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J. Biol. Chem., Vol. 276, Issue 33, 30579-30588, August 17, 2001

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-Migrating Very Low Density Lipoprotein (VLDL) Activates Smooth Muscle Cell Mitogen-activated Protein (MAP) Kinase via G Protein-coupled Receptor-mediated Transactivation of the Epidermal Growth Factor (EGF) Receptor

EFFECT OF MAP KINASE ACTIVATION ON VLDL PLUS EGF-INDUCED CELL PROLIFERATION^*

Dezheng Zhao, Jennifer Letterman, and Barbara M. Schreiber

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, April 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study examined the premise that the atherogenic lipoprotein, -migrating very low density lipoprotein (VLDL), might activate the mitogen-activated protein (MAP) kinases ERK1/ERK2, thereby contributing to the induction of smooth muscle cell proliferation in atherosclerosis. The data show that VLDL activates rabbit smooth muscle cell ERK1/ERK2. Interestingly, ERK1/ERK2 activation is mediated by G protein-coupled receptors that transactivate the epidermal growth factor (EGF) receptor. VLDL-induced MAP kinase activation depends on Ras and Src activity as well as protein kinase C. The inhibition of lysosomal degradation of VLDL has no effect on ERK1/ERK2 activation. The contribution of VLDL-induced activation of ERK1/ERK2 to smooth muscle cell proliferation was also explored. VLDL induces expression of egr-1 and c-fos mRNA. Despite its ability to stimulate early gene expression, VLDL alone is unable to inspire quiescent cells into S phase. When added in conjunction with EGF, however, stimulation of [³H]thymidine incorporation into DNA and an increase in histone gene expression are observed. Moreover, VLDL plus EGF synergistically induce cyclin D1 expression and down-regulate p27^KIP1 expression. The addition of either VLDL or EGF stimulates a robust activation of ERK1/ERK2, but the addition of both agents simultaneously sustains the activation for a longer time period. Inhibition of MAP kinase kinase, pertussis toxin-sensitive G proteins, the EGF receptor, or protein kinase C blocks VLDL plus EGF-induced proliferation, demonstrating that activation of the VLDL-induced signaling pathway results in smooth muscle cell proliferation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Elevated plasma lipids are associated with a risk for the development of atherosclerosis, a disease now believed to be an inflammatory one characterized by vascular lesions containing lipid, macrophages, T cells, and fibrous components. Smooth muscle cell migration from the medial layer of the vessel to the intima accompanied by proliferation is critical to the development of the plaque (1). A potential relationship between hyperlipidemia and disease progression has been suggested in studies showing that atherogenic lipoproteins such as low density lipoprotein (LDL)¹ and oxidized LDL induce smooth muscle cell proliferation (2-11). -migrating very low density lipoprotein (VLDL) is a cholesteryl ester-rich atherogenic lipoprotein that accumulates in the plasma of cholesterol-fed animals and humans with type III hypercholesterolemia (12). Earlier we showed that VLDL enhances smooth muscle cell growth potentiating activity of monocytes/macrophages (13). Moreover, VLDL added directly to cultured smooth muscle cells stimulates the rate of cell proliferation (14, 15).

It has been shown that smooth muscle cell proliferation can be achieved via the activation of mitogen-activated protein (MAP) kinases (16). The importance of MAP kinase activation to smooth muscle cell proliferation in vivo has been shown in balloon injury models (17-20). LDL (21, 22) as well as minimally modified and oxidized LDL (11, 22-24) have been shown to stimulate MAP kinases in smooth muscle cells, suggesting a potential link to their growth-promoting ability. Treatment with LDL results in the activation of protein kinase C and MAP kinase as well as the induction of the cell cycle-related genes c-fos, c-myc (5), and early growth response gene-1 (egr-1; Ref. 25). Interestingly, it has been shown that the activation of MAP kinase can induce not only cell proliferation but differentiation as well, highlighting the importance of measuring biological responses downstream of MAP kinase activation (26-30).

The MAP kinases ERK1/ERK2 (p44^MAPK and p42^MAPK, respectively) are stimulated in pathways initiated by extracellular stimuli that activate receptor tyrosine kinases such as the epidermal growth factor (EGF) receptor or nontyrosine kinase receptors including G protein-coupled receptors. Phosphorylation of the EGF receptor can activate Ras, commencing the Raf-1MAP kinase kinase (MEK)MAP kinase cascade. Pertussis toxin-sensitive G protein-coupled receptors such as those for lysophosphatidic acid (LPA), thrombin, and 2-adrenergic agonists stimulate the MAP kinase pathway by Gi-mediated Ras activation as shown in Rat-1 cells (31). LPA-mediated activation of MAP kinase via a pertussis toxin-sensitive transactivation of the EGF receptor has also been described (32, 33), and Ras-independent activation of MAP kinase through protein kinase C has been noted (34).

Although lipoproteins have been shown to activate MAP kinases, the molecular events upstream of MAP kinases leading to their activation remain uncertain. Moreover, the possibility that the atherogenic lipoprotein VLDL activates MAP kinases has not been explored. We sought to determine whether VLDL activates MAP kinase and, if so, what the targets upstream of MAP kinase might be. Interestingly, the data show that VLDL activates the MAP kinases ERK1/ERK2 via a G protein-coupled receptor that transactivates the EGF receptor, is Ras- and Src-dependent, and involves protein kinase C. Additional experiments examining the effect of VLDL on the proliferation of quiescent aortic smooth muscle cells showed that despite its ability to activate MAP kinase, VLDL alone does not stimulate quiescent cells to enter the S phase of the cell cycle. In combination with EGF, however, serum-deprived aortic smooth muscle cells enter S phase. Moreover, the stimulation of proliferation is mediated through a sustained activation of MAP kinase. To our knowledge, this is the first report demonstrating that a lipoprotein can transactivate the EGF receptor via activation of a pertussis toxin-sensitive G protein-coupled receptor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Aortic Smooth Muscle Cells-- Neonatal rabbit aortic smooth muscle cells were isolated as described previously (14, 15). Experiments were performed by plating into first or second passage at a density of 2 × 10⁴/cm² (except for Ras expression studies in which cells were seeded at 0.52 × 10⁴/cm²) in Dulbecco's modified Eagle's Medium (J.R.H. Biosciences, Lenexa, KS) supplemented with 3.7 g/liter NaHCO₃, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM minimum Eagle's medium nonessential amino acids (Life Technologies, Inc.), and 1 mM minimum Eagle's medium sodium pyruvate solution (Life Technologies, Inc.) (DMEM) containing 10% fetal bovine serum (FBS; Sigma). After an overnight incubation, the cells were rendered quiescent by washing twice with Puck's saline (140 mM NaCl, 5.4 mM KCl, 1.1 mM KH₂PO₄, 1.1 mM Na₂HPO₄, and 6.1 mM glucose, pH 7.4) and incubating for 3 days in DMEM with 0.5% FBS.

Isolation and Characterization of VLDL-- VLDL was isolated as described previously by density gradient ultracentrifugation from serum of male New Zealand White rabbits fed a diet rich in cholesterol and peanut oil (13, 14). Protein concentrations were determined as described previously (13, 14). VLDL preparations were tested routinely for thiobarbituric acid-reacting substances as described previously (14).

Effect of VLDL on Activity of MAP Kinase-- Neonatal rabbit aortic smooth muscle cells were seeded at a density of 2 × 10⁴ cells/cm² in DMEM containing 10% FBS. After an overnight incubation, the cultures were washed twice with Puck's saline and then incubated in DMEM containing 0.5-1% FBS for 3 days, at which time the cells were treated with VLDL, EGF (Sigma), LPA (Sigma), basic fibroblast growth factor (bFGF; Life Technologies, Inc.), phorbol-12-myristate-13-acetate (PMA; Sigma), or FBS as indicated. Pretreatment with the following inhibitors was also implemented: PD98059 (MEK inhibitor; Calbiochem), AG1478 (EGF receptor tyrosine kinase inhibitor; Calbiochem), pertussis toxin (G protein-coupled receptor inhibitor; List Biological Laboratories, Inc., Campbell, CA), bisindolylmaleimide I (protein kinase C inhibitor; Calbiochem), calphostin C (protein kinase C inhibitor; Calbiochem), or PP2 (specific Src family tyrosine kinase inhibitor; Calbiochem). In addition, the cells pretreated with chloroquine prior to the addition of VLDL were studied (see below). Total cell protein was isolated and subjected to Western blot analysis using antibodies directed against the active phosphorylated forms of ERK1/ERK2 or total ERK1/ERK2 (see below). Immunoprecipitation was carried out with an antibody against the EGF receptor followed by Western blot analysis using a monoclonal antibody against phosphotyrosine.

Western Blot Analysis-- Cells were washed twice with cold PBS and then lysed in protein extraction buffer (25 mM Tris Cl, pH 7.4, 50 mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 mM EDTA, 0.2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The extracts were incubated on ice for 15 min and centrifuged at 13,000 rpm (Eppendorf centrifuge) at 4 °C to remove insoluble material. Protein concentrations were determined by the BCA method (Pierce) according to manufacturer instructions using bovine serum albumin as a standard. Equal amounts of protein (~30 µg/lane) were subjected to SDS-polyacrylamide gel electrophoresis according to Laemmli (35). Stacking gels contained 4% acrylamide/bisacrylamide (29:1) and separating gels contained either 8% (for immunoprecipitated EGF receptor) or 12% (all other Westerns) acrylamide/bisacrylamide. Electrophoresis was carried out at 200 V in running buffer (25 mM Tris, 250 mM glycine, and 0.1% SDS). The samples were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schüll) using transfer buffer (48 mM Tris, pH 8.3, 39 mM glycine, and 20% methanol) at 100 V for 1 h at 4 °C with constant stirring. After transfer, nitrocellulose membranes were equilibrated in TBST (25 mM Tris, pH 8.0, 125 mM NaCl, and 0.1% Tween 20) for 10 min at room temperature and blocked with TBST containing 4% nonfat dry milk (Nestle Foods, Glendale, CA) for 1 h. The nitrocellulose membranes were incubated for 1 h at room temperature with the primary antibodies that were diluted in TBST containing 4% milk. After washing twice with TBST containing 4% milk for 10 min, horseradish peroxidase anti-mouse-conjugated secondary antibody (Sigma) diluted in TBST containing 4% milk (1:1000) was applied for 1 h. The blots were washed once with TBST containing 4% milk for 10 min and then twice with TBST for 10 min. Bound horseradish peroxidase was detected using ECL reagents (Amersham Pharmacia Biotech) according to manufacturer instructions. The primary antibodies (diluted 1:1000) used included mouse monoclonal antibodies against the active forms of ERK1/ERK2 (New England Biolabs) as well as phosphotyrosine, Ras, cyclin D1, and p27^KIP1 antibodies from Transduction Laboratories (Lexington, KY) and a rabbit immunoaffinity-purified IgG that recognizes all forms (active as well as inactive) of ERK1/ERK2 (Upstate Biotechnology, Lake Placid, NY).

Immunoprecipitation-- Cells were washed twice with cold PBS and incubated on ice for 10 min with 1 ml of radioimmune precipitation buffer (20 mM Tris, pH 7.5, 0.15 M NaCl, 1% Triton X-100, 1 mM Na₃VO₄, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 µg/ml pepstatin). Lysates were centrifuged at 13,000 rpm (Eppendorf centrifuge, 4 °C for 10 min) to remove insoluble material. The protein concentration was determined by the BCA method. Five hundred micrograms of protein were incubated with 1 µg of rabbit polyclonal antibody against the C terminus of the EGF receptor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 °C and then with protein A-conjugated agarose beads overnight with constant shaking at 4 °C. The beads were washed three times with radioimmune precipitation buffer, and half of each sample was subjected to Western blot analysis using a monoclonal antibody against phosphotyrosine.

[¹⁴C]Oleic Acid Incorporation into Cholesteryl Ester-- Quiescent smooth muscle cells were incubated with VLDL in the presence or absence of chloroquine. All cells received [¹⁴C]oleic acid complexed to albumin (0.43 µCi/ml) and were incubated for 24 h. Lipids were extracted and [¹⁴C]cholesteryl oleate accumulation was analyzed by thin layer chromatography essentially as described (14). The data are expressed as nmol of [¹⁴C]cholesteryl oleate/mg of protein.

Construction of a Dominant Negative Ras Expression Vector-- DNA restriction endonucleases, DNA polymerase I large fragment (Klenow), and T4 DNA ligase were purchased from New England Biolabs. The dominant negative ras mutant (ras-17N prepared by Feig and Cooper (36) was kindly provided by Dr. Debabrata Mukhopadhyay, Beth Israel Hospital, Boston, MA) was precut with EcoRI and BamHI and ligated into pBPST-4 (see below) that was digested with EcoRI and BamHI. lacZ was also ligated into pBPST-4. The ras-17N- and -galactosidase-expressing vectors are referred to as pBPST-Ras-17N and pBPST-lacZ, respectively.

The pBST-4 expression plasmid was generated originally to make stable transfectants and incorporates a tetracycline (tet)-inducible system as shown previously by Paulus et al. (37). Preliminary studies showed that expression levels in transient transfections were so high that experiments were performed on transiently transfected cultures, and thus the inducible nature of the vector was not taken advantage of in these studies. A tet-inducible retroviral vector was constructed from the tet-inducible expression system containing two plasmids, pUHD15-1 and pUHD10-3 (38), and the puromycin resistance gene (puro), which was isolated from pBabe-puro (39) with HindIII and ClaI, and then blunt-ended with the Klenow enzyme. The puro fragment was inserted in a sense orientation by blunt-end ligation into the pBabe-puro that was precut with BamHI and SalI to make plasmid pBabe-puro-puro. The puro gene in pBabe-puro-puro was removed with HindIII and ClaI and replaced with the following fragment containing HindIII, SnaBI, and ClaI restriction sites to create plasmid pBPS-SnaBI: 5'-AGCTTTACGTAAT-3' and 3'-AATGCATTAGC-5'.

The pBPS-SnaBI was cut with SnaBI, dephosphorylated with calf intestinal phosphatase, and then ligated with the tet-regulated transcription activator (tTA) gene, which was isolated from pUHD15-1 with EcoRI and BamHI and blunt-ended with Klenow enzyme. The new plasmid was named pBPST. To generate pBPST-4, a 0.88-kb fragment containing the tet-inducible promoter tetO-CMV was extracted from pUHD10-3 with HinpI I and inserted in reverse orientation to the internal SV40 promoter into pBPST, which was digested with ClaI.

Transient Transfection-- Transfection was performed using Effectene transfection reagent according to manufacturer instructions (Qiagen). Briefly, smooth muscle cells were seeded at a density of 5.2 × 10³ cells/well. After an overnight incubation, the cells were washed and incubated in 0.9 ml of DMEM with 10% FBS for 2-4 h. For each transfection, 0.8 µg of DNA (pBPST-Ras-17N or pBPST-lacZ) were added and incubated at 37 °C for 16-24 h, at which time the medium was removed, and the cells were washed with Puck's saline and incubated in serum-free DMEM for 48 h. The cells were then control-treated or treated with bFGF, EGF, LPA, or VLDL for 5 min. Total cell protein was isolated and subjected to Western blot analysis using antibodies against Ras and the active forms of ERK1/ERK2. To examine the transfection efficiency, the cells were fixed in 0.5% glutaraldehyde and incubated in 50 mM ferrocyanide, 2 mM MgCl₂, and 25 mM 5-bromo-4-chloro-3-indoyl--D-galactoside (Sigma) in PBS at 37 °C overnight. The percentage of stained (-galactosidase-positive) cells was evaluated.

Effect of VLDL and EGF on Cell Proliferation-- Quiescent smooth muscle cells were treated with VLDL, EGF (Sigma), VLDL plus EGF, or FBS for the indicated time. Total RNA was isolated and subjected to Northern blot analysis to measure the steady-state levels of expression of egr-1, c-fos, and histone H2B (see below). DNA synthesis was measured by labeling cells with [³H]thymidine and evaluating its incorporation into DNA and the percentage of radiolabeled nuclei (see below). In some cases, cells were pretreated in the absence or presence of the following inhibitors prior to the addition of reagents: PD98059, bisindolylmaleimide I, AG1478, or pertussis toxin.

Northern Blot Analysis-- Total cellular RNA was extracted, and Northern blot analysis was performed essentially as described previously (15). Equal amounts of total RNA were loaded onto each lane. Equal loading was evaluated by ethidium bromide staining (data not shown). After transfer, the blots were hybridized with a denatured DNA probe that was radiolabeled with [-³²P]dCTP (6 × 10³ Ci/mmol; DuPont) by the random-primed method using DNA polymerase I Klenow fragment according to manufacturer instructions (Roche Molecular Biochemicals). Blots were exposed to film with intensifying screens at 80 °C. Blots were probed for histone H2B, rat egr-1, which was provided kindly by Dr. Vikas Sukhatme of Harvard Medical School (Boston, MA) (40), and mouse c-fos cDNA, which was provided kindly by Dr. Michael Karin of the University of California San Diego (La Jolla, CA) (41).

Analysis of [³H]Thymidine Incorporation into DNA-- Cells plated into 96-well flat-bottom tissue culture plates were treated as described above; once rendered quiescent, the cells were incubated with reagents for 20 h, at which time the medium was removed, and the cells were washed with serum-free DMEM. The cells were radiolabeled for 4 h with 100 µl of [methyl-³H]thymidine (20 µCi/ml, 5 Ci/mmol; PerkinElmer Life Sciences) in serum-free DMEM. Cells were harvested, and [³H]thymidine incorporation into DNA was assessed as described previously (15). Alternatively, cells similarly treated in 6-well trays were evaluated for the percentage of radiolabeled nuclei as follows: the [³H]thymidine-containing medium was removed, and the cells were washed three times with cold PBS and then fixed for 2 min with 100% methanol. The cells were exposed to a photographic emulsion (1:1 Kodak NBT-2/H₂O; Eastman Kodak Co.) for 3 days at room temperature in the dark, at which time the radiolabeled nuclei were visualized after counterstaining of the cells with hematoxylin. The percentage of radiolabeled nuclei was determined by counting both positive and negative nuclei and expressing the number of positives as a percentage of the total.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

VLDL Activates the MAP Kinase Pathway-- To determine whether VLDL activates MAP kinase, the phosphorylation of MAP kinase isoforms ERK1/ERK2 was monitored in neonatal rabbit aortic smooth muscle cells. Fig. 1a shows that VLDL activated ERK1/ERK2 in a dose-responsive fashion at concentrations ranging from 3-100 µg/ml VLDL. Total levels of ERK1/ERK2 were similar in control as well as VLDL-treated samples (Fig. 1b). At a concentration of 30 µg/ml, VLDL induced a time-dependent activation of ERK1/ERK2 with a maximal response noted after 5 min in the presence of the lipoprotein (Fig. 1c). Phosphorylated ERK1/ERK2 began to decrease at 15 min and returned to baseline more than 6 h later. To determine whether VLDL-induced MAP kinase activation is mediated through the classical MAP kinase cascade, PD98059, a MEK-specific inhibitor, was used. The data in Fig. 1d show that pretreatment of the cells with PD98059 (20 µM) for 30 min blocked baseline activity and completely blocked the VLDL-induced phosphorylation of ERK1/ERK2.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   VLDL activates MAP kinase. a, smooth muscle cells were treated with increasing concentrations of VLDL (3-100 µg/ml) for 5 min. Total cell protein was extracted and subjected to Western blot analysis using an antibody against phosphorylated ERK1/ERK2. b, smooth muscle cells were treated as described for a. The samples were subjected to Western blot analysis using an antibody against total ERK1/ERK2. c, smooth muscle cells were treated with VLDL (30 µg/ml) for various times (5 min-15 h). Phosphorylated ERK1/ERK2 was measured as described for a. d, smooth muscle cells were preincubated in the absence () or presence (+) of PD98059 (20 µM) for 30 min and then incubated in the absence () or presence (+) of VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described for a.

VLDL Activates a Pertussis Toxin-sensitive G Protein-coupled Receptor That Transactivates the EGF Receptor-- To analyze the contribution of pertussis toxin-sensitive G proteins to the VLDL-induced MAP kinase activation, the effect of pertussis toxin on the phosphorylation of ERK1/ERK2 by VLDL was examined. The data in Fig. 2a show that pretreatment with pertussis toxin (100 ng/ml) for 24 h inactivated pertussis toxin-sensitive G protein-coupled receptors as evidenced by a loss of LPA-induced MAP kinase activation. As expected, the activation by EGF was unaffected by pertussis toxin treatment. Interestingly, the data clearly show that the VLDL-induced activation of MAP kinase is mediated by G protein-coupled receptors, because its effect was diminished by the addition by pertussis toxin.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Pertussis toxin-sensitive G proteins mediate VLDL-induced EGF receptor transactivation. a, smooth muscle cells were preincubated in the absence () or presence (+) of pertussis toxin (100 ng/ml) for 24 h and then control-treated or treated with EGF (20 ng/ml), LPA (25 µM), or VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend. b, smooth muscle cells were preincubated in the absence () or presence (+) of AG1478 (1 µM) for 30 min and then control-treated or treated with bFGF (20 ng/ml), EGF (20 ng/ml), LPA (25 µM), or VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend. c, smooth muscle cells were preincubated in the absence () or presence (+) of pertussis toxin (100 ng/ml) for 24 h and then control-treated or treated with EGF (20 ng/ml) or VLDL (30 µg/ml) for 5 min. Cell lysates were immunoprecipitated with an antibody against the EGF receptor. The immunoprecipitates were subjected to Western blot analysis using an antibody against phosphotyrosine. d, smooth muscle cells were preincubated in the absence () or presence (+) of pertussis toxin (100 ng/ml) for 24 h and/or AG1478 (1 µM) for 30 min and then control-treated or treated with VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend.

In a preliminary study, Western blot analysis of VLDL-treated cells was performed using an antibody against phosphotyrosine residues. The data (not shown) suggested that a 170-kDa protein was phosphorylated in response to VLDL. The EGF receptor is 170 kDa and has been shown to be transactivated by LPA (32). To determine directly whether VLDL activated the EGF receptor, AG1478, a specific EGF receptor tyrosine kinase inhibitor, was used. The cells were pretreated with the inhibitor, and then bFGF, EGF, LPA or VLDL, was added for 5 min, at which time MAP kinase activity was assessed. The data in Fig. 2b show that low levels of MAP kinase activity in control cultures were inhibited by AG1478, suggesting that baseline activity results from activation of the EGF receptor. As expected, bFGF-induced activation of MAP kinase was unaffected by AG1478. The inhibitor was shown to be effective in that MAP kinase activation by EGF was completely abolished in its presence. LPA-induced activation of smooth muscle cell MAP kinase was partially mediated by EGF receptor tyrosine kinase activity, as shown previously in Rat-1 fibroblasts (31). Finally, the data show that AG1478 partially inhibited the ability of VLDL to activate MAP kinase, furthering the hypothesis that it transactivates the EGF receptor.

To pursue the possibility that pertussis toxin-sensitive G proteins mediate the VLDL-induced EGF receptor transactivation, smooth muscle cells were treated with pertussis toxin for 24 h and then with EGF or VLDL for 5 min. The EGF receptor was immunoprecipitated, and then Western blot analysis was performed with an antibody against phosphotyrosine residues to visualize activated receptors. The data in Fig. 2c show that VLDL induced tyrosine phosphorylation of the EGF receptor. As expected, phosphorylation of the EGF receptor by EGF was unaffected by pertussis toxin. Confirmation of the activation of the EGF receptor was achieved by the demonstration that pertussis toxin completely blocked the EGF receptor tyrosine phosphorylation that was induced by VLDL.

To determine whether the MAP kinase activation by a pertussis toxin-sensitive G protein-coupled receptor is entirely mediated through transactivation of the EGF receptor, the cells were pretreated either with pertussis toxin, AG1478, or pertussis toxin plus AG1478, and then VLDL was added and MAP kinase activation was measured. The data (Fig. 2d) show that pertussis toxin had no additional effect on the partial inhibition by AG1478, suggesting that the pathway to VLDL-mediated MAP kinase activation by a G protein-coupled receptor was entirely through transactivation of the EGF receptor.

VLDL Activates Src Kinase-- To determine whether Src is involved in VLDL-induced MAP kinase activation, smooth muscle cells were pretreated with the specific Src family tyrosine kinase inhibitor PP2. The data in Fig. 3 show that PP2 inhibited MAP kinase activation by LPA but had no effect on EGF-induced activation of MAP kinase at low doses, as expected. Furthermore, the data show that pretreatment with the inhibitor significantly decreased the activation of MAP kinase by VLDL.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   VLDL-induced MAP kinase activation is Src-dependent. Smooth muscle cells were preincubated in the absence () or presence (+) of PP2 (5 µM) for 30 min and then control-treated or treated with LPA (25 µM), VLDL (30 µg/ml), or EGF (20 ng/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend.

Protein Kinase C Mediates VLDL-induced MAP Kinase Activation-- Protein kinase C was shown to be activated by LDL (5). To determine whether protein kinase C is involved in VLDL-induced MAP kinase activation, smooth muscle cells were pretreated with a protein kinase C inhibitor, bisindolylmaleimide I, for 30 min and then treated with EGF, PMA, or VLDL. The data in Fig. 4a show that as expected, the inhibitor did not affect EGF-induced MAP kinase activation but completely blocked PMA-induced MAP kinase activation (42, 43). Interestingly, VLDL-induced MAP kinase activation was substantially blocked by pretreatment with the inhibitor. To further substantiate the role of protein kinase C and to see if the activation of MAP kinase partially or fully depended on protein kinase C, the cells were pretreated with either increasing doses of bisindolylmaleimide I, with the protein kinase C inhibitor calphostin C, or with PMA and then treated with VLDL. The data in Fig. 4b show that although bisindolylmaleimide I only partially inhibited the VLDL-induced activation of MAP kinase (except with very high doses of the inhibitor), pretreatment with calphostin C at doses known to specifically inactivate protein kinase C completely blocked the VLDL-induced activation of MAP kinase. Likewise, cells that were depleted of protein kinase C by overnight treatment with PMA were virtually devoid of the active forms of MAP kinase.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Protein kinase C mediates VLDL-induced MAP kinase activation. a, smooth muscle cells were preincubated in the absence () or presence (+) of bisindolylmaleimide I (1 µM) for 30 min and then control-treated or treated with EGF (20 ng/ml), PMA (100 ng/ml), or VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend. b, smooth muscle cells were preincubated with various concentrations of bisindolylmaleimide I (5, 10, and 20 µM) for 30 min or with calphostin C (0.5 and 2.5 µM) or PMA (100 and 500 ng/ml) overnight and then control-treated or treated with VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend.

Dominant Negative Ras Mutant Blocks VLDL-induced MAP Kinase Activation-- To determine whether Ras is required for VLDL-induced MAP kinase activation, a dominant negative mutant form of Ras with an asparagine substituted for serine at position 17 (36) was expressed as described under "Experimental Procedures." Ras expression was determined by Western blotting. Controls were provided by cells transfected with the same plasmid expressing -galactosidase (pBPST-lacZ) instead of ras-17N. Initial experiments were performed with pBPST-lacZ to determine transfection efficiency; although it did vary somewhat from experiment to experiment, it was never lower than 20% and was sometimes as high as 60%. Most importantly, in cultures transfected with pBPST-Ras-17N, the levels of Ras were much higher than those of endogenous Ras (Fig. 5a), suggesting that this approach could be used to determine whether VLDL activates Ras (the additional lanes show that the expression of ras-17N was equivalent in untreated and bFGF-, EGF-, LPA-, or VLDL-treated cultures). To determine the effect of loss of Ras activity on VLDL-induced MAP kinase activation, the levels of phosphorylated ERK1/ERK2 were evaluated in cells transiently transfected with pBPST-Ras-17N and pBPST-lacZ. The data in Fig. 5b show that the dominant negative mutant inhibited baseline MAP kinase activity and that the function of the Ras mutant was displayed by the inhibition of MAP kinase activation by bFGF, EGF, and LPA, all previously shown to activate MAP kinase in a Ras-dependent fashion (31, 44). Finally, the data clearly show that VLDL-induced stimulation of MAP kinase was inhibited by expression of the Ras mutant, demonstrating that it is a Ras-dependent activation.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Dominant negative mutant ras-17N blocks VLDL-induced MAP kinase activation. a, smooth muscle cells seeded at low density were transfected with pBPST-Ras-17N or pBPST-lacZ and then incubated in serum-free medium for 2 days. The cells were then control-treated or treated with bFGF (20 ng/ml), EGF (20 ng/ml), LPA (25 µM), or VLDL (30 µg/ml) for 5 min. Total cell protein was extracted and subjected to Western blot analysis using an antibody against Ras. b, smooth muscle cells were treated as described for a. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend.

VLDL-induced MAP Kinase Activation Is Independent of its Degradation and Internalization-- VLDL binds to LDL receptors and/or LDL receptor-related protein (LRP) and undergoes receptor-mediated endocytosis, subsequently being degraded in lysosomes. Free cholesterol released from lysosomes is esterified into cholesteryl ester in the endoplasmic reticulum (45, 46). To determine whether degradation of VLDL is critical to its ability to activate MAP kinase, chloroquine was used to inhibit lysosomal function. Chloroquine (10 µM) inhibited the VLDL-induced esterification of cholesterol (Fig. 6a). It had no effect on VLDL-induced MAP kinase activation, however, suggesting that lysosomal degradation of the lipoprotein is not required (Fig. 6b).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   VLDL-induced MAP kinase activation is independent of its lysosomal degradation. a, smooth muscle cells were incubated for 24 h in the absence () or presence (+) of chloroquine (10 µM) and VLDL (0, 20, and 40 µg/ml). All cultures received [14C]oleic acid complexed to albumin (0.43 µCi/ml). Cultures were harvested, and [14C]cholesteryl ester levels were determined. b, smooth muscle cells were preincubated in the absence (0) or presence (10 and 20 µM) of chloroquine for 30 min and then incubated in the absence () or presence (+) of VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend.

Induction of egr-1 and c-fos by VLDL Is Sensitive to Inhibition of the MAP Kinase Cascade-- Because the activation of MAP kinase has been associated with cell proliferation and differentiation, it is important to measure biological responses downstream of MAP kinase activation (26-30). To determine the potential role of the signaling cascade induced by VLDL, its contribution to smooth muscle cell proliferation was assessed. The induction of immediate early genes such as egr-1 and c-fos is crucial for growth factor-stimulated cell proliferation, and we first determined whether VLDL can induce egr-1 and c-fos expression. Cultures were treated with 30 µg/ml VLDL, and the expression of egr-1 and c-fos was examined by Northern blot analysis. As shown in Fig. 7a, VLDL induced a rapid increase in both egr-1 and c-fos mRNA, with a maximal level achieved at 30 min. Fig. 7b shows that the induction of egr-1 and c-fos was dose-dependent, with maximal expression occurring at a lipoprotein concentration of 50-100 µg/ml VLDL. The role of the MAP kinase cascade in the induction of egr-1 and c-fos by VLDL was examined by determining the effect of PD98059 on expression of the genes. Pretreatment of smooth muscle cells with PD98059 (20 µM) for 30 min dramatically decreased the induction of both genes by VLDL (Fig. 7c).

View larger version (57K): [in this window] [in a new window]   Fig. 7.   VLDL induces smooth muscle cell expression of egr-1 and c-fos mRNA. a, quiescent smooth muscle cells were treated with VLDL (30 µg/ml) for the time indicated. Total RNA was harvested, and steady-state levels of egr-1 and c-fos mRNA were assessed by Northern blot analysis. b, quiescent smooth muscle cells were treated with the indicated concentration of VLDL for 30 min, at which time total RNA was harvested, and steady-state levels of egr-1 and c-fos mRNA were assessed by Northern blot analysis. c, quiescent smooth muscle cells were preincubated in the absence () or presence (+) of the MEK inhibitor PD98059 (20 µM) for 30 min and then incubated in the absence () or presence (+) of VLDL (30 µg/ml) for 30 min. Total RNA was harvested, and steady-state levels of egr-1 and c-fos mRNA were assessed by Northern blot analysis (10 µg/lane).

VLDL Plus EGF Synergistically Promote Smooth Muscle Cell Proliferation-- The effect of VLDL on the proliferation of quiescent smooth muscle cells was examined. Quiescent cells were treated with either VLDL or EGF (20 ng/ml), VLDL plus EGF, or increasing concentrations of FBS (v/v). The cells were incubated for 20 h, at which time [³H]thymidine was added as described under "Experimental Procedures." Four hours later, the cells were harvested, and the level of [³H]thymidine incorporation into DNA was determined. The data in Fig. 8 show that [³H]thymidine incorporation in the control cultures was very low and that the cells responded with a proliferative burst to the addition of FBS. (Additional experimentation demonstrated that EGF was effective in the 5-25 ng/ml range (data not shown). Moreover, Fig. 8 shows that the addition of VLDL or EGF alone elicited only small increases in [³H]thymidine incorporation. The stimulation of [³H]thymidine incorporation into DNA induced by treatment of the cells with both 100 µg/ml VLDL and EGF was robust. To further verify the proliferative capacity of this regimen, its effect on histone expression was determined by Northern blot analysis of total cellular RNA. The data in Fig. 9 show that as expected, the addition of 10% FBS resulted in release of the cells from growth arrest as demonstrated by the increase in steady-state levels of mRNA expression of the S phase-specific histone H2B. Likewise, treatment with VLDL plus EGF resulted in an increase in the expression of steady-state levels of histone mRNA, although either agent alone was not sufficient to alter its expression. Similar experiments in which the percentage of [³H]thymidine-labeled nuclei was examined substantiated these results as shown in Table I, i.e. initially very few of the nuclei were radiolabeled (1.3%) confirming that the cells were quiescent, and the addition of 10% FBS or the simultaneous addition of VLDL plus EGF stimulated a substantial increase in that number (31.0 and 24.1%, respectively).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   VLDL plus EGF stimulate DNA synthesis. Quiescent smooth muscle cells were control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml), VLDL plus EGF. Inset, quiescent smooth muscle cells were treated with increasing concentrations of FBS. After incubation for 20 h, the cells were radiolabeled with [3H]thymidine (20 µCi/ml) for an additional 4 h and harvested for determination of the incorporation of [3H]thymidine into DNA as described under "Experimental Procedures." The data are expressed as mean cpm/well ± S.D. (n = 5).

View larger version (44K): [in this window] [in a new window]   Fig. 9.   VLDL plus EGF induce expression of histone mRNA. Quiescent smooth muscle cells were control-treated or treated with VLDL (100 µg/ml), EGF (13 ng/ml), VLDL plus EGF, or 10% FBS. After incubation for 24 h, total RNA was harvested, and steady-state levels of histone H2B mRNA were assessed by Northern blot analysis (15 µg/lane).

View this table: [in this window] [in a new window]   Table I Effect of VLDL plus EGF on [3H]thymidine labeling of nuclei

VLDL Plus EGF Synergistically Induce Cyclin D1 Expression and Down-regulate p27^KIP1 Expression-- To determine whether VLDL plus EGF induce cyclin D1 expression, quiescent cells were treated, and the expression of cyclin D1 was examined by Western blot analysis. The data in Fig. 10a show that VLDL plus EGF synergistically induced cyclin D1 expression; however, either agent alone was ineffective. The effect of VLDL plus EGF on p27^KIP1 expression was examined by Western blot analysis, which shows that similar to 10% FBS, VLDL plus EGF down-regulated the expression of p27^KIP1 protein; however, VLDL alone or EGF alone had no effect on this parameter (Fig. 10b). The data on [³H]thymidine incorporation, histone mRNA, cyclin D1, and p27^KIP1 expression show that neither VLDL nor EGF alone are capable of inducing quiescent smooth muscle cells into S phase.

View larger version (41K): [in this window] [in a new window]   Fig. 10.   Sustained activation of ERK1/ERK2 by VLDL plus EGF is associated with an induction of cyclin D1 expression and a decrease of p27KIP1 expression. a, quiescent smooth muscle cells were control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml), VLDL plus EGF, or 10% FBS for the indicated time. Total cell protein was extracted and subjected to Western blot analysis using an antibody against cyclin D1. b, smooth muscle cells were treated as described for a. Total cell protein was extracted and subjected to Western blot analysis using a mouse monoclonal antibody against p27KIP1. c, quiescent smooth muscle cells were control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml), VLDL plus EGF, or 10% FBS for the indicated time. Phosphorylated ERK1/ERK2 was measured as described in the Fig. 1a legend.

It has been suggested that cell proliferation is induced only when MAP kinase activation is sustained. Because VLDL or EGF alone do activate MAP kinase but do not induce the cells to enter S phase, it was of interest to determine the levels of activation of MAP kinase by each agent alone as well as in combination. As shown in Fig. 10c, VLDL strongly activated the MAP kinases ERK1/ERK2 after 5 min of treatment as reported above. MAP kinases were strongly activated after 5 min of treatment with EGF as well, but by 7 h after the addition of either agent alone, the levels of phosphorylated ERK1/ERK2 were dramatically decreased, and by 17 h they were close to baseline levels. Interestingly, the response to the addition of VLDL plus EGF was also highest at early time points; however the activation of MAP kinase was still evident even at 17 h at levels equivalent to those induced by 10% FBS.

Pertussis Toxin-sensitive G Proteins, the EGF Receptor, and Protein Kinase C-mediated MAP Kinase Activation Are Responsible for the Mitogenic Effect of VLDL Plus EGF-- To determine whether MAP kinases and their upstream signaling molecules are critical for VLDL-induced DNA synthesis, quiescent smooth muscle cells were pretreated with PD98059, pertussis toxin, AG1478, or bisindolylmaleimide I and then treated with 100 µg/ml VLDL, 20 ng/ml EGF, or VLDL plus EGF for 20 h, at which time the incorporation of [³H]thymidine into DNA was examined. The data in Fig. 11 show that the inhibition of MEK, pertussis toxin-sensitive G proteins, the EGF receptor, or protein kinase C significantly blocked VLDL plus EGF-induced DNA synthesis, indicating a critical role of MAP kinase activation on the mitogenic effect of VLDL.

View larger version (31K): [in this window] [in a new window]   Fig. 11.   Inhibition of MAP kinase activation blocks DNA synthesis induced by EGF plus VLDL. Quiescent smooth muscle cells were preincubated in the absence (no inhibitor) or presence of PD98059 (50 µM), AG1478 (1 µM), or bisindolylmaleimide I (1 µM) for 30 min or pertussis toxin (100 ng/ml) for 24 h. The cells were then control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml), or VLDL plus EGF. Inset, quiescent smooth muscle cells were treated with increasing concentrations of FBS. After incubation for 20 h, the cells were radiolabeled with [3H]thymidine (20 µCi/ml) for an additional 4 h and harvested for determination of the incorporation of [3H]thymidine into DNA as described under "Experimental Procedures." The data are expressed as mean cpm/well ± S.D. (n = 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These experiments show that VLDL activates smooth muscle cell MAP kinase, and the activation is mediated by a G protein-coupled receptor that transactivates the EGF receptor. VLDL-induced activation of MAP kinase is mediated by protein kinase C and is Ras-dependent. Previous workers noted that native and oxidized LDL stimulate smooth muscle cell MAP kinase in a protein kinase C-dependent manner (22). Interestingly, MAP kinase was stimulated by LDL and oxidized LDL in U-937 macrophage-like cells; however, G protein-coupled receptors or protein kinase C were not involved (47). Others showed that oxidized LDL-induced proliferation of macrophages, however, resulted from the activation of protein kinase C via pertussis toxin-sensitive G proteins and the internalization of lysophosphatidylcholine (48). Sachinidis et al. (21) demonstrated that LDL-stimulated smooth muscle cell proliferation involves the activation of MAP kinase via a G protein-coupled receptor, which seems to be different from the classical lipoprotein receptor. Our results with chloroquine suggest that the VLDL-induced MAP kinase pathway does not involve lysosomal processing of the lipoprotein.

Cross-talk of signaling pathways was suggested by studies in which transactivation of the EGF receptor by tumor necrosis factor- was noted in human endothelial cells (49). EGF receptor transactivation by thrombin, endothelin and LPA receptors (32, 33), and the m1 muscarinic acetylcholine receptor (50) as well as the angiotensin II type I receptor (51) has been demonstrated. A novel finding of the present study is that the VLDL-induced activation of MAP kinase can be significantly inhibited by AG1478, a specific EGF receptor tyrosine kinase inhibitor. Moreover, pertussis toxin inhibited VLDL-induced tyrosine phosphorylation of the EGF receptor, indicating that pertussis toxin-sensitive G proteins mediate EGF receptor transactivation, thereby leading to MAP kinase activation. To our knowledge, this is the only report of lipoprotein-induced transactivation of the EGF receptor via activation of a G protein-coupled receptor. Our data also show that LPA-induced MAP kinase activation required transactivation of the EGF receptor in smooth muscle cells as was shown in Rat-1 cells (32). Interestingly, EGF receptor transactivation by LPA in GD25 fibroblast cells (52) and PC12 cells (53) was not shown, suggesting that involvement of the EGF receptor in G protein-coupled receptor-induced MAP kinase activation is cell type-dependent.

Incomplete inhibition of VLDL-induced MAP kinase activation by AG1478 and pertussis toxin indicates that the MAP kinase activation may not be exclusively mediated via the G protein-coupled receptor-mediated transactivation of the EGF receptor pathway and must involve other upstream transducers. Because the activation is completely inhibited by calphostin C, it is likely that another pathway that goes through protein kinase C is responsible.

A potential intermediate in the G protein-coupled receptor-mediated transactivation of the EGF receptor could be the Src family kinases. Src has been shown to be recruited to a complex consisting of G protein subunit, -arrestin, and a G protein-coupled receptor (54) where it is tyrosine-phosphorylated and activated after stimulation of a G protein-coupled receptor in Cos-7 cells (53, 55). A constitutively active Src mutant stimulated tyrosine phosphorylation of the EGF receptor (56); however, there is conflicting evidence that Src is not required for LPA-induced MAP kinase activation in Rat-1 or Cos-7 cells (57). Our data using the Src kinase inhibitor PP2 demonstrates that the activation of MAP kinase by VLDL depends on the activity of Src.

Agonist-stimulated MAP kinase can be Ras-dependent or Ras-independent. For example, it was noted that activation of MAP kinase by angiotensin II differed in cardiac fibroblasts and cardiac myocytes such that the activation was pertussis toxin-sensitive and Ras-dependent in fibroblasts, whereas MAP kinase activation in myocytes was Ras-independent and insensitive to treatment with pertussis toxin but strongly repressed by inhibitors of protein kinase C activity (58). The present study demonstrates that Ras is at least partially responsible for VLDL-induced MAP kinase activation in aortic smooth muscle cells, because a dominant negative Ras mutant blocked activation. Our results strongly suggest that VLDL induces the RasRaf-1MEKMAP kinase cascade. Oxidized LDL stimulated smooth muscle cell Ras-GTP formation and MAP kinase (23); however, the relationship between the two remains to be investigated. Sachinidis et al. (21) found LDL-stimulated MAP kinase activation to be independent of Raf-1 phosphorylation in smooth muscle cells, and our data provide no direct evidence for Raf-1 involvement. Angiotensin II was shown to stimulate MAP kinase by a Ras-independent pathway in rat smooth muscle cells (59), whereas other workers showed that angiotensin II-mediated activation of MAP kinase in rat smooth muscle cells was pertussis toxin-insensitive, Ras-dependent, and mediated by phospholipase C (60). Takahashi et al. (59) proffer the explanation that phenotypic differences between smooth muscle cell cultures may be responsible for the variations in results. This highlights the importance of characterizing the cells under study and the recognition that receptor expression as well as availability of downstream targets are likely to vary not only with cell type but with changes in phenotype. The cells examined in this report were deprived of serum prior to the addition of VLDL, rendering the cells quiescent. It would be of interest to determine whether changes in the proliferative/differentiation state of the cells contribute to the ability of VLDL to activate MAP kinase and/or the pathway mediating such activation.

Deciphering the contribution of atherogenic lipoproteins to smooth muscle cell proliferation is important to understand the development of the atherosclerotic plaque as well as restenosis after balloon angioplasty. The effect of atherogenic lipoproteins on smooth muscle cell proliferation was the subject of a number of past investigations (4, 5, 61). Our laboratory showed that VLDL increased the proliferative rate of neonatal aortic smooth muscle cells that were actively proliferating in media containing lipid-deficient serum (14, 15). Others studying high density lipoprotein (HDL)- and LDL-induced human smooth muscle cell proliferation noted synergy with other growth factors including platelet-derived growth factor (PDGF), EGF, insulin-like growth factor-I (IGF), and bFGF (5, 10), although Resink et al. (10) found that LDL and high density lipoprotein stimulated proliferation even in the absence of additional growth factors. Our data extend this work to show that although the atherogenic lipoprotein VLDL does not stimulate proliferation of quiescent smooth muscle cells alone, when added in combination with EGF this lipoprotein increased both total [³H]thymidine incorporation into DNA as well as the percentage of [³H]thymidine-labeled nuclei. Moreover, the expression of histone mRNA confirmed that VLDL plus EGF stimulated entry of the cells into the S phase of the cell cycle. The dependence of the induction of cell proliferation on the pathway activating MAP kinase was shown by inhibiting G protein-coupled receptors, the EGF receptor, protein kinase C, or MEK, which resulted in a dramatic decrease in VLDL plus EGF-induced cell proliferation. [³H]thymidine incorporation into DNA was inhibited ~80% by pertussis toxin, making it likely that the synergistic action of VLDL plus EGF on smooth muscle cell proliferation largely depends on activation of a G protein-coupled receptor by VLDL.

Cell cycle progression results from processes leading to the binding of AP-1 transcription factors (consisting of dimers of the Fos and Jun families), leading to the activation of target genes such as cyclin D1, which is the regulatory subunit of cyclin-dependent kinases essential for G₁ progression (62, 63). Chatterjee et al. (23) found that oxidized LDL but not native LDL stimulated Ras, MAP kinase, and c-fos expression in human aortic smooth muscle cells. Somewhat different results were noted by Kusuhara et al. (22), who found that native as well as oxidized LDL preparations stimulated MAP kinase in smooth muscle cells; however, neither LDL nor oxidized LDL induced c-fos expression. Our results with VLDL suggest that the native lipoprotein stimulates MAP kinase and a transient expression of c-fos and egr-1; however, this was unable to sustain cell cycle progression in the absence of EGF. We showed that cyclin D1 expression and ultimately G₁ progression depended on the synergistic action of VLDL plus EGF. It has been suggested that the activation of MAP kinases is essential for growth factor-stimulated induction of cyclin D1 and cell cycle progression (64). That sustained activation of MAP kinase is required for progression of cells to S phase has been suggested in a number of studies. Auge et al. (11) demonstrated that oxidized LDL-induced smooth muscle cell proliferation was mediated by an activation of the sphingomyelin-ceramide pathway, which induces a sustained activation of MAP kinase. Both LDL and minimally modified LDL induced mesangial cell phosphorylation of platelet-derived growth factor and EGF receptors, activated Ras, and resulted in sustained (up to 24 h) activation of MAP kinase and cell proliferation (65). A sustained activation of MAP kinase by LPA in Rat-1 cells seems essential for the induction of cell proliferation (66, 67). These authors noted that nonmitogenic doses of LPA stimulated a transient activation of MAP kinase, which induced c-fos expression; however, mitogenic doses were necessary for sustained MAP kinase activation and robust expression of Fra-1, Fra-2, c-Jun and JunB, leading to cell proliferation. Although c-fos expression was induced in a MAP kinase-dependent manner by nonmitogenic stimuli in CCL39 cells, only agents that were able to sustain activation of MAP kinase induce late AP-1 genes and cyclin D1 expression, leading to cell cycle entry (64, 68). Our results support the notion of sustained MAP kinase activation leading to cell proliferation in our system in that the activation of MAP kinase by VLDL plus EGF was sustained for a longer period of time than was evident when either agent was used alone. Sustained activation of MAP kinase correlated with an increase in cyclin D1 expression and decrease in p27^KIP1, the latter previously shown to be involved in the regulation of smooth muscle cell proliferation (69-73). One must be cautious, however, in assigning categorical significance to the time of activation of MAP kinase, because it represents a measure of the convergence of multiple signaling pathways and is also a point of divergence of additional myriad of downstream signals. It is important to measure cell functionality, e.g. cell proliferation rather than MAP kinase activation, to be certain of the role of a particular agonist in cell/organ functionality. Bornfeldt et al. (30) found that platelet-derived growth factor-induced MAP kinase activation stimulated cell proliferation in cyclooxygenase-2-negative human smooth muscle cells; however, the opposite effect on proliferation was noted when cells expressing cyclooxygenase-2 were treated with platelet-derived growth factor despite a similar increase in MAP kinase activation. These authors proffer the explanation that target availability at the time of stimulation dictates among varying functional responses potentially elicited by MAP kinase activation. In this manner, a signal pathway can serve multiple functions, and therefore it is important to study functional responses downstream of MAP kinase activation.

The contribution of oxidation to the proliferative capacity of LDL remains controversial. Heery et al. (8) found that only oxidized LDL stimulated smooth muscle cell proliferation. Resink et al. (10) found that native and oxidized LDL were equally effective in stimulating [³H]thymidine incorporation into DNA, and Kusuhara et al. (22) noted that both native as well as oxidized LDL preparations stimulated MAP kinase in a protein kinase C-dependent manner; however, oxidized LDL was more effective. Although it seemed that a lipoprotein-associated lipid moiety was responsible for the activation, interestingly, neither thiobarbituric acid-reacting substances nor lipid peroxide content correlated with MAP kinase activation, suggesting that neither aldehydes nor lipid peroxides are responsible. Although the VLDL preparations used in this study were protected from oxidation as reported previously (14), one cannot rule out the possibility that some oxidation has occurred.

In summary, the data show that VLDL activates the MAP kinase ERK1/ERK2 in smooth muscle cells. The activation is mediated by a G protein-coupled receptor that transactivates the EGF receptor and depends on the activation of Src and Ras as well as protein kinase C. Moreover, VLDL stimulates egr-1 and c-fos expression in quiescent smooth muscle cells, and the stimulation is sensitive to inhibition of the MAP kinase cascade. The addition of VLDL plus EGF activated a sustained MAP kinase response and induced cell proliferation, which depends on the activation of pertussis toxin-sensitive G protein-coupled receptors, EGF receptors, protein kinase C, and the classical MAP kinase cascade.

	ACKNOWLEDGEMENTS

We would like to thank Rosemarie Moscaritolo, Daniel Pine, and Valerie Verbitzki for their exceptional technical assistance. In addition, we thank Drs. Judith Foster and Barbara Slack of the Boston University School of Medicine for their expert advice in preparing this manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AG-9006 and HL-13262.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 Biochemistry, 715 Albany St., Boston, MA 02118. Tel.: 617-638-5094; Fax: 617-638-5339; E-mail: schreibe@biochem.bumc.bu.edu.

Published, JBC Papers in Press, May 25, 2001, DOI 10.1074/jbc.M103761200

	ABBREVIATIONS

The abbreviations used are: LDL, low density lipoprotein; VLDL, -migrating very low density lipoprotein; MAP, mitogen-activated protein; EGF, epidermal growth factor; MEK, MAP kinase kinase; LPA, lysophosphatidic acid; DMEM, Dulbecco's modified Eagle's Medium supplemented with 3.7 g/liter NaHCO₃, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM minimum Eagle's medium nonessential amino acids and 1 mM minimum Eagle's medium sodium pyruvate solution; FBS, fetal bovine serum; bFGF, basic fibroblast growth factor; PMA, phorbol-12-myristate-13-acetate; tet, tetracycline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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