Mitogen-activated protein kinase phosphatase 1 activity is necessary for oxidized phospholipids to induce monocyte chemotactic activity in human aortic endothelial cells.

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-alpha-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.

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)(13)(14). Mice lacking receptors for MCP-1 and the murine orthologue for the IL-8 receptor, Gro-␣ receptor, are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions (15,16). MM-LDL induces MCP-1 (17) and , and blocking MCP-1 production inhibits MM-LDLinduced 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-PAPCmediated induction of MCP-1 and IL-8 remain to be elucidated.
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.

EXPERIMENTAL PROCEDURES
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) ϩ 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 g 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) 32 P-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 l 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 g/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 experi-mental condition and analyzed for MKP-1 as described below. The oligonucleotides used were: antisense, 5Ј-CCCACTTCCATGAC-CATGG-3Ј; sense, 5Ј-CCATGGTCATGGAAGTGGG-3Ј; and random, 5Ј-GCAGTGCCTGTTGTTGGATTG-3Ј.
Western Analysis-Protein samples (30 -50 g) 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 peroxidaseconjugated 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 ϫ 10 5 cells/cm 2 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 ϫ 10 5 /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 Ϯ 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 ϫ 10 4 /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 Ϯ 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 ϳ10-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 l 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 -70) containing the expected peak of activity were dried by SpeedVac and tested for chemotactic activity. Peak chemotactic activity was found to elute at ϳ50% 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 g/ml and incubated for 1 h at 37°C. The samples were centrifuged for 1 min at 8000 ϫ 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 Ͻ 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 g/ml of ox-PAPC for 1, 2, and 4 h was subtracted from mRNA pooled from HAEC treated with 50 g/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 g/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 M) 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 3Јuntranslated 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-PAPCtreated 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-PAPCinduced 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-PAPCtreated 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. DISCUSSION 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-PAPCmediated 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 dualspecificity 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), H 2 O 2 in astrocytes (36), gluca-gon 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 stressactivated 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-Bdependent 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.
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 ␣ (43), IL-1 (44), and lipopolysaccharide (45). Among the three distinct MAPK pathways (extracellular signal-regulated kinase, c-Jun NH 2 -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 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 g/ml neutralizing monoclonal antibody to MCP-1 (mono.␣MCP-1), or (iii) 0.5 g/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 Ͻ 0.05. HPF, high power field. 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.
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.