The Growth-Promoting Effect of Low-Density Lipoprotein May Be Mediated by a Pertussis Toxin-Sensitive Mitogen-Activated Protein Kinase Pathway

  1. Agapios Sachinidis,
  2. Stefan Seewald,
  3. Petra Epping,
  4. Claudia Seul,
  5. Yon Ko and
  6. Hans Vetter
  1. Medizinische Universitäts-Poliklinik, 53111 Bonn, Germany
     
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    Abstract

    Low-density lipoprotein (LDL) is known to be a mitogenic factor for vascular smooth muscle cells (VSMCs), fibroblasts, and endothelial cells. In the current study, we describe possible intracellular mechanisms by which LDL elicits its mitogenic effects. Stimulation of VSMCs with LDL resulted in a pertussis-toxin (PTX)-sensitive stimulation of the 44-kDa mitogen-activated protein (MAP) kinase (p44mapk) and 42-kDa MAP kinase (p42mapk) isoforms as well as in a PTX-sensitive increase in intracellular free Ca2+ concentration ([Ca2+]i). Binding of the LDL-induced increase in [Ca2+]i to the intracellular Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester resulted in a 2-fold increase in the phosphorylated p44mapk and p42mapk isoforms but did not influence the LDL effect of VSMC DNA synthesis. PD 98059, a MAP kinase kinase inhibitor, remarkably attenuated the LDL-induced activation of MAP kinases and DNA synthesis. Treatment of normal human skin fibroblasts and human fibroblasts isolated from patients with familial hypercholesterolemia homozygote class 1 mutations, which are not able to produce the classic LDL receptor, resulted also in a PTX-sensitive increase in cell DNA synthesis and stimulation of the p44mapk and p42mapk isoforms in both cell types. These results demonstrate that the mitogenic effect of LDL is mediated by a PTX-sensitive Gi-coupled receptor that is independent of its classic receptor and involves activation of MAP kinase isoforms. Furthermore, the mitogenic effect of LDL may be mediated by the activation of the MAP kinase pathway. In contrast, the LDL-induced increase in [Ca2+]i may be implicated in this process only in conjugation with other signaling components.

    VSMCs hypertrophy, and proliferation may participate in the pathophysiology of cardiovascular diseases (1). Atherosclerotic lesions result from an excessive, inflammatory-fibroproliferative response to various forms of injury to the endothelium and smooth muscle within the arterial wall (2). The major physiological function of LDL is to deliver cholesterol into cells via binding to its classic receptor (3). LDL is considered to be the main atherogenic class of lipoproteins (4). In addition to its physiological role as a cholesterol transport vehicle and its regulatory function for cholesterol homeostasis, there is evidence that LDL causes general cellular activation and cell proliferation. In this context, it has been shown that LDL stimulates the phosphoinositide catabolism (5), elevates [Ca2+]i, activates the Na+/H+ exchanger (6), and promotes the expression of immediate-early growth response genes such as c-fos (5) and egr-1 (7) in VSMCs. Furthermore, several laboratories reported that LDL per se and in combination with classic growth factors such as PDGF exerts mitogenic effects on VSMCs (6, 8-10) and endothelial cells (9, 11). Recently, an atypical LDL binding site on human VSMCs was characterized that is independent of its classic receptor and may mediate the LDL-induced phosphoinositide catabolism in VSMCs (12).

    PDGF is a potent serum factor that promotes the proliferation of fibroblasts, glial cells, and VSMCs (13, 14) and thus is postulated to play an important role in the pathogenesis of atherosclerosis (1, 2). It is established that the classic growth factor PDGF-BB propagates its mitogenic signals via autophosphorylation of its respective PDGF-β receptor on tyrosine residues, resulting in tyrosine phosphorylation of different substrate proteins such as PLC-γ1, p21ras GTPase activating protein, and PI3 kinase, carrying SH2 domains that are capable of binding to specific regions of the PDGF-β receptor containing autophosphorylated tyrosine residues (15). There are several reports demonstrating that the PLC-γ pathway induced by potent VSMC mitogens such as PDGF and FGF might not be essential for cell DNA synthesis (16, 17). A more important mitogenic signal seems to be activation of the PI3 kinase that phosphorylates the 3 position of the inositol ring of phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-biphosphate to produce their respective PI3 phosphorylated metabolites (18, 19); therefore, the PI3 kinase was an interesting candidate to investigate whether it is involved in the LDL-induced DNA synthesis. It has recently been recognized that further transmission of growth signals from the receptor to the nucleus is mediated by sequentially activated protein kinases. The activation of MAP kinases, in particular, the 42-kDa (p42mapk) and the 44-kDa (p44mapk) isoforms, seems to be a key step in growth signal transduction through tyrosine kinase receptors such as the PDGF-β receptor (20, 21).

    In this context, it is assumed that MAP kinases and PI3 kinase activate S6 protein kinases, in particular the 70-kDa (p70rsk) and the 90-kDa kinase (p90rsk), encoded by the rsk gene family. Stimulation of the S6 protein seems to be the requisite for mitogenic signal transduction. Furthermore, it is postulated that activation of MAP kinases is involved in the growth factor-induced expression of immediate-early genes such as c-fos (20, 21).

    It is believed that activation of the MAP kinase isoforms occurs by MEK via threonine and tyrosine phosphorylation, and Raf-1 kinase (a 74-kDa protein kinase encoded by the proto-oncogene raf-1) may be the kinase responsible for activation of MEK (20, 21). In this context, it is assumed that activation of the MAP kinase occurs via a cascade that requires the activation of p21ras, p21rasGTPase activating protein, Raf-1 kinase, and MEK. It is believed that activation of Raf-1 kinase occurs via phosphorylation of several serine and threonine residues of the molecule by the activated c-Ras via an unknown mechanism. It has been proposed that activation of c-Ras occurs by Grb2/Sos. Grb2 is activated by the SH2-adaptor protein Shc, which is activated by tyrosine-phosphorylation in response to growth factors. Alternatively, this cascade may also stimulated by G proteins via activation of MEK kinase independently of the Raf-1 cascade (20, 21).

    In contrast to PDGF-BB, agonists such as thrombin and LPA propagate their mitogenic effect in fibroblasts through a PTX-sensitive Gi protein pathway (22-24).

    To elucidate whether activation of the MAP kinase pathway is implicated in LDL-induced DNA synthesis, the effect of LDL on the phosphorylation of p42mapk and p44mapkisoforms and DNA synthesis in VSMCs was investigated in the presence and absence of the selective MEK inhibitor PD 98059 [(2–2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one] (25). To show whether the effect of LDL on cell growth is mediated by a PTX-sensitive Gi protein, the effects of PTX on LDL- or LPA-induced DNA synthesis and intracellular transduction pathways were investigated. Finally, to demonstrate whether these effects are mediated by the classic LDL receptor, the effect of LDL on MAP kinase activity and DNA synthesis in cultured normal human fibroblasts and human fibroblasts isolated from patients with FH homozygote class 1 mutations was examined. These cells are not able to produce LDL receptor protein as determined by reaction with monoclonal antibodies (3).

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    Experimental Procedures

    Materials.

    Fura-2/pentaacetoxymethyl ester, MAPTAM, and PD 98059 were obtained from Calbiochem (Bad Soden, Germany). Anti-α-smooth muscle actin was obtained from Sigma Chemical (Deisenhofen, Germany). FITC-conjugated F(ab′)2fragment of goat anti-mouse immunoglobulins was obtained from Dako Diagnostika (Hamburg, Germany). DMEM, Ham’s F-10, and phosphate-buffered saline were obtained from GIBCO BRL (Eggestein, Germany). FCS was obtained from Boehringer-Mannheim Biochemica (Mannheim, Germany). PDGF-BB was a gift from Prof. Dr. Jürgen Hoppe (Department of Physiological Chemistry, University of Würzburg, Germany) and was prepared as previously described (14). [3H]Methyl-thymidine, Hybond N+ membranes, and ECL Western blotting detection system were obtained from Amersham (Little Chalfont, UK). PhosphoPlusmapk Antibody Kit was obtained from New England Biolabs (Beverly, MA). Sepharose-coupled anti-phosphotyrosine and anti-phosphothreonine antibodies were obtained from Sigma. Monoclonal anti-PI3 kinase and anti-Raf (c-Raf-1) antibodies were obtained from Transduction Laboratories (Lexington, KY). Lipidophor system for lipoprotein electrophoresis was obtained from Immuno AG (Vienna, Austria).

    Isolation and culture of VSMCs.

    Rat aortic VSMCs were isolated from thoracic aortae of Wistar-Kyoto rats (6–8 weeks old, Charles River Wiga GmbH, Sulzfeld, Germany) by enzymatic dispersion using a slight modification of the method of Chamley et al.(26) as previously described (27). Cells were cultured in DMEM supplemented with 10% FCS, nonessential amino acids, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37° in the Steri-Cult incubator (Forma Scientific, Göttingen, Germany) in a humidified atmosphere of 95% air/5% CO2. Cells (3 × 106) were grown in 75-cm2flasks to confluence over 4–5 days. The purity of VSMC cultures was confirmed by immunocytochemical localization of smooth muscle-specific α-smooth muscle actin using FITC-conjugated monoclonal anti-α-smooth muscle actin antibodies plus a second FITC-conjugated F(ab′)2 fragment of goat anti-mouse immunoglobulins. Experiments were performed using cells between passages 5 and 20.

    Culture of fibroblasts.

    Normal and FH-homozygote fibroblasts were obtained from Human Genetic Mutant Cell Repository Institute for Medical Research (Camden, NJ) and cultured over several passages after detachment of the confluent cells with Puck’s Saline A physiological solution containing 0.04% trypsin/0.02% EDTA buffer. The cells were allowed to grow as described for VSMCs.

    LDL isolation.

    LDL (d = 1.019–1.063 g/ml) was isolated from the plasma of four normocholesterolemic subjects (serum cholesterol < 6.2 mm) by KBr density-gradient ultracentrifugation according to Redgrave et al. (28). The LDL fraction was dialyzed against 0.15 m NaCl containing 1 mm EDTA. No oxidation of LDL was observed for ≥4 weeks after LDL preparation as assessed by measurement of malondialdehyde according to the thiobarbituric acid method as previously described (11). Quantification of LDL was performed by determination of the protein component according to the method of Bradford (29). The purity of LDL was examined with a commercially available lipoprotein electrophoresis kit (Lipidophor AII In 12; Immuno AG) after 1% agarose electrophoresis according to the manufacturer’s protocol as previously described (30).

    Measurement of [Ca2+]i.

    VSMCs were cultured onto round glass microscope slides (diameter, 12 mm) under normal tissue culture conditions until confluence. Cells were incubated with 2 μm Fura-2/pentaacetoxymethyl ester at 37° for 20 min in HEPES buffer supplemented with 1% bovine serum albumin (w/v). Just before measurements were made, the cell monolayer was rinsed with HEPES buffer without bovine serum albumin, containing 1 mmCaCl2, and the glass slide was positioned diagonally in the cuvette. Measurements were performed in HEPES buffer containing 1 mm CaCl2. The Ca2+/Fura-2 fluorescence was measured at 37° in a Perkin-Elmer LS50 fluorescence spectrofluorometer (Überlingen, Germany) at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 505 nm. Maximum (Rmax) and minimum (Rmin) fluorescence values were determined by the addition of digitonin (30 μm) followed by the addition of 1% Triton X-100 (v/v) and Tris base/EGTA at a final concentration of 100 mm Tris base/25 mm EGTA. Fluorescence was corrected for cellular autofluorescence. Fluorescence signals were calibrated according to Grynkiewicz et al. (31) using the following equation: [Ca2+]i =Kd × (R − Rmin)/(Rmax − R) × (Sf2/Sb2), where the Kd value for the Fura-2/Ca2+ complex at 37° is assumed to be 224 nm; Sf2 is the 380 nm-exited fluorescence in the absence of Ca2+ (EGTA added), and Sb2 is the 380 nm-excited fluorescence in the presence of saturating Ca2+ (1 mmCa2+).

    Gel electrophoresis and immunostaining.

    VSMCs were seeded onto 24-well culture plates (4 × 105cells/well; well diameter, 12 mm) and cultivated in culture medium until confluent. Medium was then replaced by serum-free medium consisting of a mixture of DMEM and Ham’s F-10 medium (1:1). After another 32-hr cultivation in serum-free medium, cells were stimulated for different time periods with LDL. After removal of the medium, cells were lysed with a 1-ml buffer containing 137 mm NaCl, 20 mm Tris·HCl, pH 6.7, 2% SDS, 2% mercaptoethanol, and 1 mm sodium orthovanadate. After 10 min at 0°, cell lysates were centrifuged at 14,000 × g for 2 min. Then, cell lysates were mixed with 80 μl of Sepharose-coupled anti-phosphotyrosine antibody to immunoprecipitate PI3 kinase and Sepharose-coupled anti-phosphothreonine antibody to immunoprecipitate Raf-1 kinase and shaken for ≥2 hr at 4°. Tyrosine- and threonine-phosphorylated proteins were eluted with 100 μl of lysis buffer containing 5 mm phenylphosphate. Then, 20 μl was mixed with sample buffer and heated for 5 min at 95°. After separation of the proteins in a 7.5% SDS-polyacrylamide gel, proteins were transferred to a PVDF membrane overnight at 100 mA in a buffer containing 25 mmTris base, 192 mm glycine, and 20% methanol, pH 8.3. The protein transfer was checked using Ponceau S. Enhanced chemiluminescence detection of PI3 kinase was performed as previously described using monoclonal anti-PI3-kinase (1:5,000) and secondary anti-mouse antibodies (1:10,000) (32).

    MAP kinase protein analysis was performed by chemiluminescence Western blotting as described in the manufacturer’s instructions for the PhosphoPlusmapk Antibody Kit (New England Biolabs). After cell stimulation with LDL, the medium was removed, and the cells were lysed with the SDS sample buffer containing 62.5 mm Tris·HCl, pH 6.8, 2% SDS (w/v), 10% glycerol, and 50 mm dithiothreitol. Aliquots were used for protein determinations with the BioRad (Richmond, CA) protein assay according to the method of Bradford (27). Then, 0.1% bromphenol blue (w/v) was added to the aliquots; 30 μg of protein was analyzed by SDS-PAGE in a 10% acrylamide gel with a thickness of 0.75 mm through use of the Mini Gel Protean System (BioRad).

    Proteins were transferred to a PVDF membrane as described above. Membrane blocking and antibody incubations were performed according to the manufacturer’s instructions for the PhosphoPlusmapk Antibody Kit using a phosphospecificmapk rabbit polyclonal IgG primary antibody and the alkaline phosphatase-conjugated anti-rabbit secondary antibody. The primary antibody recognized p42mapkand p44mapk only when catalytically activated by phosphorylation at Tyr204 (33). Chemiluminescence detection of the active p42mapk and p44mapkwas performed as described in the manufacturer’s instructions for the Phototope Western detection system.

    Determination of DNA synthesis.

    The effect of LDL on [3H]thymidine incorporation into cell DNA was assessed as previously described (27).VSMCs or fibroblasts were seeded onto 24-well culture plates and grown to confluence. The medium was replaced by serum-free medium consisting of a mixture of DMEM and Ham’s F-10 medium (1:1). After another 32-hr cultivation in serum-free medium, stimuli were added to the cells. Cultures were exposed to the stimulating agents for 20 hr before 3 μCi/ml [3H]thymidine was added to the serum-free medium. Four hours later, experiments were terminated by aspiration of the medium and subjection of the cultures to sequential washes with phosphate-buffered saline containing 1 mmCaCl2, 1 mmMgCl2, 10% trichloroacetic acid, and ethanol/ether (2:1, v/v). Acid-insoluble [3H]thymidine was extracted into 250 μl/dish 0.5 m NaOH, and 100 μl of this solution was mixed with 5 ml of scintillant (Ultimagold; Packard, Groningen, The Netherlands) and quantified using a liquid scintillation counter (LS 3801; Beckman, Düsseldorf, Germany). Then, 50 μl of the residual solution was prepared for the determination of protein using the BioRad protein assay according to the method of Bradford (29).

    Statistics.

    Values are expressed as the arithmetic mean ± standard deviation. Statistical analysis of the data was performed using the one-factor analysis of variance Scheffé Ftest (StatView 512+, Version 1.0; Apple Computer, Cupertino, CA). Triplicate wells were analyzed for each [3H]thymidine incorporation experiment, and each experiment was performed independently at least three times. A value of p < 0.05 was considered to be statistically significant.

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    Results

    Analysis of lipoproteins after agarose electrophoresis.

    The purity of LDL isolated by KBr density-gradient ultracentrifugation method (28) was examined using the Lipidophor reagent kit. Lipoprotein electrophoresis was performed in 1% agarose gel according to the manufacturer’s protocol. As shown in Fig.1 (lane 3), LDL isolated by the density-gradient ultracentrifugation method was not contaminated with HDL or VLDL. In addition, the purity of HDL (lane 2) and VLDL (lane 4) isolated by this method (28) was very high.

    Figure 1
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    Figure 1

    Analysis of lipoproteins by 1% agarose electrophoresis. After isolation of lipoproteins by KBr density-gradient ultracentrifugation (quantification of lipoproteins was performed by determination of the protein component), lipoproteins were analyzed by 1% agarose electrophoresis according to manufacturer’s protocol (Lipidophor System). After electrophoresis, lipoproteins were visualized with a solution containing 6.3 mm phosphotungstic acid, 182 mmMgCl2, 70 mmol NaCl, and 12 mm NaN3according to manufacturer’s protocol. Lane 1, mixture of lipoproteins containing 4 μg of LDL, 10 μg of HDL, and 0.5 μg of VLDL. Lane 2, 30 μg of HDL. Lane 3, 12 μg of LDL.Lane 4, 1.5 μg of VLDL.

    Effect of LDL on [Ca2+]i in PTX-treated and MAPTAM-treated VSMCs.

    As demonstrated in Fig.2a, LDL (100 μg/ml) induced a maximal elevation in [Ca2+]i from 40 to 140 nm, with a peak occurring at 10 sec. Thereafter, [Ca2+]i declined toward a stable value of 65 nm within 40 sec. Preincubation of the cells for 32 hr with PTX (100 ng/ml) resulted in a remarkable reduction of the maximal effect of LDL at 10 sec to 60 nm (Fig. 2b). Evaluation of four experiments was performed by calculating the maximal increase in [Ca2+]i at 10 sec. LDL at a concentration of 100 μg/ml caused an increase in [Ca2+]i from 42 ± 10 (basal value) to 145 ± 15 nm. The effect of LDL in PTX-treated cells was significantly reduced to 60 ± 15 nm (mean ± standard deviation, four experiments,p < 0.05). The ability of MAPTAM, which is known to be a potent intracellular chelator of Ca2+, to bind the LDL-induced increase in [Ca2+]i was examined after stimulation of MAPTAM-treated cells with 100 μg/ml LDL. As demonstrated in Fig. 2c, the LDL-induced increase in intracellular free Ca2+ was completely abolished in MAPTAM-treated VSMCs. MAPTAM was not able to abolish the effect of the detergent digitonin, which allows equilibration of extracellular and intracellular Ca2+.

    Figure 2
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    Figure 2

    Effect of LDL on [Ca2+]iin VSMCs. a, Confluent VSMCs on slides were precultured for 32 hr in serum-free medium (representative tracing from four independent experiments). LDL (100 μg/ml) was applied to Fura-2-loaded VSMCs, and changes in fluorescence were monitored. After subtraction of autofluorescence, changes in 340/380-nm excitation wavelength ratio by the emission wavelength of 505 nm were converted into corresponding levels of [Ca2+]i. b, After preincubation of the cells in serum-free medium for 32 hr in the presence of 100 ng/ml PTX, Fura-2-loaded VSMCs were stimulated with LDL (100 μg/ml). c, After preincubation of the cells in serum-free medium for 31 hr, MAPTAM at a concentration of 20 μm was added in the serum-free medium (one representative tracing from three experiments). After 60 min, cells were loaded with Fura-2 and then stimulated with LDL (100 μg/ml) and digitonin (30 μm).

    Effect of LDL on the phosphorylation of MAP kinase isoforms in VSMCs and human skin fibroblasts.

    Activation of p44mapk and p42mapk is accompanied by phosphorylation of the Tyr204 residue (33). When VSMCs were stimulated with LDL (100 μg/ml), maximal stimulation of the p44mapk and p42mapkoccurred at 5 min (Fig. 3A,top). Densitometric analysis of the blot revealed that at 5 min, LDL induced an 8-fold increase in both isoforms above control levels (Fig. 3A, bottom). No stimulation of either isoform could be observed after 30 min. Parallel cell stimulation with PDGF-BB for 5 min resulted in a 12-fold increase in the phosphorylated isoforms (Fig. 3A, top). As demonstrated in Fig. 3B (top left), the LDL-induced phosphorylation of each isoform was dose dependent. Densitometric analysis of the blot indicated that the maximal effect was achieved with 20 μg/ml LDL (Fig. 3B,bottom). To elucidate whether the LDL-induced increase in [Ca2+]i is implicated in the activation of the MAP kinase isoforms, VSMCs were preincubated for 60 min with 20 μm MAPTAM, which is known to be a potent intracellular chelator of intracellular Ca2+. As shown in Fig. 3B (top right and center), pretreatment of VSMCs with MAPTAM resulted in a 2-fold increase in the LDL-dependent effects on phosphorylation of the MAP kinase isoforms (=100%).

    Figure 3
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    Figure 3

    Effect of LDL on the phosphorylation of the p44mapk and p42mapk at Tyr204 in VSMCs and human skin fibroblasts. VSMCs (A–C), normal fibroblasts (D), and FH fibroblasts (E) were seeded onto 24-well culture plates (4 × 105 cells/well; well diameter, 12 mm) and cultivated in culture medium until confluence. Then, the medium was replaced by serum-free medium. After another 32-hr cultivation, cells were stimulated with LDL (100 μg/ml), PDGF-BB (50 ng/ml), and LPA (5 μg/ml). Cells were lysed, and 30 μg of protein were analyzed by SDS-PAGE. MAP kinase was detected after blotting onto PVDF membranes by a specific MAP kinase antibody that recognizes the catalytically activated p42mapk and p44mapk.Diagrams below blots, densitometric analyses of the blots. A, Time-dependent phosphorylation of p42mapk and p44mapk in VSMCs by LDL (100 μg/ml). B, Left, dose-dependent phosphorylation of p42mapk and p44mapk by LDL in VSMCs. Right, last diagram, densitometric analysis from three blots derived from three experiments. VSMCs were preincubated with 20 μmMAPTAM for 60 min before stimulation with LDL or PDGF-BB for 5 min. C,First blot, time-dependent phosphorylation of p42mapk and p44mapk in VSMCs by LPA (5 μg/ml). C, Second blot, last diagram, densitometric analysis from three blots derived from three independent experiments. VSMCs were pretreated with PTX (100 ng/ml) and then stimulated with LDL (100 μg/ml), PDGF-BB (50 ng/ml), LPA (5 μg/ml), and 5% FCS for 5 min. D, Time-dependent phosphorylation of p42mapk and p44mapk in normal human skin fibroblasts by LDL (100 μg/ml). E, Time-dependent phosphorylation of p42mapk and p44mapk by LDL (100 μg/ml) in human skin FH fibroblasts.

    As demonstrated in Fig. 3C (first blot) and the diagram below (densitometric analysis of the blot), stimulation of VSMCs with LPA (5 μg/ml) resulted in a rapid time-dependent phosphorylation of Tyr204 with a maximum at 5 min. Stimulation of PTX-pretreated VSMCs with LDL (100 μg/ml) or LPA (5 μg/ml) resulted in an almost complete inhibition of the phosphorylation of the MAP kinase isoforms (Fig. 3C, second blot, last diagram). Treatment of VSMCs with PTX for 32 hr did not influence the PDGF-BB-induced stimulation of the MAP kinase isoforms, whereas ∼40% of the effect of FCS was inhibited (Fig. 3C, second blot).

    Fig. 3 (D and E) shows the time course of the MAP kinase activation in normal human skin fibroblasts and FH fibroblasts that lacked LDL receptors, respectively. LDL induced in both cell types a remarkable stimulation of both MAP kinase isoforms. Maximal phosphorylation occurred at 5 min (8-fold above control in normal and 12-fold in FH fibroblasts), but the activation profile in FH fibroblasts was sharper than that for normal fibroblasts. Parallel stimulation of the normal and FH fibroblasts with PDGF-BB (50 ng/ml) for 5 min resulted in a 8-fold (Fig. 3D) and 18-fold (Fig. 3E) increase in the phosphorylated isoforms, respectively.

    Effect of LDL on PI3 kinase.

    Activation PI3 kinase is accompanied by phosphorylation of several tyrosine residues. As indicated in Fig.4, analysis of anti-tyrosine-precipitated proteins using anti-PI3 kinase antibodies showed that LDL (100 μg/ml) failed to stimulate phosphorylation of the PI3 kinase. In contrast, parallel stimulation with PDGF-BB (50 ng/ml) induced a remarkable phosphorylation of PI3 kinase at 3 and 5 min (Fig. 4).

    Figure 4
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    Figure 4

    Effect of LDL on PI3 kinase tyrosine phosphorylation in VSMCs (one representative blot from three independent experiments). Confluent cells in 75 cm2 were preincubated in 5 ml of serum-free medium for 32 hr. Cells were stimulated with 50 ng/ml PDGF-BB or 100 μg/ml LDL for different time periods. The cells were lysed, and tyrosine-phosphorylated proteins were bound using an anti-phosphotyrosine antibody coupled to Sepharose. Proteins (3 μg) were analyzed by 7.5% SDS-PAGE. After electrophoresis and Western blotting, PI3 kinase was detected by the enhanced chemiluminescence method using anti-PI3 kinase monoclonal antibodies.

    Effect of LDL on the phosphorylation of Raf-1 kinase.

    Activation of the protein kinase Raf-1 is accompanied by phosphorylation of several serine/threonine residues. As demonstrated in Fig. 5, analysis of anti-threonine-precipitated proteins using anti-Raf-1 antibodies showed that LDL (100 μg/ml) failed to stimulate threonine phosphorylation of Raf-1.

    Figure 5
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    Figure 5

    Effect of LDL on Raf-1 kinase threonine phosphorylation in VSMCs (one representative blot from three independent experiments). Confluent cells in 75 cm2 were preincubated in 5 ml of serum-free medium for 32 hr. Cells were stimulated with 100 μg/ml LDL for different time periods. The cells were lysed, and threonine-phosphorylated proteins were bound using an anti-threonine antibody coupled to Sepharose. Proteins (3 μg) were analyzed by 7.5% SDS-PAGE. After electrophoresis and Western blotting, Raf-1 kinase was detected by the enhanced chemiluminescence method using anti-Raf-1 kinase monoclonal antibodies.

    Effect of LDL on DNA synthesis in VSMCs and human fibroblasts.

    As demonstrated in Fig. 6A, treatment of the cells with PTX (100 ng/ml) for 32 hr resulted in a complete inhibition of the LDL- and LPA-induced [3H]thymidine incorporation but failed to inhibit the effect of PDGF-BB. To demonstrate whether binding of [Ca2+]i with MAPTAM influences VSMC DNA synthesis, the effect of LDL on [3H]thymidine incorporation in MAPTAM-treated cells was investigated. As demonstrated in Fig. 6B, blocking of the LDL-induced increase in [Ca2+]i did not influence LDL-induced VSMC DNA synthesis. Similar results were obtained using normal (Fig. 6C) and FH (Fig. 6D) fibroblasts. Again, treatment of both types of fibroblasts with PTX for 32 hr resulted in a complete inhibition of the LDL- and LPA-induced DNA synthesis in both cell types.

    Figure 6
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    Figure 6

    Effect of LDL on cell DNA synthesis in VSMCs and human fibroblasts. Confluent VSMCs (A and B), normal fibroblasts (C), and FH fibroblasts (D) were precultured onto 24-well plates for 32 hr in serum-free medium in the presence and absence of 100 ng/ml PTX or 20 μm PD 98059 (E). Then, cells were stimulated with LDL (100 μg/ml), PDGF-BB (50 ng/ml), and/or LPA (5 μg/ml), respectively. After another 20 hr of incubation, cells were exposed to 3 μCi/ml [3H]thymidine. Four hours later, the reaction was terminated, and cell protein and [3H]thymidine incorporation into cell DNA was quantified. B, Confluent VSMCs (on 24-well plates) were precultured for 31 hr in serum-free medium. After another 1 hr of incubation with 20 μm MAPTAM, cells were stimulated with LDL (100 μg/ml). After 20 hr of incubation, cells were exposed to 3 μCi/ml [3H]thymidine. Four hours later, the reaction was terminated, and cell protein and [3H]thymidine incorporation into cell DNA was quantified. Results are from a representative experiment performed in triplicate wells and are expressed as mean ± standard deviation. ∗,p < 0.05 for PDGF-BB, LPA, and LDL versus control. ∗∗, p < 0.05 for the PTX plus LPA versus the LPA. ∗∗, p < 0.05 for PTX plus LDL versus LDL. ∗∗.p < 0.05 for LDL plus MAPTAM versus LDL.

    Effect of PD 98059 on LDL-induced phosphorylation of p44mapk and p42mapk and DNA synthesis in VSMCs.

    Finally, to ensure that the mitogenic effect of LDL is mediated by activation of the MAP kinase pathway, the effect of LDL on the MAP kinase activation and DNA synthesis was investigated after treatment of VSMCs with PD 98059. As shown in Fig.7, treatment of VSMCs with the specific MEK inhibitor PD 98059 remarkably inhibited the LDL-induced phosphorylation of both MAP kinase isoforms (Fig. 7). Treatment of the cells with 20 μm PD 98059 resulted in a 71 ± 6% inhibition of phosphorylation of the p44mapkisoform and 65 ± 6% inhibition of phosphorylation of the p44mapk isoform. In accordance with these findings, treatment of the cells with PD 98059 caused a 62% inhibition of the LDL-induced DNA synthesis in VSMCs (Fig.8). Furthermore, PD 98059 induced a 32% inhibition of basal DNA synthesis.

    Figure 7
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    Figure 7

    Effects of PD 98059 on the LDL-induced phosphorylation of p44mapk and p42mapk at Tyr204 in VSMCs. VSMCs were seeded onto 24-well culture plates (4 × 105 cells/well; well diameter, 12 mm) and cultivated in culture medium until confluent (densitometric analysis of three blots derived from three independent experiments). Then, the medium was replaced by serum-free medium. After another 32 hr cultivation in the presence and absence of PD 98059, cells were stimulated with LDL (100 μg/ml) for 5 min. Cells were lysed, and 30 μg of protein were analyzed by SDS-PAGE. MAP kinase was detected after blotting onto PVDF membranes by a specific MAP kinase antibody that recognizes the catalytically activated p42mapk and p44mapk.

    Figure 8
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    Figure 8

    Effect of PD 98059 on the LDL-induced DNA synthesis in VSMCs. Confluent VSMCs were precultured onto 24-well plates for 32 hr in serum-free medium in the presence and absence of 20 μm PD 98059. Then, cells were stimulated with LDL (100 μg/ml). After another 20 hr of incubation, cells were exposed to 3 μCi/ml [3H]thymidine. Four hours later, the reaction was terminated, and cell protein and [3H]thymidine incorporation into cell DNA was quantified.∗, p < 0.05 for LDL versus control. ∗∗, p < 0.05 for PD 98059 versus control. ∗∗, p < 0.05 for PD 98059 plus LDL versus LDL.

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    Discussion

    We found that the Raf-1 pathway is not involved in LDL-induced stimulation of MAP kinase isoforms. Pretreatment of VSMCs with PTX abolished the LDL-induced activation of MAP kinases and significantly reduced LDL-induced increase in [Ca2+]i. A Gi-dependent stimulation of MAP kinase without involvement of the Raf-1 pathway has been repeatedly described in different cell types and species (21). Recently, we found that stimulation of PTX-treated cells with LPA (5 μg/ml) resulted in 80% inhibition of the maximal LPA-induced increase in [Ca2+]i occurring at ∼15 sec (34). In contrast, PTX treatment of VSMCs did not influence the PDGF-BB-induced increase in [Ca2+]i.1In accordance with these findings, the mitogenic effect of LDL on the PTX-treated cells was completely abolished. Similarly, we found that the LPA-induced activation of MAP kinase isoforms as well as the stimulation of DNA synthesis in VSMCs and human fibroblasts is mediated by a PTX-sensitive G protein. Because intrinsic GTPase activity of the Gi subfamily can be inhibited by PTX via ADP-ribosylation of specific residues, it can be postulated that LDL promotes DNA synthesis in VSMCs via a Gi-mediated pathway. Entire inactivation of PTX-sensitive Gis was shown by [32P]ADP-ribosylation of isolated VSMC membranes from PTX-treated and untreated cells as previously described (27). Activation of VSMCs with LDL was not associated with phosphorylation by the PI3 kinase. LDL also failed to stimulate tyrosine phosphorylation of PLC-γ1.2 Furthermore, treatment of VSMCs with PTX had no effect on the PDGF-BB-induced stimulation of MAP kinase isoforms, [Ca2+]i, and DNA synthesis. These findings suggest that the LDL-induced intracellular mechanisms causing an increase in DNA synthesis are distinct from those induced by other potent VSMC mitogens, such as PDGF-BB.

    It has been repeatedly described that LPA and thrombin activate the PI turnover-signaling system in hamster lung fibroblasts (CCL39) and Rat-1 cells through activation of phospholipase C. Furthermore, it has been demonstrated that the mitogenic effect of both agonists in CCL39 and Rat-1 cells is strongly attenuated by PTX treatment, suggesting that Gis are involved in the mitogenic effect of both agonists (24). Moreover, both agonists caused a PTX-sensitive activation of p21ras. On the basis of these results, it has been proposed that both agonists promote DNA synthesis via a Gi-regulated p21ras pathway and that activation of the PI-signaling system by LPA or thrombin is not involved in their growth-promoting effects (24). The concept of this novel mitogenic signaling pathway mediated by certain Gis is supported by the findings of Goodemotoet al. (35) that a PTX-sensitive G protein is also involved in the mitogenic signaling pathway of sphingosine-1-phosphate in Swiss 3T3 fibroblasts and that sphingosine-1-phosphate-induced stimulation of the PI turnover-signaling system is not essential for its mitogenic effects. Indeed, mitogenic signal transduction through a PTX-sensitive Gi/p21ras pathway is established for several agonists; thus, it is likely that a Gi-regulated/p21ras pathway is involved in the mitogenic signaling pathway of LDL.

    One interesting observation was that prevention of the LDL-induced increase in [Ca2+]i by the intracellular chelator of Ca2+, MAPTAM, resulted in a 2-fold increase in the phosphorylated MAP kinase isoforms but not in an attenuation of LDL-induced DNA synthesis. Because it is believed that MAP kinase phosphatase-1 dephosphorylates and inactivates MAP kinases (36), we speculate that the LDL-induced rapid increase in [Ca2+]i is implicated in the regulation of a MAP kinase phosphatase-1.

    Furthermore, we have shown that treatment of VSMCs with MAPTAM resulted in an abolishment of the LDL-induced transient increase in [Ca2+]i but failed to inhibit LDL-induced DNA synthesis. On the basis of this finding, we may suppose that the LDL-induced transient increase in [Ca2+]i alone is not sufficient to stimulate DNA synthesis and that conjugation with other signaling components, such as MAP kinase, may be necessary for a mitogenic response.

    We demonstrated that LDL induces elevation in [Ca2+]i and stimulates the Na+/H+ antiport in normal and FH fibroblasts (37). In the current study, we have shown that LDL induces activation of p44mapk and p42mapk as well as DNA synthesis in normal and FH fibroblasts, which failed to produce classic LDL receptor via a PTX-sensitive pathway. Furthermore, inhibition of MEK by PD 98059 resulted in an inhibition of the activation of the MAP kinase isoforms as well as cell DNA synthesis. Thus, we may postulate that the intracellular signaling transduction pathway and the mitogenic effect of LDL are independent of its classic receptor but are mediated by a putative Gi-coupled receptor. Stimulation of this receptor causes a Raf-1-independent stimulation of p44mapk and p42mapk that results in an increase in DNA synthesis.

    There is some evidence supporting our concept that the mitogenic signaling pathway of LDL is not mediated by its classic receptor but instead through a putative Gi-coupled receptor. For example, an atypical LDL binding site on human VSMCs with aKd value of 50 μg/ml has been characterized that may mediate the LDL-induced phosphoinositide catabolism in VSMCs (12). The authors demonstrated that the atypical low affinity receptor in these cells may mediate the LDL-induced signal transduction, including stimulation of the phosphoinositide catabolism and elevation in [Ca2+]i(12). Thus, the effects of LDL on MAP kinase activation and cell DNA synthesis may be mediated by this atypical LDL receptor.

    We suggest our findings are relevant to in vivo physiology and pathophysiology. In this context, it has been proposed that most of circulating LDL is transported through vascular endothelium by transcytosis via plasmalemma vesicles, which deliver LDL to other cells of the vascular wall (38). It is likely that LDL contributes physiologically to VSMCs or fibroblasts growth via a MAP kinase pathway. Cardiovascular risk factors such as hypertension and hypercholesterolemia induce an elevation in LDL transport from blood in the rat aortic intima (39). Furthermore, injection of animals with vasoactive agonists such as serotonin, angiotensin II, and catecholamines resulted in an increased transfer of LDL from blood to rat arterial vessels (40). Thus, it is conceivable that under pathophysiological conditions, elevated LDL in the aortic intima leads to abnormal VSMC growth via a MAP kinase pathway and thus may contribute to the development and acceleration of cardiovascular diseases. Because it is believed that minimally oxidized LDL is involved in the pathogenesis of atherosclerosis and native LDL can be minimally oxidized by endothelial cells, it is likely that minimally oxidized LDL is able to exert similar effects in VSMCs.

    In summary, we attempted to elucidate the intracellular pathways involved in the LDL-induced DNA synthesis and postulate that like LPA, the growth-promoting effect of LDL is mediated by a putative Gi-coupled receptor and probably involves activation of MAP kinases.

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    Footnotes

    • Send reprint requests to: PD Dr. A. Sachinidis, Medizinische Universitäts-Poliklinik, Wilhelmstr. 35–37, 53111 Bonn, Germany. E-mail: umm501{at}ibm.rhrz.uni-bonn.de

    • ↵1 A. Sachinidis, S. Seewald, P. Epping, C. Seul, Y. Ko, and H. Vetter, unpublished observations.

    • ↵2 A. Sachinidis, S. Seewald, P. Epping, C. Seul, Y. Ko, and H. Vetter, unpublished observations.

    • This work was supported by Deutsche Forschungsgemeinschaft Grant Sa568/2–1.

    • Abbreviations:
      VSMC
      vascular smooth muscle cell
      LDL
      low-density lipoprotein
      HDL
      high-density lipoprotein
      VLDL
      very low-density lipoprotein
      PDGF
      platelet-derived growth factor
      LPA
      lysophosphatidic acid
      DMEM
      Dulbecco’s modified Eagle’s medium
      MAP
      mitogen-activated protein
      PLC
      phospholipase C
      FCS
      fetal calf serum
      PI3
      phosphatidylinositol-3
      PTX
      pertussis toxin
      SDS
      sodium dodecyl sulfate
      PAGE
      polyacrylamide gel electrophoresis
      MEK
      mitogen-activated protein kinase kinase
      [Ca2+]i
      intracellular free Ca2+concentration
      MAPTAM
      bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester
      FITC
      fluorescein isothiocyanate
      PVDF
      polyvinylidene difluoride
      FH
      familial hypercholesterolemia
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      EGTA
      ethylene glycol bis(α-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      • Received March 19, 1997.
      • Accepted May 22, 1997.
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    1. Molecular Pharmacology September 1, 1997 vol. 52 no. 3 389-397
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