree.26 Osmotic pressure
and UV irradiation also selectively activate the JNK pathway
but not the ERK
pathway.27 28 Ras
can activate both Raf-1 and MEKKs. Raf-1 activates ERK
but not JNK/p38, whereas MEKKs activate JNK/p38 but not
ERK.29 Thus, LDL-stimulated
Ras may activate MEKKs to a greater extent than Raf-1.
Alternatively, other LDL-induced signaling pathways may inhibit Raf-1
signaling. cAMP inhibition of ERK activation by preventing
Ras-dependent activation of Raf-1 has been
reported.30 Indeed,
LDL increased cAMP-responsive elementbinding protein binding and
protein kinase A activation in
ECs.8 Recently, the
phosphatidylinositol 3-kinase (PI3K)Akt pathway was shown to inhibit
the Raf-MEK-ERK pathway through Akt phosphorylation of
Raf-1.31 32 The
involvement of the PI3K-Akt pathway in the LDL-mediated EC activation
remains to be studied. Regarding upstream signaling, Cdc42 and Rac-1,
in addition to Ras, can also selectively activate JNK and
p38.33 34 Thus,
LDL may also activate JNK/p38 through Rho family
GTPase-mediated signal transduction.
The guanine nucleotide exchange factors are
important in Ras activation in response to growth factors, shear
stress, and cytokines. In many instances, docking proteins such
as Grb2/Sos are necessary for Ras activation. Sos is a cytoplasmic
Ras-GEF that is constitutively associated with the adaptor protein
Grb2. When Grb2 interacts with a
tyrosine-phosphorylated membrane receptor, it positions
Sos at the plasma membrane where it can promote activation of Ras.
Alternatively, a tyrosine-phosphorylated Shc may serve
as a bait for Grb2 docking and Sos activation. We detected no membrane
translocation of these docking proteins in HUVECs at up to 6 hours of
LDL exposure (Zhu et al, unpublished observation). Thus, LDL may
activate Ras in ECs through a process independent of growth
factor activation. Conversely, the elevated cellular
cholesterol level resulting from LDL exposure could promote
the membrane translocation of caveolin-1 and Ras, which is a
caveolin-binding signaling molecule, into caveolae in human
ECs.12 Thus, caveolin may
play a role in LDL-mediated Ras activation.
E-selectin is an important adhesion molecule that is
transiently and specifically expressed in ECs on stimulation with
cytokines and
LDL.6 35
E-selectin can serve as a marker for endothelial
activation. ATF2 was reported to play an important role in the
activation of the E-selectin
promoter.10 LDL increases
E-selectin at both the mRNA and protein
levels6 8 and can
increase the E-selectin promoter activity (Zhu et al, unpublished
observation). Here, we report that a p38 kinase inhibitor
attenuates LDL-induced E-selectin at the mRNA level. Thus, it appears
likely that LDL activates the E-selectin promoter in ECs
through the ATF-2dependent mechanism. The blocking effects of RasN17
on ATF-2 and c-Jun phosphorylation and AP-1
transactivation provide further evidence for the involvement of Ras in
this transcriptional activation.
Collectively, the results of the present and previous
studies show that LDL activates ECs predominantly through
Ras-JNK/p38 signaling pathways. This supports our working hypothesis
that LDL perturbs the cell membrane and activates
membrane-associated proteins (eg, Ras), which, in turn,
activate the JNK/p38 pathways. Subsequently, c-Jun and ATF-2
are activated and form a c-Jun/ATF-2 dimer to induce c-Jun
expression via the TRE site at the Jun promoter. Accumulation of c-Jun
by de novo synthesis consistently elevates the level of AP-1,
which further chronically induces other specific cellular genes
regulated by AP-1 (eg, ICAM-1 and E-selectin), leading to EC
activation.
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Acknowledgments
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This study was supported in part by
NIH grant HL-43023 (to M.B.S.),
HL-60789 (to J.Y.-J.S.), and American
Heart Association, Western
States Affiliate grant 98-252 (to Y.Z.).
J.Y.-J.S. is an Established
Investigator of the American Heart
Association.
Received March 15, 2001;
accepted April 27, 2001.
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