From the Atherosclerosis Research Unit,
Department of Medicine, and § Department of
Molecular and Medical Pharmacology, University of California,
Los Angeles, California 90095-1679
Received for publication, December 26, 2000, and in revised form, February 21, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Entrapment and oxidation of low density
lipoproteins (LDL) in the sub-endothelial space is a key process in the
initiation of atherosclerotic lesion development. Functional changes
induced by oxidized lipids in endothelial cells are early events
in the pathogenesis of atherosclerosis.
Oxidized-L- Entrapment and oxidation of low density lipoproteins
(LDL)1 in the
sub-endothelial space and the subsequent interactions between endothelial cells and monocytes is a key process in the initiation of
atherosclerotic lesion development (1, 2). Minimally modified/oxidized-LDL (MM-LDL) contains biologically active molecules that are capable of inducing endothelial cells to produce inflammatory agents such as chemokines, adhesion molecules, and growth factors. These inflammatory molecules promote the recruitment and adhesion of
monocytes to the endothelial cells (2). Recently, several biologically
active oxidized phospholipids have been identified in MM-LDL and in
atherosclerotic lesions of animal models (3-8). Oxidized-L- The chemotactic factors monocyte chemoattractant protein 1 (MCP-1) and
interleukin 8 (IL-8) are important determinants of endothelial/monocyte
interactions (11). MCP-1 has been shown to contribute to the
progression of atherosclerosis in animal models (12-14). Mice lacking
receptors for MCP-1 and the murine orthologue for the IL-8 receptor,
Gro- To study the functional changes that occur in endothelial cells exposed
to ox-PAPC, we recently performed a subtraction screening procedure and cloned several
ox-PAPC-induced genes from human aortic endothelial cells
(HAEC).2 One of the genes
isolated is mitogen-activated protein kinase phosphatase 1 (MKP-1).
MKP-1 belongs to a family of inducible nuclear dual-specificity
phosphatases. The dual-specificity phosphatases are able to
dephosphorylate both threonine/serine and tyrosine residues (20).
MKP-1, the first identified mammalian dual-specificity phosphatase, was
initially cloned and characterized as an oxidative stress and heat
shock-inducible gene (21). Since a number of kinases involved in the
mitogen-activated protein kinase (MAPK) and stress-activated protein
kinase pathways are regulated by phosphorylation of serine/threonine
and tyrosine residues, MKP-1 and its family members are considered to
play a regulatory role in the MAPK and stress-activated protein kinase
signaling pathways. Although there is evidence both in vitro
and in vivo that members of the family of MKP proteins can
regulate MAPKs and stress-activated protein kinases, definitive proof
is still lacking in mammalian systems. MKP-1 knockout mice develop
normally and are fertile (22). Furthermore, cells cultured from MKP-1
knockout mice are not impaired in either MAPK activation or
inactivation (22).
In this paper, we report the characterization of MKP-1 expression and
synthesis as an ox-PAPC-induced gene in HAEC. MKP-1 is rapidly and
transiently induced after ox-PAPC addition to HAEC. Our data suggest
that (i) MKP-1 activity is necessary for the production of monocyte
chemotactic activity in ox-PAPC-treated HAEC and (ii) MKP-1 plays a
role in the ox-PAPC-induced signaling pathways that result in the
production of MCP-1 protein by ox-PAPC-treated HAEC.
Materials--
All cell culture reagents and oligonucleotides
were purchased from Life Technologies, Inc., and all laboratory
chemical supplies were purchased from Fisher. PAPC was obtained from
Avanti Polar Lipids (Alabaster, AL). Oxidized-PAPC (ox-PAPC) was
prepared as described previously (5).
Cell Culture--
HAEC were isolated and cultured as described
previously (17) and were used at passage levels of four to six. Unless
stated otherwise, 80% confluent HAEC were shifted to M199 medium
containing 10% lipoprotein-deficient serum the day before the
experiments. Monocytes were isolated by a modification of the Recalde
method, as described previously (23), from the blood of normal
volunteers after obtaining written consent under a protocol approved by
the Human Research Subject Protection Committee of the University of
California, Los Angeles.
Suppression Subtractive Hybridization--
Reverse transcription
of poly(A)+ RNA and generation of subtracted cDNA
molecules were performed using the polymerase chain reaction Select
cDNA subtraction kit (CLONTECH, Palo Alto, CA) according to the manufacturer's protocol. Subsequent cloning and analysis of the subtracted cDNA library is detailed in a manuscript in preparation.2
Northern Analysis--
Total RNA from cell cultures was
purified using RNeasy kit (Qiagen Inc., Valencia, CA). Ten µg of
total RNA was subjected to electrophoresis on 1% agarose gel,
transferred to Hybond-N membranes (Amersham Pharmacia Biotech), and
hybridized with random-primed (Amersham Pharmacia Biotech)
32P-labeled cDNA probes for human MKP-1 and
glyceraldehyde-3-phosphate dehydrogenase. Electrophoresis and
hybridization protocols were described previously (24).
Antisense Assays--
HAEC were set up in 6-well plates.
Phosphorothioate oligonucleotides were used at a final concentration of
100 nM for all antisense transfection experiments.
Appropriate amounts of the oligonucleotides were diluted in 200 µl of
serum-free M199 medium in 0.5-ml Eppendorf tubes. Three microliters of
SuperFect reagent (Qiagen) was added to each tube and incubated at room
temperature for 15 min to allow SuperFect reagent-DNA complex
formation. During the incubation the HAEC were washed with
phosphate-buffered saline and supplemented with 0.8 ml of complete M199
medium. The transfection complexes were added to the wells and
incubated for 2 h. The cultures were washed in PBS and
supplemented with complete M199 medium. 18 h later the
transfection protocol was repeated, and cultures received 50 µg/ml
ox-PAPC. Six hours later, supernatants were collected and tested for
monocyte adhesion activity and monocyte chemotactic activity.
Additionally, total cellular protein was isolated from each
experimental condition and analyzed for MKP-1 as described below. The
oligonucleotides used were: antisense, 5'-CCCACTTCCATGACCATGG-3';
sense, 5'-CCATGGTCATGGAAGTGGG-3'; and random,
5'-GCAGTGCCTGTTGTTGGATTG-3'.
Western Analysis--
Protein samples (30-50 µg) were
electrophoresed on 10% SDS-polyacrylamide electrophoresis gels and
electroblotted onto Hybond ECL nitrocellulose membranes using a semidry
transfer apparatus (Bio-Rad). Membranes were blocked with Tris-buffered
saline, 5% nonfat dried milk for 60 min, washed, and incubated with
primary and secondary antibodies for 2 and 1 h, respectively.
Antibodies to human MKP-1 were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The secondary antibody, horseradish
peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech), was
used at a 1:4000 dilution. The membranes were washed extensively with
Tris-buffered saline, 0.1% Tween after secondary antibody incubation
and detected using the ECL Western blotting kit (Amersham Pharmacia
Biotech) according to the manufacturer's suggested protocol.
Monocyte Adhesion Assay--
HAEC were plated at 2 × 105 cells/cm2 in 48-well tissue culture plates
and allowed to grow, forming a monolayer of confluent HAEC in 2 days.
Cell culture supernatants (0.5 ml) from various experiments to be
tested for monocyte adhesion were added to fresh HAEC monolayers and
incubated for 4 h at 37 °C. The cells were washed, and a
suspension of human peripheral blood monocytes (4 × 105/well) was added. After 10 min, loosely adherent
monocytes were washed off, and the remaining monolayers were fixed in
0.1% glutaraldehyde. The adherent monocytes were counted in nine
standardized high power microscopic fields and represented as mean ± S.D. Lipopolysaccharide was used as a positive control in all experiments.
Monocyte Chemotaxis Assay--
HAEC supernatants collected from
various experimental conditions were tested for monocyte chemotactic
activity as described previously (25). Briefly, the supernatants were
added to a standard Neuroprobe chamber (Neuroprobe, Cabin John, MD).
Monocytes (5 × 104/well) were added in the upper
wells of the chamber and incubated for 1 h at 37 °C. The
chamber was disassembled, and the nonmigrated monocytes were removed.
The membrane was air-dried, fixed with 1% glutaraldehyde, and stained
with 0.1% crystal violet dye. The number of migrated monocytes was
determined microscopically and expressed as the mean ± S.D. of 12 standardized high power fields counted for quadruplicate wells. In all
assays two to three different dilutions of the supernatants were used
to determine the monocyte chemotactic activity.
Fractionation of HAEC Supernatants by HPLC--
HAEC
supernatants from antisense and phosphatase inhibitor experiments were
concentrated ~10-fold by centrifugation through Centricon (10 K) filters, desalted by gel filtration, and brought to 5%
acetonitrile and 0.1% trifluoroacetic acid for fractionation by
HPLC.
HPLC was performed on a C18 column (Vydac, Hesperia, CA) equilibrated
with acetonitrile/water/ trifluoroacetic acid (5% solvent B). Solvent
A consisted of 0.1% trifluoroacetic acid in water, and solvent B
consisted of 0.085% trifluoroacetic acid in acetonitrile. Ten minutes
after injection, the sample was eluted with a linear gradient (5-100%
B) for 80 min and monitored at 215 and 280 nm. A total of 144 fractions
(0.5 ml) were collected for each HPLC run. Individual fractions were
dried in a SpeedVac (Savant, Farmingdale, NY), resuspended in 250 µl
of 0.1% bovine serum albumin in phosphate-buffered saline, and assayed
for monocyte chemotactic activity. In preliminary studies all 144 fractions were collected and analyzed for chemotactic activity. In
subsequent experiments, only fractions (64) containing the expected
peak of activity were dried by SpeedVac and tested for chemotactic
activity. Peak chemotactic activity was found to elute at ~50% B, in
agreement with Valente et al. (26).
MCP-1 Protein Determinations--
MCP-1 protein in HAEC culture
supernatants was quantified by standard ELISA procedures. MCP-1 ELISA
kit was purchased from BIOSOURCE International
Inc. (Camarillo, CA), and the assays were performed according to the
manufacturer's suggested protocols. All samples and standards were run
in triplicate, and each experimental sample was measured in three
different dilutions (1:5, 1:10, and 1:20 (v/v)). The amount of MCP-1 in
the samples was determined from the standard curves obtained from the
ELISA kits.
MCP-1-neutralizing Antibody Experiments--
Neutralizing
monoclonal antibody to MCP-1 (Antigenix America Inc., Huntington
Station, NY), neutralizing polyclonal antibody to MCP-1 (26), preimmune
rabbit serum (26), or monoclonal antibody to apoJ (Quidel, San Diego,
CA) were added to HAEC supernatants at 0.5 µg/ml and incubated for
1 h at 37 °C. The samples were centrifuged for 1 min at
8000 × g and assayed for chemotactic activity.
Pre-immune rabbit serum (26) and monoclonal antibody to apoJ were used
as control antibodies for the MCP-1-neutralizing polyclonal and
monoclonal antibodies, respectively.
Other Methods--
Protein concentrations were determined using
the Bradford reagent (Bio-Rad). Statistical significance was determined
by analysis of variance. The analyses were first carried out in the
"Excel" application program, followed by a paired Student's
t test to identify significantly different means.
Significance is defined as p < 0.05.
Ox-PAPC Induces the Expression of MKP-1 in HAEC--
To identify
genes that are induced by ox-PAPC in HAEC, mRNA pooled from HAEC
treated with 50 µg/ml of ox-PAPC for 1, 2, and 4 h was
subtracted from mRNA pooled from HAEC treated with 50 µg/ml PAPC
for 1, 2, and 4 h using the suppression subtractive hybridization
technique (27). We identified several genes in HAEC that are induced by
ox-PAPC,2 including MKP-1 and IL-8, which are highly
induced by 4 h (Fig. 1A).
Control and PAPC treatment for 4 h does not show any expression of
MKP-1 (Fig. 1A). MKP-1 induction by ox-PAPC is seen as early as 15 min after induction, reaches a peak at 4 h (Fig.
1B), and then returns to control levels by 12 h (data
not shown). MKP-1 induction measured at the 4-h time point is
dose-dependent (Fig. 1C), and MKP-1 expression
is also detected at ox-PAPC concentrations of 10 and 25 µg/ml at
longer exposures (data not shown). Moreover, among the three
biologically active components of ox-PAPC, only POVPC and PEIPC induce
MKP-1 expression in HAEC (Fig. 1D).
Sodium Orthovanadate Prevents the Synthesis of Monocyte Chemotactic
Activity by ox-PAPC-treated HAEC--
To determine whether MKP-1 plays
a role in ox-PAPC-induced endothelial-monocyte interactions, we first
examined the effect of sodium orthovanadate on the accumulation of
monocyte chemotactic activity in ox-PAPC-treated HAEC culture
supernatants. Sodium orthovanadate is a specific inhibitor of tyrosine
phosphatases, including MKP-1 (28-30). HAEC were treated with either
ox-PAPC alone or in the presence of sodium orthovanadate (10 µM) for 4 h. The supernatants were tested for
monocyte chemotactic activity. Sodium orthovanadate prevents
ox-PAPC-induced production of monocyte chemotactic activity by HAEC
(Fig. 2). Supernatants from HAEC treated
with sodium orthovanadate alone contain monocyte chemotactic activity
similar to control HAEC supernatants (data not shown).
Antisense Oligonucleotides to MKP-1 Inhibit the Production of
Monocyte Adhesion Activity and Monocyte Chemotactic Activity in
ox-PAPC-treated HAEC--
We next examined the effect of selective
inhibition of MKP-1 on the production of monocyte adhesion activity and
monocyte chemotactic activity by ox-PAPC-treated HAEC. Antisense
oligonucleotides targeted to different regions of rat MKP-1 cDNA
have been previously used to successfully block the expression of rat
MKP-1 protein in vascular smooth muscle cells (31). Two of the
antisense oligonucleotides targeted to the translational start site and
a 3'-untranslated region resulted in the best inhibition of rat MKP-1
expression. We designed antisense oligonucleotides targeted to the same
two regions on the human MKP-1 cDNA sequence for our experiments.
HAEC were pretreated with either sense or antisense oligonucleotides to
MKP-1 and then incubated with or without ox-PAPC for 4 h.
Untreated HAEC do not express MKP-1 message and protein; however, MKP-1
message and protein are induced after stimulation with ox-PAPC (Figs.
1A and 3A). Pretreatment of HAEC with antisense oligonucleotides to MKP-1 inhibits the expression of MKP-1 protein in
ox-PAPC-treated HAEC (Fig.
3A). Sense oligonucleotides do
not effect MKP-1 protein expression in ox-PAPC-treated HAEC (Fig. 3A). In the absence of ox-PAPC, supernatants from control
HAEC, HAEC pretreated with sense oligonucleotides MKP-1, and HAEC
pretreated with antisense oligonucleotides to MKP-1 contain similar
levels of monocyte adhesion activity (Fig. 3B, upper
panel) and monocyte chemotactic activity (Fig. 3B,
lower panel). The addition of ox-PAPC induces both monocyte
adhesion activity (Fig. 3B, upper panel) and
monocyte chemotactic activity (Fig. 3B, lower
panel) in supernatants from control HAEC as well as in
supernatants from HAEC pretreated with sense oligonucleotides to MKP-1.
However, ox-PAPC does not induce monocyte adhesion activity (Fig.
3B, upper panel) or monocyte chemotactic activity
(Fig. 3B, lower panel) in HAEC pretreated with
antisense oligonucleotides to MKP-1.
Sodium Orthovanadate and Antisense Oligonucleotides to MKP-1
Prevent the Production of MCP-1 in ox-PAPC-treated HAEC
Supernatants--
We next examined ox-PAPC-induced synthesis of MCP-1
in HAEC in the presence of sodium orthovanadate or after pretreatment with antisense oligonucleotides to MKP-1. ox-PAPC induces the production of MCP-1 protein in HAEC. Sodium orthovanadate inhibits ox-PAPC-induced MCP-1 protein synthesis (Fig.
4, upper panel). Furthermore,
HAEC pretreated with antisense oligonucleotides to MKP-1 do not show an
induction of MCP-1 protein after ox-PAPC treatment (Fig. 4, lower
panel). Our data suggest that sodium orthovanadate and antisense
oligonucleotides to MKP-1 inhibit monocyte chemotactic activity in
ox-PAPC-treated HAEC by inhibiting the production of MCP-1 protein.
MCP-1 Is Responsible for More than 70% of the Monocyte Chemotactic
Activity Associated with Supernatants from ox-PAPC-treated
HAEC--
We previously reported that antibodies to baboon aortic
smooth muscle cell chemotactic factor block MM-LDL-induced production of monocyte chemotactic activity by artery wall co-cultures (17). Smooth muscle cell chemotactic factor was later shown to contain monocyte chemoattractant protein 1 (26). To determine whether MCP-1 is
the major component of ox-PAPC-induced monocyte chemotactic activity in
HAEC supernatants, we tested both the polyclonal antibodies to smooth
muscle cell chemotactic factor as well as a commercially available
neutralizing monoclonal antibody to MCP-1 for its ability to inhibit
monocyte chemotactic activity in supernatants from ox-PAPC-treated
HAEC. Preimmune rabbit serum (26) and monoclonal antibody to apoJ were
used as control antibodies for the polyclonal and monoclonal
antibodies, respectively, and they did not change monocyte chemotactic
activity in the supernatants tested (data not shown). Both the
polyclonal antibody to smooth muscle cell chemotactic factor and the
neutralizing monoclonal antibody to MCP-1 completely block monocyte
chemotactic activity of recombinant human MCP-1 (1 ng/ml) (Fig.
5). Monocyte chemotactic activity from
ox-PAPC-treated HAEC supernatants is inhibited by more than 70% when
incubated with the polyclonal antibody and more than 60% when
incubated with the neutralizing monoclonal antibody to MCP-1 (Fig.
5).
We next examined whether monocyte chemoattractants other than MCP-1
present in the oxidized lipid-treated supernatants contribute to
monocyte chemotactic activity. Supernatants from phosphatase inhibitor
studies (Fig. 2) and antisense oligonucleotide experiments (Fig.
3B, lower panel) were subjected to HPLC
fractionation. A total of 144 fractions were collected, and individual
fractions were analyzed for monocyte chemotactic activity. Supernatants from ox-PAPC-treated HAEC give a single peak for monocyte chemotactic activity between fractions 64 and 70 (data not shown) corresponding to
the elution profile for MCP-1 protein under conditions documented by
Valente et al. (26). Our results suggest that (i) MCP-1 is the main chemoattractant factor responsible for the monocyte
chemotactic activity present in ox-PAPC-treated HAEC supernatants, and
(ii) MKP-1 is required for the production of active MCP-1 protein by ox-PAPC-treated HAEC.
The migration of circulating monocytes into the vessel wall is an
important step in the pathology of atherosclerosis. There is
considerable evidence that oxidized lipids present in MM-LDL play an
important role in the recruitment of monocytes in the aortic
environment (1). Ox-PAPC and its biologically active components, POVPC,
PGPC, and PEIPC, play a major role in MM-LDL-mediated activation of
endothelial cells as well as MM-LDL-mediated promotion of
endothelial/monocyte interactions. However, the molecular mechanisms by
which MM-LDL and ox-PAPC bring about these important changes are not
well understood. We recently performed a subtraction cloning procedure
and identified several genes whose expression is induced by ox-PAPC in
HAEC.2 Interestingly, the types of genes induced by ox-PAPC
include signaling molecules, transcription factors, extracellular
matrix protein, chemokines, and several unknown genes. MKP-1 is one of the genes we isolated from our screen whose expression is rapidly (within 15 min after ox-PAPC addition) and transiently induced. In an
attempt to characterize the functional relevance of MKP-1 in
ox-PAPC-mediated functional changes in endothelial cells, we studied
the effect of both a chemical inhibitor of MKP-1 activity (sodium
orthovanadate) as well as antisense oligonucleotides targeted to MKP-1
on the induction of monocyte chemotactic activity by ox-PAPC-treated
HAEC. Our data suggest that MKP-1 plays a significant role in the
early signaling events mediated by ox-PAPC. Moreover, these studies
also provide evidence for the first time that a nuclear non-receptor
type dual-specificity phosphatase plays a role in the production of
MCP-1.
Regulation and Function of MKP-1--
MKP-1 is the
archetypal member of the dual-specificity protein phosphatase family.
MKP-1 expression is rapidly induced by growth factors in quiescent
fibroblasts (32), cellular stress (28), macrophage colony-stimulating
factor in bone marrow-derived macrophages (33), arachidonic acid and
LDL in vascular smooth muscle cells (34, 35),
H2O2 in astrocytes (36), glucagon in
hepatocytes (37), adhesion to fibronectin in human umbilical vein
endothelial cells (38), and bacterial toxins in Caco-2 cells (39).
MKP-1 thus appears to play a central role in a number of signal
transduction pathways. Furthermore, the induction of MKP-1 has been
shown to be dependent on the protein kinase C pathway (33, 35).
The family of dual-specificity protein phosphatases can be further
classified into two sub-groups (40). MKP-1 belongs to the sub-group of
dual-specificity phosphatases, which are inducible nuclear
dual-specificity phosphatases encoded by immediate early genes (40).
The second sub-group is characterized by their constitutive and
cytoplasmic expression (40). Since a number of kinases involved in the
MAPK and stress-activated protein kinase pathways are regulated by
phosphorylation of serine/threonine and tyrosine residues, MKP family
members are considered to play a regulatory role in the MAPK and
stress-activated protein kinase signaling pathways. Although there is
evidence both in vitro and in vivo that members of the sub-group of MKP proteins that are not inducible and are cytoplasmic regulate MAPKs and stress-activated protein kinases, definitive proof is still lacking in mammalian systems for the MKP-1
sub-group.
Inhibition of Monocyte Chemotactic Activity and MCP-1 Protein
Synthesis--
Blocking MKP-1 activity with sodium orthovanadate or
blocking MKP-1 synthesis using antisense oligonucleotides inhibits the production of monocyte chemotactic activity (Figs. 2 and 3) and MCP-1
(Fig. 4) by ox-PAPC-treated HAEC. Sodium orthovanadate can also block
the activity of other tyrosine phosphatases, and therefore its effect
on the monocyte chemotactic activity and MCP-1 protein synthesis may be
mediated by more than one phosphatase. However, the level of inhibition
of monocyte chemotactic activity and MCP-1 protein in the antisense
oligonucleotide experiments is similar to that we obtained using sodium
orthovanadate, suggesting that MKP-1 may be the main phosphatase
involved in ox-PAPC-induced MCP-1 production.
Differential Induction of MKP-1 by the Biologically Active
Components of ox-PAPC--
We noted an interesting difference in MKP-1
induction by the individual biologically active components of ox-PAPC.
Although all three oxidized phospholipids, POVPC, PGPC, and PEIPC, are capable of inducing monocytes to bind to endothelial cells (9), only
POVPC and PEIPC induce MKP-1 expression, and PGPC has no effect (Fig.
1D). On the other hand, unlike POVPC and PEIPC, PGPC induces
neutrophils to bind to endothelial cells (9). POVPC inhibits
lipopolysaccharide-mediated induction of neutrophil binding and
expression of E-selectin protein and mRNA in a protein kinase A-dependent manner, resulting in down-regulation of
NF- Chemotactic Factors--
MCP-1 and IL-8 play important roles in a
number of cardiovascular diseases including atherosclerosis (41). MCP-1
is induced in a number of cell types including endothelial cells (42)
and smooth muscle cells (43) by a number of pro-inflammatory stimuli including tumor necrosis factor
In summary, our data suggest that MKP-1 is necessary for
ox-PAPC-induced induction of monocyte chemotactic activity by HAEC. The
majority of the monocyte chemotactic activity in ox-PAPC-treated HAEC
is mediated by MCP-1, and MKP-1 is required for the synthesis of MCP-1
protein. Understanding the signaling and molecular mechanisms underlying this paradigm and identification of selective MKP-1 inhibitors will result in major therapeutic targets for a number of
inflammatory diseases including atherosclerosis.
-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC), a major component of minimally modified/oxidized-LDL (MM-LDL) mimics the biological activities assigned to MM-LDL both in vitro in a co-culture model as well as in
vivo in mice. We hypothesized that ox-PAPC initiates gene
expression changes in endothelial cells that result in enhanced
endothelial/monocyte interactions. To analyze the gene expression
changes that oxidized lipids induce in endothelial cells, we used a
suppression subtractive hybridization procedure to compare
mRNA from PAPC-treated human aortic endothelial cells (HAEC) with
that of ox-PAPC-treated cells. We report here the identification of a
gene, mitogen-activated protein kinase phosphatase 1 (MKP-1),
that is rapidly and transiently induced in ox-PAPC-treated
HAEC. Inhibition of MKP-1 using either the phosphatase inhibitor sodium
orthovanadate or antisense oligonucleotides prevents the accumulation
of monocyte chemotactic activity in ox-PAPC-treated HAEC supernatants.
Furthermore, we show that decreased monocyte chemotactic activity in
HAEC treated with sodium orthovanadate or MKP-1 antisense
oligonucleotides is due to decreased MCP-1 protein. Our results
implicate a direct role for MKP-1 in ox-PAPC-induced signaling pathways
that result in the production of MCP-1 protein by ox-PAPC-treated
HAEC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC) and three of its components, 1-palmitoyl-2
(5-oxovaleroyl)- sn-glycero-3-phosphocholine (POVPC),
1-palmitoyl-2-glutaroyl- sn-glycero-3-phosphocholine
(PGPC), and 1-palmitoyl-2 (5, 6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC) (6,
9-10), induce monocyte binding to endothelial cells and play a major
role in the activation of endothelial cells by MM-LDL. However, the
target receptors, the signaling pathways, and the molecular mechanisms
by which MM-LDL and its biologically active components (i) initiate
functional changes in endothelial cells, (ii) induce monocyte
chemotactic activity, and (iii) enhance endothelial/monocyte
interactions are not known.
receptor, are less susceptible to atherosclerosis and have
fewer monocytes in vascular lesions (15, 16). MM-LDL induces MCP-1 (17)
and IL-8 (18), and blocking MCP-1 production inhibits MM-LDL-induced
monocyte chemotactic activity (17). More recently, ox-PAPC and its
components have been shown to induce the synthesis of MCP-1 and IL-8 in
HAEC, and the transcription factor peroxisome proliferator activator receptor-
(PPAR-
) was shown to play a role in the oxidized
phospholipid-mediated induction of MCP-1 and IL-8 (18). However, the
details of signaling pathways and the elements responsible for
ox-PAPC-mediated induction of MCP-1 and IL-8 remain to be elucidated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
[in a new window]
Fig. 1.
Induction of MKP-1 expression in
ox-PAPC-treated HAEC. A, ox-PAPC, but not PAPC, in HAEC
induces MKP-1 expression. Confluent cultures of HAEC were treated with
ox-PAPC (50 µg/ml) or PAPC (50 µg/ml). Four hours later the cells
were lysed, and 10 µg of total RNA from each condition was subjected
to electrophoresis and transferred to a nitrocellulose membrane. The
immobilized RNA was hybridized with radiolabeled MKP-1, IL-8, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.
B, MKP-1 induction after ox-PAPC treatment of HAEC is rapid.
At various time points (as indicated in the figure) after the addition
of PAPC (50 µg/ml) and ox-PAPC (50 µg/ml), HAEC were lysed, and
total RNA was isolated and analyzed by Northern for the expression of
MKP-1. C, dose response of MKP-1 expression in
ox-PAPC-treated HAEC. Confluent cultures of HAEC were treated for
4 h with various concentrations (as indicated in the figure) of
ox-PAPC or PAPC. Total RNA was isolated and analyzed for the expression
of MKP-1. D, induction of MKP-1 mRNA by the biologically
active components of ox-PAPC. Confluent cultures of HAEC were treated
for 4 h with 5 µg/ml POVPC, PGPC, or PEIPC. Total RNA was
isolated and subjected to Northern analysis for MKP-1. CONT,
control.
View larger version (15K):
[in a new window]
Fig. 2.
Sodium orthovanadate inhibits ox-PAPC-induced
accumulation of monocyte chemotactic activity in HAEC
supernatants. HAEC were either left untreated or were treated with
ox-PAPC (50 µg/ml) in the presence or absence of sodium orthovanadate
(10 µM) for 4 h. Supernatants were collected,
filtered, and analyzed for monocyte chemotactic activity as described
under "Experimental Procedures." Similar results were obtained in
three separate experiments. The asterisk indicates a
p value < 0.05. HPF, high power
field.
View larger version (24K):
[in a new window]
Fig. 3.
Inhibition of MKP-1 prevents ox-PAPC-induced
endothelial-monocyte interactions in HAEC. A, MKP-1
protein is induced in ox-PAPC-treated HAEC, and antisense
oligonucleotides directed against MKP-1 prevent the accumulation of
MKP-1 in ox-PAPC-treated HAEC. HAEC were transfected with either
antisense or sense phosphorothioate oligonucleotides (100 nM) to human MKP-1. Control (CONT) and
transfected cells were either left untreated or treated with ox-PAPC
(50 µg/ml) for an additional 6 h. After the treatments, cell
lysates were prepared and analyzed by Western blotting for MKP-1
protein expression. B, supernatants from ox-PAPC-treated
HAEC pretreated with antisense oligonucleotides to MKP-1 do not promote
monocyte adhesion and monocyte chemotaxis. HAEC were transfected with
either antisense or sense phosphorothioate oligonucleotides (100 nM) to human MKP-1. Control and transfected cells were
either left untreated or treated with ox-PAPC (50 µg/ml) for an
additional 6 h. After the treatments, cell supernatants were
collected and analyzed for monocyte adhesion (upper panel)
and monocyte chemotaxis (lower panel). The results were
similar in three independent experiments. The asterisk
indicates a p value < 0.05. LPS,
lipopolysaccharide.
View larger version (18K):
[in a new window]
Fig. 4.
Sodium orthovanadate and antisense
oligonucleotides to MKP-1 inhibit accumulation of MCP-1 protein in the
supernatants of ox-PAPC treated HAEC. A, HAEC were
either left untreated or treated with ox-PAPC (50 µg/ml) in the
presence or absence of sodium orthovanadate (10 µM) for
4 h. Supernatants were collected and analyzed for MCP-1 protein
accumulation by ELISA. Similar results were obtained in three separate
experiments. B, antisense oligonucleotides to MKP-1 inhibit
accumulation of MCP-1 protein in the supernatants of ox-PAPC-treated
HAEC. HAEC were transfected with antisense phosphorothioate
oligonucleotides (100 nM) to human MKP-1. After
transfections, control and transfected cells were either left untreated
or treated with ox-PAPC (50 µg/ml) for an additional 6 h. Cell
supernatants were analyzed for MCP-1 protein by ELISA. An
asterisk indicates a p value < 0.05.
View larger version (26K):
[in a new window]
Fig. 5.
MCP-1 is responsible for more than 70% of
the monocyte chemotactic activity associated with supernatants from
ox-PAPC-treated HAEC. Supernatants from HAEC that were either
untreated (NA), untreated and supplemented with recombinant
MCP-1 (1 ng/ml), ox-PAPC-induced, or lipopolysaccharide
(LPS)-induced were incubated with either (i) no antibody,
(ii) 0.5 µg/ml neutralizing monoclonal antibody to MCP-1
(mono. MCP-1), or (iii) 0.5 µg/ml
neutralizing polyclonal antibody to MCP-1 (26)
(poly.
MCP-1) at 37 °C. One hour later the
samples were analyzed for monocyte chemotactic activity. The
asterisks indicate a p value < 0.05. HPF, high power field.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-dependent transcription (9). PGPC, on the other
hand, induces the expression of both E-selectin and vascular cell
adhesion molecule-1 (VCAM-1) in endothelial cells (9). The authors of
these studies conclude that POVPC and PGPC might act through different
receptors (9). Therefore, the signaling mechanisms and resulting
activation of chemotactic factors may be different between POVPC and
PGPC.
(43), IL-1 (44), and
lipopolysaccharide (45). Among the three distinct MAPK pathways
(extracellular signal-regulated kinase, c-Jun NH2-terminal
kinase, and p38 kinase), the p38 kinase pathway has been shown to be
critical for tumor necrosis factor
-induced expression of MCP-1 in
human umbilical vein endothelial cells (46) and IL-1-induced expression
of MCP-1 in human mesangial cells (44). More interestingly, recent
findings suggest that direct binding of MKP-1 to p38 promotes the
catalytic activation of MKP-1 both in vitro and in
vivo (19). Since MKP-1 is necessary for MCP-1 protein synthesis in
ox-PAPC-treated HAEC (Fig. 4B), we are currently studying
the effect of p38 kinase inhibitors on MKP-1 activation and MCP-1
production in ox-PAPC-treated HAEC. We are also investigating the role
of MKP-1 in the production of IL-8 by ox-PAPC-treated HAEC. The signal
transduction mechanisms utilized by MM-LDL and ox-PAPC are not known
and are currently under investigation in our laboratory. Future studies
directed at the role of MKP-1 in the p38 kinase pathway might determine the mechanisms by which ox-PAPC induces MCP-1 synthesis. Moreover, endothelial and other cell types derived from the viable and fertile MKP-1 knockout mouse (22) would also be valuable tools to further our
understanding of the signal transduction pathways involved in oxidized
lipid-induced signaling.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant HL 30568, a Tobacco Related Disease Research Project grant from the State of California, and the Laubisch, Castera, and M. K. Gray Fund at UCLA.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 Medicine and Dept. of Molecular and Medical Pharmacology, University of California Los Angeles, 650 Charles E. Young Dr. South, A8-131, CHS, Los Angeles, CA 90095. Tel.: 310-206--3915; Fax: 310-206-3605; E-mail: sreddy@mednet.ucla.edu.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M011663200
2 S. T. Reddy, V. Grijalva, S. Hama, K. H. Hassan, R. Mottahedeh, D. J. Wadleigh, M. Navab, and A. M. Fogelman, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LDL, low density lipoprotein; MM-LDL, mildly oxidized-LDL; MAPK, mitogen-activated protein kinase; MKP-1, MAPK phosphatase 1; MCP-1, monocyte chemoattractant protein 1; IL-8, interleukin 8; apo, apolipoprotein; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; ox-PAPC, oxidized-PAPC; POVPC, 1-palmitoyl-2 (5-oxovaleroyl)-sn-glycero-3-phosphocholine; PGPC, 1palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine; PEIPC, 1-palmitoyl-2 (5, 6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine; HAEC, human aortic endothelial cells; HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Navab, M.,
Berliner, J. A.,
Watson, A. D.,
Hama, S. Y.,
Territo, M. C.,
Lusis, A. J.,
Shih, D. M.,
Van Lenten, B. J.,
Frank, J. S.,
Demer, L. L.,
Edwards, P. A.,
and Fogelman, A. M.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
831-842 |
2. |
Berliner, J. A.,
Navab, M.,
Fogelman, A. M.,
Frank, J. S.,
Demer, L. L.,
Edwards, P. A.,
Watson, A. D.,
and Lusis, A. J.
(1995)
Circulation
91,
2488-2496 |
3. | Watson, A. D., Navab, M., Hama, S. Y., Sevanian, A., Prescott, S. M., Stafforini, D. M., McIntyre, T. M., La Du, B. N., Fogelman, A. M., and Berliner, J. A. (1995) J. Clin. Invest. 95, 774-782[Medline] [Order article via Infotrieve] |
4. | Watson, A. D., Berliner, J. A., Hama, S. Y., La Du, B. N., Faull, K. F., Fogelman, A. M., and Navab, M. (1995) J. Clin. Invest. 96, 2882-2891[Medline] [Order article via Infotrieve] |
5. |
Watson, A. D.,
Leitinger, N.,
Navab, M.,
Faull, K. F.,
Horkko, S.,
Witztum, J. L.,
Palinski, W.,
Schwenke, D.,
Salomon, R. G.,
Sha, W.,
Subbanagounder, G.,
Fogelman, A. M.,
and Berliner, J. A.
(1997)
J. Biol. Chem.
272,
13597-13607 |
6. |
Watson, A. D.,
Subbanagounder, G.,
Welsbie, D. S.,
Faull, K. F.,
Navab, M.,
Jung, M. E.,
Fogelman, A. M.,
and Berliner, J. A.
(1999)
J. Biol. Chem.
274,
24787-24798 |
7. |
Leitinger, N.,
Watson, A. D.,
Hama, S. Y.,
Ivandic, B.,
Qiao, H-H.,
Huber, J.,
Faull, K. F.,
Grass, D. S.,
Navab, M.,
Fogelman, A. M.,
de Beer, F. C.,
Lusis, A. J.,
and Berliner, J. A.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1291-1298 |
8. | Subbanagounder, G., Watson, A. D., and Berliner, J. A. (2000) Free Radic. Biol. Med. 28, 1751-1761[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Leitinger, N.,
Tyner, T. R.,
Oslund, L.,
Rizza, C.,
Subbanagounder, G.,
Lee, H.,
Shih, P. T.,
Mackman, N.,
Tigyi, G.,
Territo, M. C.,
Berliner, J. A.,
and Vora, D. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12010-12015 |
10. |
Subbanagounder, G.,
Leitinger, N.,
Schwenke, D. C.,
Wong, J. W.,
Lee, H.,
Rizza, C.,
Watson, A. D.,
Faull, K. F.,
Fogelman, A. M.,
and Berliner, J. A.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
2248-2254 |
11. | Gerszten, R. E., Garcia-Zepeda, E. A., Lim, Y. C., Yoshida, M., Ding, H. A., Gimbrone, M. A., Jr., Luster, A. D., Luscinskas, F. W., and Rosenzweig, A. (1999) Nature 398, 718-723[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Aiello, R. J.,
Bourassa, P. A.,
Lindsey, S.,
Weng, W.,
Natoli, E.,
Rollins, B. J.,
and Milos, P. M.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1518-1525 |
13. | Gu, L., Okada, Y., Clinton, S. K., Gerard, C., Sukhova, G. K., Libby, P., and Rollins, B. J. (1998) Mol. Cell 2, 275-281[Medline] [Order article via Infotrieve] |
14. |
Gosling, J.,
Slaymaker, S.,
Gu, L.,
Tseng, S.,
Zlot, C. H.,
Young, S. G.,
Rollins, B. J.,
and Charo, I. F.
(1999)
J. Clin. Invest.
103,
773-778 |
15. | Dawson, T. C., Kuziel, W. A., Osahar, T. A., and Maeda, N. (1999) Atherosclerosis 143, 205-211[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Boisvert, W. A.,
Santiago, R.,
Curtiss, L. K.,
and Terkeltaub, R. A.
(1998)
J. Clin. Invest.
101,
353-363 |
17. | Navab, M., Imes, S. S., Hama, S. Y., Hough, G. P., Ross, L. A., Bork, R. W., Valente, A. J., Berliner, J. A., Drinkwater, D. C., Laks, H., and Fogelman, A. M. (1991) J. Clin. Invest. 88, 2039-2046[Medline] [Order article via Infotrieve] |
18. |
Lee, H.,
Shi, W.,
Tontonoz, P.,
Wang, S.,
Subbanagounder, G.,
Hedrick, C. C.,
Hama, S.,
Borromeo, C.,
Evans, R. M.,
Berliner, J. A.,
and Nagy, L.
(2000)
Circ. Res.
87,
516-521 |
19. | Hutter, D., Chen, P., Barnes, J., and Liu, Y. (2000) Biochem. J. 352, 155-163[CrossRef][Medline] [Order article via Infotrieve] |
20. | Guan, K. L., Broyles, S. S., and Dixon, J. E. (1991) Nature 350, 359-362[CrossRef][Medline] [Order article via Infotrieve] |
21. | Keyse, S. M., and Emslie, E. A. (1992) Nature 359, 644-647[CrossRef][Medline] [Order article via Infotrieve] |
22. | Dorfman, K., Carrasco, D., Gruda, M., Ryan, C., Lira, S. A., and Bravo, R. (1996) Oncogene 13, 925-931[Medline] [Order article via Infotrieve] |
23. | Fogelman, A. M., Sykes, K., Van Lenten, B. J., Territo, M. C., and Berliner, J. A. (1988) J. Lipid Res. 29, 1243-1247[Abstract] |
24. | Reddy, S. T., Winstead, M., Tischfield, J. A., and Herschman, H. R. (1997) J. Biol. Chem. 271, 13591-13596[CrossRef] |
25. |
Navab, M.,
Hama, S. Y.,
Cooke, C. J.,
Anantharamaiah, G. M.,
Chaddha, M.,
Jin, L.,
Subbanagounder, G.,
Faull, K. F.,
Reddy, S. T.,
Miller, N. E.,
and Fogelman, A. M.
(2000)
J. Lipid Res.
41,
1481-1494 |
26. | Valente, A. J., Graves, D. T., Vialle-Valentin, C. E., Delgado, R., and Schwartz, C. J. (1988) Biochemistry 27, 4162-4168[Medline] [Order article via Infotrieve] |
27. |
Diatchenko, L.,
Lau, Y. F.,
Campbell, A. P.,
Chenchik, A.,
Moqadam, F.,
Huang, B.,
Lukyanov, S.,
Lukyanov, K.,
Gurskaya, N.,
Sverdlov, E. D.,
and Siebert, P. D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6025-6030 |
28. | Gordon, J. A. (1991) Methods Enzymol. 201, 477-482[Medline] [Order article via Infotrieve] |
29. | Charles, C. H., Sun, H., Lau, L. F., and Tonks, N. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5292-5296[Abstract] |
30. | Lin, W., and Hsu, Y. (2000) Cell. Signalling 12, 457-461[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Duff, J. L.,
Monia, B. P.,
and Berk, B. C.
(1995)
J. Biol. Chem.
270,
7161-7166 |
32. | Lau, L. F., and Nathans, D. (1985) EMBO J. 4, 3145-3151[Abstract] |
33. |
Valledor, A. F.,
Xaus, J.,
Marques, L.,
and Celada, A.
(1999)
J. Immunol.
163,
2452-2462 |
34. |
Metzler, B.,
Hu, Y.,
Sturm, G.,
Wick, G.,
and Xu, Q.
(1998)
J. Biol. Chem.
273,
33320-33326 |
35. |
Metzler, B.,
Li, C.,
Hu, Y.,
Sturm, G.,
Ghaffari-Tabrizi, N.,
and Xu, Q.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1862-1871 |
36. | Tournier, C., Thomas, G., Pierre, J., Jacquemin, C., Pierre, M., and Saunier, B. (1997) Eur. J. Biochem. 244, 587-595[Abstract] |
37. | Schliess, F., Kurz, A. K., and Haussinger, D. (2000) Gastroenterology 118, 929-936[Medline] [Order article via Infotrieve] |
38. | Kim, F., and Corson, M. A. (2000) Biochem. Biophys. Res. Commun. 273, 539-545[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Kojima, S.,
Yanagihara, I.,
Kono, G.,
Sugahara, T.,
Nasu, H.,
Kijima, M.,
Hattori, A.,
Kodama, T.,
Nagayama, K. I.,
and Honda, T.
(2000)
Infect. Immun.
68,
2791-2796 |
40. | Keyse, S. M. (2000) Curr. Opin. Cell Biol. 12, 186-192[CrossRef][Medline] [Order article via Infotrieve] |
41. | Sasayama, S., Okada, M., and Matsumori, A. (2000) Cardiovasc. Res. 45, 267-269[CrossRef][Medline] [Order article via Infotrieve] |
42. | Cushing, S. D., Berliner, J. A., Valente, A. J., Territo, M. C., Navab, M., Parhami, F., Gerrity, R., Schwartz, C. J., and Fogelman, A. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5134-5138[Abstract] |
43. |
De Keulenaer, G. W.,
Ushio-Fukai, M.,
Yin, Q.,
Chung, A. B.,
Lyons, P. R.,
Ishizaka, N.,
Rengarajan, K.,
Taylor, W. R.,
Alexander, R. W.,
and Griendling, K. K.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
385-391 |
44. | Rovin, B. H., Wilmer, W. A., Danne, M., Dickerson, J. A., Dixon, C. L., and Lu, L. (1999) Cytokine 11, 118-126[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Hu, H. M.,
Tian, Q.,
Baer, M.,
Spooner, C. J.,
Williams, S. C.,
Johnson, P. F.,
and Schwartz, R. C.
(2000)
J. Biol. Chem.
275,
16373-16381 |
46. |
Goebeler, M.,
Kilian, K.,
Gillitzer, R.,
Kunz, M.,
Yoshimura, T.,
Brocker, E. B.,
Rapp, U. R.,
and Ludwig, S.
(1999)
Blood
93,
857-865 |