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Endocrinology Vol. 142, No. 11 4852-4860
Copyright © 2001 by The Endocrine Society

ARTICLES

Cooperation between Low Density Lipoproteins and IGF-I in the Promotion of Mitogenesis in Vascular Smooth Muscle Cells

Belén González, Santiago Lamas and Elvira M. Melián

Endocrinology Division (B.G., E.M.M.), Hospital Carlos III, Instituto de Salud Carlos III, Madrid 28029, Spain; and Centro de Investigaciones Biológicas and Instituto "Reina Sofía" de Investigaciones Nefrológicas (S.L.), Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain

Address all correspondence and requests for reprints to: Elvira M. Melián, Endocrinology Service, Hospital Carlos III, Instituto de Salud Carlos III, C/Sinesio Delgado 10-12, Madrid 28029, Spain. E-mail: emelian{at}hciii.insalud.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Low density lipoproteins (LDL) are an independent risk factor for atherosclerosis and show synergism with some growth factors in vascular smooth muscle cell (VSMC) proliferation. IGF-I has mitogenic actions on VSMC, which, in turn, show enhanced expression of IGF-I and its receptor when exposed to hypercholesterolemic diets in vivo. To investigate the molecular basis of a possible interaction between LDL and the IGF-I signaling system in VSMC, we used A10 cells, where synergism between both factors in DNA synthesis was demonstrated. IGF-I activates phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated MAPK pathways in A10 cells, although insulin receptor substrate-1 (IRS-1)-associated PI3 kinase is more closely linked to IGF-I induced proliferation. LDL, in pathophysiological concentrations, affect the IGF-I signaling pathway at multiple levels: 1) they induce phosphorylation of IGF-I receptor ß and IRS-1 in a time- and dose-dependent manner; 2) they up-regulate IRS-1-associated PI3 kinase/Akt activation in response to IGF-I at early times; and 3) they show additive effects with IGF-I on extracellular signal-regulated MAPK 1/2 phosphorylation. These actions are not present in very low density lipoprotein treatments. Taken together, these results indicate specific cooperation between LDL and the IGF-I signaling pathways and may represent a more general mechanism through which proatherogenic lipoproteins modulate VSMC response to growth factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A HIGH LEVEL of low density lipoproteins (LDL) is a well-established and independent risk factor for atherosclerosis (1). Lipoproteins and other cardiovascular risk factors play integral roles in activating arterial wall cells, including vascular smooth muscle cells (VSMC) that, in turn, promote local inflammatory events responsible for advanced atherosclerotic lesions (2). Indeed, dietary lipids modify VSMC differentiation in vivo (3); and LDL, even at low concentrations, stimulate VSMC proliferation and have synergistic effects, with some growth factors, on DNA synthesis in VSMC (4, 5). Direct effects of lipoproteins on the lipid bilayer, inducing phosphorylation and activation of membrane receptors, have been suggested (6, 7, 8). However, the molecular mechanisms of these interactions and their impact on downstream intracellular pathways remain obscure.

IGF-I is present in high concentrations in the circulation and is locally produced by cells of the cardiovascular system, where it acts as a growth promoter, thus being involved in several cardiovascular diseases (9, 10, 11). Normal-to-elevated IGF-I levels have been described in obese humans and animal models, where dyslipemia with increased LDL is generally present (12, 13). Moreover, IGF-I and IGF-I receptor (IGF-IR) expression is increased in VSMC of atherosclerotic subjects and changes in the lipid composition of diets alter local IGF-I production in VSMC (9, 14, 15). Actions of IGF-I are mediated through the IGF-IR, a transmembrane tyrosine kinase that, by activation of insulin receptor substrate-1 (IRS-1) and Shc docking proteins, activates two primary intracellular systems: phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated MAPK (ERK) 1/2 MAPK pathways (16, 17, 18). The IGF-IR is abundantly expressed in VSMC, where the relevance of IRS-1-associated PI3 kinase activation for mitogenic IGF-I actions has been recently demonstrated (19).

The aim of the present study was to investigate the consequences of increased LDL concentrations on the IGF-I signaling system in VSMC at the molecular level. We have used A10 cells, isolated from rat fetal aorta. These cells offer some advantages over primary cultures, including morphological and biochemical stability after multiple passages (20) and specific induction of IGF-IR by LDL exposure (21). We demonstrate that A10 cells show a synergistic proliferative response to native LDL and IGF-I. Furthermore, we provide molecular evidence for the interaction of LDL with the IGF-I signaling pathway at multiple levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All chemicals and reagents were purchased from Sigma (Madrid, Spain) unless specified otherwise. Human IGF-I was purchased from R & D Systems (Minneapolis, MN). IGF-IRß-subunit and 4G10-antiphosphotyrosine were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). IRS-1 and p85 PI3 kinase antibodies were from Upstate Biotechnology, Inc. (New York, NY). Phosphospecific activated and control antibodies for PKB/Akt and ERK 1/2 kinases were from New England Biolabs, Inc. (Beverly, MA). Horseradish peroxidase-linked antirabbit and antimouse antibodies were from DAKO Corp. (Glostrup, Denmark). IGF-IR oligos, protein A-Sepharose 6MB, and isotopes were from Amersham Pharmacia Biotech (Barcelona, Spain). MTS assay (Celltiter 96) was from Promega Corp. (Madison, WI). LY294002 and wortmannin were from Calbiochem (Darmstadt, Germany). FBS, DMEM, and antibiotics were from BioWhitthaker, Inc. (Walkersville, MD).

Lipoprotein preparation and modification
Lipoproteins were isolated by sequential ultracentrifugation [very low density lipoproteins (VLDL), density < 1.006; LDL, density = 1.019–1.063 g/ml] of EDTA-anticoagulated plasma obtained from healthy normolipemic volunteers (22). Protein concentrations of lipoprotein preparations were determined using the Lowry method. The presence of potential contaminant IGF-I was excluded by Western blot. All lipoproteins were stored at 4 C and were used for experiments within 2 wk after preparation.

Cell culture
Rat A10 VSMC, derived from thoracic aorta of fetal rats, were donated by Dr. F. Mayor from Centro de Biologia Molecular, Madrid. Cells were grown in low (5 mM) or high (25 mM) glusose DMEM supplemented with glutamine (4 mM), penicillin, streptomycin, and 10% FBS at 37 C. The medium was changed every third day until confluency. To subculture the cells, confluent monolayers were washed with DMEM (serum free), treated with 0.1% trypsin-0.04% EDTA, placed in an equal volume of medium, and centrifuged at 1200 rpm for 5 min. Before stimulation experiments, medium was changed to serum-free DMEM (SFM) for 48 h. This SFM was replaced with SFM together with the appropriate stimulus for various times. Passages 20–28 were used for experimental purposes.

[3H]-Thymidine incorporation assay
To determine the rate of DNA synthesis, A10 cells were grown in 24-well plates, incubated 48 h in SFM at 37 C, rinsed three times with DMEM, and exposed to the desired stimulus. [3H]-Thymidine was pulsed for the last 2 h of exposure. Each treatment was added to quadruplicate wells. Cells were washed twice with PBS, twice with methanol, and twice with cold 5% trichloroacetic acid for 10 min at 4 C and were solubilized in 500 µl of 0.3 M NaOH at room temperature. The solubilized DNA was harvested for liquid scintillation counting.

MTS assay
A10 growth was determined by the MTS method. This assay is based on the cellular reduction of MTS by the mitochondrial dehydrogenase of viable cells to a yellow formazan product that can be measured spectrophotometrically. Briefly, after each experiment, 20 µl MTS were added to each well, followed by an additional 4 h of incubation at 37 C. Absorbance was then measured with a multiwell spectrophotometer at 490 nm. Several experiments were previously performed to establish a correlation between cell number and the formation of MTS formazan.

Antisense oligonucleotides and transfection assays
A previously described 20-oligomer antisense oligodeoxinucleotides (ODN) against IGF-IR, targeting 2 bp 5' to the ATG site, was used (23). Transfections were done by lipofection incubating cells with the ODNs-lipofectamine complex for 5 h in SFM and then letting cells recover in DMEM with 10% FBS for 24 h before experimental procedures. Final concentrations of ODNs assayed in cells were 32–130 nM.

Western immunoblotting analysis
Cell lysates were separated on an SDS-PAGE (10%), transferred to a PVDF membrane (Immobilon P, 0.45-mµ pore size; Millipore Corp., Bedford, MA), and blocked in Tris-buffered saline Tween 20 (5% powdered milk). Membranes were incubated with a 1:1000 solution of the antibody for 2 h in PI3 kinase blotting and overnight in PKB/Akt and ERK 1/2 blotting. Blots were then washed with buffer A (Tris-buffered saline) and buffer B (Tris-buffered saline Tween 20) and incubated with a 1/2000 dilution of horseradish-peroxidase-linked antirabbit secondary antibody for 1 h, followed by further washing. Enhanced chemiluminescence was performed according to the enhanced chemiluminescence manufacturer’s instructions (Amersham Pharmacia Biotech).

Immunoprecipitation
Equal amounts of cell lysates (HEPES, 50 mM, pH 7.4; sodium pyrophosphate, 10 mM; NaF, 100 mM; EDTA, 2 mM; Na3VO4, 2 mM; 1% Triton; 10% glicerol; 0.5 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin) were incubated with the indicated antibodies, at 4 C, according to the manufacture’s instructions. Protein A-sepharose (50 µl) was then added for 1 h at 4 C and followed by 3 washes with lysis buffer and two with PBS. Beads were treated with Laemmli buffer, boiled, and separated by SDS-PAGE, followed by Western blotting.

Assay of PI3 kinase activity
Cells were washed, lysed, and incubated with IRS-1 antibody, followed by incubation with protein-A sepharose. After washing, the activity of PI3 kinase present in the resuspended immunoprecipitate was determined using phosphatidylinositol (20 µg) and [{gamma}-32P]ATP (24).

Statistical analysis
Representative experiments for three to four independent ones or the mean ± SEM for all data are shown. Unpaired t test or appropriate nonparametric tests were used for analysis of differences between various treatments. P values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A10 cells show synergism between IGF-I and LDL on VSMC proliferation
To establish whether A10 cells were an appropriate model to evaluate interactions between native LDL and IGF-I on VSMC proliferation, we first characterized the IGF-I response of [3H]-thymidine in these cells. Fig. 1AGo shows the time course of maximal rate of DNA synthesis induced by IGF-I (the 5-nM dose was previously established as the most effective for mitogenesis of A10 cells in 24-h exposures). Treatment of growth-arrested A10 cells with 5 nM IGF-I induced a 6- to 7-fold increase vs. control, at 16 h of exposure (P < 0.001). In keeping with expectations, when cell growth activity was determined by the MTS assay, there was a significant increase in cell number after exposure to 5 nM IGF-I, compared with control cells, at 24 h of treatment (161 ± 7% vs. 100 ± 2%, P < 0.001). Antisense ODNs eliminated IGF-IR expression in A10 cells and blunted the mitogenic response to IGF-I (Fig. 1BGo). We next examined the effects of different LDL concentrations on the previously characterized IGF-I induced proliferation. As shown in Fig. 2AGo, the presence of LDL doses of 25 and 50 µg/ml synergistically enhanced the IGF-I response on thymidine incorporation (P < 0.001 vs. theoretical additive value of IGF-I plus LDL). Moreover, LDL significantly increased thymidine incorporation in A10 cells by itself, with a maximal stimulation at 50 µg/ml (P < 0.001 vs. control cells). VLDL did not modify either basal or IGF-I induced proliferative response. Cell growth at 24 h, as measured by the MTS assay, was increased in an additive way by the combined treatment of LDL and IGF-I [C, 100 ± 2%; LDL, 50 µg/ml (140 ± 20%); IGF-I, 5 nM (174 ± 10%); and IGF-I, 5 nM, plus LDL, 50 µg/ml (210 ± 5%), P < 0.01 vs. IGF-I alone].



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  		Figure 1. Mitogenic effects of IGF-I on A10 VSMC. A, Time-course study of the maximal effects of IGF-I on DNA synthesis. After synchronization, cells were incubated, in the presence of 5 nM IGF-I, for the indicated times. [3H]-Thymidine incorporation was determined as described in Materials and Methods. Values are mean ± SEM of three separate experiments, each one done in quadruplicate. *, P < 0.001 vs. control cells. B, Effects of antisense ODNs against IGF-IR on IGF-IR protein and IGF-I induced DNA synthesis. A10 cells were transfected with lipofectAMINE (control, c), antisense (AS) or sense (S) ODNs directed against IGF-IR and, after a recovery period of 24 h, treated with IGF-I for 16 h. [3H]-Thymidine incorporation and IGF-IR protein expression (inset blot) of a representative experiment, from a total of three, is shown.

 


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  		Figure 2. Lipoprotein effects on basal and IGF-I induced A10 cell proliferation. Effects of lipoproteins on basal and IGF-I-induced DNA synthesis. After synchronization, cell cultures were exposed to 5 nM IGF-I or vehicle in the presence or absence of different lipoprotein concentrations. [3H]-thymidine was present for the last 2 h, and its incorporation was measured 16 h after IGF-I or vehicle treatment. Values are mean ± SEM of six separate experiments, each one done in quadruplicate. *, P < 0,001 vs. control; {dagger}, P < 0.001 vs. theoretical additive effect of IGF-I plus LDL. Where error bars are not visible, they are smaller than data points.

 
LDL induce tyrosine phosphorylation of IGF-IR and IRS-1 in A10 VSMC
We next evaluated LDL effects on IRS-1 and/or IGF-IRß phosphorylation, as a potentially early step of interaction of LDL with the IGF-I signaling system. As shown in Fig. 3AGo, treatment of cells during 8 min with LDL increased IGF-IRß tyrosine autophosphorylation in a dose-dependent manner, with a maximal effect at 100 µg/ml. At the 50-µg/ml dose (used currently in our experiments), there was a timedependent phosphorylation pattern, with a decay to basal levels 20 min after the stimulus. A parallel effect was seen in IRS-1 phosphorylation, as can be observed in Fig. 3BGo. VLDL (50 µg/ml), used as control, did not modify the level of IRS-1 phosphorylation.



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  		Figure 3. Effects of LDL on the tyrosine phosphorylation of the IGF-IRßsubunit and IRS-1 proteins. A, Dose- and time- dependence of the LDL effect on IGF-IRß phosphorylation. Serum-starved cells were exposed to different LDL concentrations, 8 min before cell lysates were immunoprecipitated with an IGF-IRß antibody (dose-dependence, upper part) or exposed to LDL (50 µg/ml) for the indicated times (time dependence, lower part), transferred to PDVF membranes and blotted with an antibody against antiphosphotyrosine. B, Dose- and time-dependence of the LDL effect on IRS-1 phosphorylation. Serum-starved cells were processed as in A, immunoprecipitated with an IRS-1 antibody, transferred to polyvinylidene fluoride membranes, and blotted with an antibody against antiphosphotyrosine. VLDL (50 µg/ml) was used for comparison. Representative experiments from a total of four are shown PY, Phosphotyrosine; IP, immunoprecipitating; Ab, antibody.

 
Preferential involvement of the PI3 kinase pathway in IGF-I-induced DNA synthesis of A10 VSMC
Because activation of the IRS-1-associated PI3 kinase seems to be a pivotal regulator of IGF-I-induced VSMC proliferation (19), we first characterized this IGF-I signaling pathway in A10 cells. As seen in Fig. 4AGo, 5Go nM IGF-I increased IRS-1-associated PI3 kinase activity in a time-dependent manner, with a maximal activation at 2 min of exposure. Because it has been recently described that insulin and IGF-I up-regulate p85 mRNA and protein abundance in other cell types (25), we evaluated this possibility in our model. As seen in Fig. 4BGo, p85 PI3 kinase protein levels increased after 24 h of incubation with 5 nM IGF-I. To further examine the effect of IGF-I in activating the PI3 kinase cascade, the phosphorylation of PKB/Akt was examined using a phosphospecific and a control antibody. As seen in the insert blot of Fig. 4CGo, 5Go nM IGF-I induced a robust increase in the serine phosphorylation of PKB/Akt after 10 min of stimulation. To verify the involvement of PI3 kinase in IGF-I-dependent A10 cell proliferation, LY294002, an irreversible PI3 kinase inhibitor, was used. The effectiveness of this compound in inhibiting PI3 kinase activation was evaluated by directly monitoring the phosphorylation status of PKB/Akt. LY294002 inhibited IGF-I stimulated thymidine incorporation in a dose-dependent way, with almost total inhibition at 20 µM. Even when considering that LY294002 had an effect on basal thymidine incorporation, these results suggest that the PI3 kinase pathway is a crucial regulator of IGF-I-dependent proliferation also in this cell type.



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  		Figure 4. PI3 kinase pathway involvement in IGF-I-induced DNA synthesis. Panel A, Time course of the IGF-I effect on the activation of IRS-1-associated PI3 kinase in A10 cells. After synchronization, cells were treated with 5 nM IGF-I for the indicated times. Cell lysates (0.5 mg protein) were immunoprecipitated with IRS-1 antibody, and precipitates were assayed for PI3 kinase activity. A representative experiment of four is shown. Panel B, Long-term effects of IGF-I on p85 protein levels. Serum-starved cells were treated with or without 5 nM IGF-I for 24 h. A representative blot of three is shown. C, Control; Pos C, positive control. Panel C, Effects of PI3 kinase inhibition on IGF-I-induced DNA synthesis and PKB/Akt activation. Serum-starved cells were pretreated with or without different concentrations of LY294002 (LY) in the presence (black bars) or absence (gray bars) of 5 nM IGF-I for 16 h. [3H]thymidine incorporation was determined as described in Materials and Methods. Values are mean ± SEM of four separate experiments, each one done in triplicate. *, P < 0.001 vs. control cells in same experimental conditions. The inset blot represents PKB/Akt activation after 10 min of IGF-I addition and its inhibition with 20 µM LY294002. A blot that is representative of four is shown. PIP, Phosphatidylinositol phosphate; PKB/Akt, protein kinase B/Akt.

 


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  		Figure 5. ERK 1/2 kinase pathway involvement in IGF-I-induced DNA synthesis. A, Time course of the IGF-I effect on the phosphorylation of ERK 1/2 in A10 cells. After synchronization, cells were treated with 5 nM IGF-I for the indicated times. Western blotting for total and phosphorylated forms was performed. A representative blot of four is shown. B, Effects of ERK 1/2 inhibition on IGF-I-induced DNA synthesis. Serum-starved cells were pretreated with or without 10 µM PD98059 in the presence (black bars) or absence (gray bars) of 5 nM IGF-I for 16 h. [3H]-Thymidine incorporation was determined as described in Materials and Methods. Values are mean ± SEM of four separate experiments, each one done in triplicate. *, P < 0.001 vs. control cells (C) in same experimental conditions. The inset blot represents ERK 1/2 activation after 10 min of IGF-I addition and its inhibition by 10 µM PD98059. A blot, representative of three, is shown.

 
ERKs are the other classical MAPK known to be involved in IGF-I-dependent cell replication (26). We next studied its activation after IGF-I treatment and the effect of its inhibition on IGF-I-stimulated DNA synthesis. Fig. 5AGo shows the time-dependent phosphorylation of ERK 1/2, after IGF-I treatment, clearly peaking at 10 min and decreasing to control levels after 30 min. As seen in Fig. 5BGo, pretreatment of cells with 10 µM PD98059, an specific inhibitor of the ERK 1/2 pathway, suppressed ERK phosphorylation in our system, decreasing basal and IGF-I induced levels of [3H]-thymidine incorporation. However, this inhibitor only partially reduced IGF-I-stimulated DNA synthesis in A10 cells (Fig. 5BGo). Doses of 20–50 µM PD98059 were toxic for our cells in basal conditions.

LDL differentially interacts with IGF-I-stimulated pathways in A10 VSMC
We then tested whether LDL would modify the previously characterized IGF-I-stimulated pathways, by evaluating IGF-I effects on PKB/Akt and ERK 1/2 phosphorylation in the absence or presence of LDL. As can be observed in Fig. 6AGo, IGF-I induced a fast PKB/Akt phosphorylation, maximal at 10 min but undetectable after 120 min. LDL did not induce PKB/Akt phosphorylation, by itself, but its presence significantly increased PKB/Akt phosphorylation in response to IGF-I at 10 and 30 min (P < 0.001). In contrast, when the MAPK pathway was studied, we observed that LDL was able to increase ERK 1/2 phosphorylation by itself. An additive effect with IGF-I on ERK activation, at the times where maximal IGF-I activation of ERK was overt, was also detected (Fig. 6BGo).



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  		Figure 6. Effects of LDL on basal and IGF-I-induced PKB/Akt and ERK 1/2 phosphorylation. A, Effect of LDL on basal and IGF-I-induced PKB/Akt phosphorylation. Serum-starved cells were treated with 5 nM IGF-I in the presence or absence of LDL (50 µg/ml) for the indicated times. A representative blot and the statistical analysis of four independent experiments are shown. B, Effect of LDL on basal and IGF-I-induced ERK 1/2 phosphorylation. Serum-starved cells were treated with 5 nM IGF-I in the presence or absence of LDL (50 µg/ml) for the indicated times. A representative blot and the statistical analysis of three independent experiments are shown. {dagger}, P < 0.05; {ddagger}, P < 0.01 vs. IGF-I alone.

 
LDL specifically up-regulate IRS-1-associated IGF-I-dependent PI3 kinase activation in A10 VSMC
Because our data suggested that PI3 kinase activation was associated with proliferation, we performed in vitro PI3 kinase assays to evaluate LDL modulation of this pathway up-stream of PKB/Akt activation. We evaluated its effects in the experimental conditions previously tested for IGF-I. As seen in Fig. 7AGo, LDL significantly increased IRS-1-associated PI3 kinase activity in response to IGF-I, compared with IGF-I addition in its absence (P < 0.01), without modifying the IRS-1-associated PI3 kinase activity per se. VLDL had no stimulatory effects on basal or IGF-I-stimulated conditions. LDL did not modify the amount of p85 protein by itself or in IGF-I-stimulated cells, even after a long exposure to the stimuli (Fig. 7BGo). These results suggest the existence of cooperative signaling between IGF-I and LDL on the PI3 kinase pathway, which could be mediated, at least in part, through IRS-1. This effect could ultimately lead to an early increase in PKB/Akt activation, contributing to their synergistic effects on DNA synthesis.



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  		Figure 7. Effects of LDL on basal and IGF-I-stimulated PI3 kinase pathway. A, LDL modulation of IGF-I-induced IRS-1-associated PI3 kinase activity. Serum-starved cells were synchronized and pretreated with LDL (50 µg/ml), VLDL (50 µg/ml), or vehicle, 3 min before IGF-I (5 nM) stimulation. Cells were lysed 5 min after IGF-I addition, and lysates (0.5 mg protein) were immunoprecipitated with an IRS-1 antibody and assayed for PI3 kinase activity. Two independent controls are shown in the far left and right of the figure. A representative blot and the statistical analysis of four independent experiments are shown. {dagger}, P < 0.001 vs. IGF-I alone. B, LDL effects on basal and IGF-I-induced p85 protein expression. Serum-starved cells were treated with LDL (50 µg/ml), 5 nM IGF-I, or both for 24 h. A blot that is representative of three is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LDL cholesterol is incorporated in the vessel wall and induces phenotypic modulations of VSMC of critical relevance in the onset of atherosclerosis (2, 27). Numerous studies are ongoing to address the intimate mechanism by which atherogenic lipoproteins modulate phenotype and functionality of VSMC (27, 28, 29). In vitro lipoproteins are mitogenic and show synergistic interactions with growth factors for VSMC cell proliferation, a major step in the development of atherosclerotic lesions (2, 5, 7). Although these effects were initially assumed to be attributable to nutritional components provided by lipoproteins, as cholesterol or fatty acids, when these molecules were supplied alone to VSMC, the effects were lost (4). In fact, differential effects of lipoproteins modulating intracellular cell kinases, including growth factor receptors, have been very recently described (6, 30, 31).

Accumulating evidence now indicates that IGF-I, acting through its specific receptor in VSMC, affects its proliferation and migration; and the dysregulation of this axis can be involved in atherogenesis (11, 32). There are clinical settings in vivo, as obesity, when high levels of LDL-cholesterol could interact in VSMC with increased IGF-I, coming from circulation or locally induced by hyperlipemia (13, 15, 33). Moreover, expression of IGF-IR is increased in VSMC of atherosclerotic walls with a parallel pattern to lipid accumulation, supporting a potential autoparacrine relationship between them (14). Based on these data, we sought a suitable model of VSMC where a synergism between lipoproteins and IGF-I on DNA synthesis would justify the further investigation of a possible modulation by LDL of the IGF-I signaling pathway. We have focused on the study of IGF-I-induced cell proliferation by native LDL (thiobarbituric acid reactive values < 1 nmol/mg LDL protein). Our results show that LDL cooperate with IGF-I in the promotion of mitogenesis of A10 VSMC. This effect is specific for LDL, because VLDL do not affect basal or IGF-I-induced effects on [3H]-thymidine incorporation. Moreover, experiments with oxidized LDL with a thiobarbituric acid reactive value 15–25 times over native produced a dose-dependent decrease in A10 DNA synthesis, in agreement with the reported cytotoxic and proapoptotic actions of oxidized LDL on VSMC in vivo and in vitro (34, 35) (data not shown). The opposed actions of native and oxidized LDL on cell proliferation and IGF-I axis regulation in VSMC of rat aorta have been suggested, and our data are consistent with this notion. Whereas native LDL are mitogenic and up-regulate local IGF-I and IGF-IR production, oxidized LDL are proapoptotic and down-regulate this system (36).

Effects of LDL as inductors of phosphatidylinositol turnover have been described for various cell types, including VSMC (7, 37). According to this concept, LDL might be mitogenic and potentiate IGF-I-induced mitogenesis just by favoring phosphatidylinositol recycling. However, in this study, we find that tyrosine phosphorylation of the IGF-IRß-subunit and IRS-1 is promoted by LDL. This is a rapid, reversible, and regulated event, because it is present already at 1 min of exposure, and it is almost reversed at 20 min. It is also specific, because VLDL, used as control, did not mimic this effect. LDL did not induce IRS-1-associated PI3 kinase activation by itself, but activation of other IGF-I-dependent pathways could support the requirement for IGF-I in the intrinsic LDL signaling system recently suggested in other VSMC types (36).

We demonstrate that IGF-I activation of PI3 kinase signaling pathway, through IRS-1 in A10 cells, led to a sustained activation of PKB/Akt and is required for IGF-I-induced cell proliferation. These results agree with the overwhelming evidence on the relevant role of PI3 kinase/Akt pathway in IGF-I-dependent mitogenesis (38) and, more precisely, with recent reports on the requirement of IRS-1-associated PI3 kinase activation for proliferative actions of IGF-I in VSMC (19, 39, 40). Moreover, IGF-I also up-regulates the p85 subunit of PI3 kinase protein expression at 24 h of exposure, as recently described for the p85{alpha} PI3 kinase in other cell systems (25). We found that IGF-I-induced, IRS-1-associated PI3 kinase activity is up-regulated by LDL. Consistent with this observation, LDL induced a significant and fast potentiation in serine phosphorylation of the PI3 kinase downstream target, PKB/Akt. This effect disappeared at 3 h and was not caused by a direct action of LDL on non-IRS-1-mediated PI3 kinase activation, because this possibility was excluded by results from cells treated with LDL alone. The amplifying effect of LDL on IGF-I-induced, IRS-1-associated PI 3 kinase activation may be involved in their synergistic effect on DNA synthesis, because the presence of LDL would enhance the activity of the pathway more directly involved in the proliferative response to this growth factor in A10 cells.

ERK 1/2 phosphorylation was also induced by IGF-I in our cells in a time-dependent manner, although our data with specific inhibitors suggest that the involvement in its mitogenic effects is less relevant. In our model, LDL alone induced ERK activation according to previous data in other VSMC (41). Moreover, there was an additive effect between LDL and IGF-I in ERK phosphorylation at times when the effect of IGF-I on this pathway already declined. Because the role of MAPK activation after IGF-I treatment in proliferation of VSMC is more controversial (39), the possible consequences of these findings, in terms of LDL modulation to other IGF-I responses, should be explored.

In summary, we have characterized an interaction between LDL and the IGF-I signaling pathway in A10 VSMC (Fig. 8Go). This resulted in quantitatively significant changes of intracellular pathways within a discrete temporal frame. Recent reports suggest that changes in the balance of PI3 kinase/Akt and the MAPK pathways induced by growth factors, including IGF-I, are determinant for the phenotype and function of VSMC (42, 43). The mechanism presented here might contribute to establishing the molecular basis for the deleterious interaction of elevated levels of LDL-cholesterol with growth factors in the pathogenesis of the atherosclerotic lesion.



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  		Figure 8. LDL interactions with IGF-I signal transduction cascades in A10 VSMC. After autophosphorylation and activation of the IGF-IR, IRS-1, and Shc, molecules are bound to the intracellular region of the IGF-IRß-subunit. This leads to a coordinate activation of IRS-1associated PI3 kinase/Akt pathway (preferentially involved in IGF-I-induced proliferation in A10 cells) and MAPK (ERK1/2) pathway. LDL induce the phosphorylation of IGF-IRß-subunit and IRS-1, markedly up-regulate IRS-1-associated PI 3 kinase/Akt in response to IGF-I without affecting this pathway by themselves, and show an additive effect with IGF-I on ERK 1/2 phosphorylation. These changes affect, in a temporal frame, the balance between PI3 kinase/Akt and ERK 1/2 pathways after IGF-I stimulation. Crosses imply direct effect of LDL. Wide arrow symbols imply potentiation by LDL without effect of LDL alone. MEK, Mitogen-activated protein kinase.

 


   Acknowledgments
 
We are grateful to Dr. L. Boscá for his help with PI3 kinase activity determination and for helpful comments. We thank Dr. J. Mostaza and Dr. C. LaHoz for their helpful suggestions regarding lipoprotein isolation. Finally, we thank Dr. F. Sanchez-Franco for his support during the development of this work.


   Footnotes
 
Belén González is a predoctoral fellow from Instituto de Salud Carlos III (99/4213). This work was supported by Grants SAF 98-0003 (to E.M.) and SAF 2000-0149 (to S.L.), from the Plan Nacional de Investigación y Desarrollo, Spain, and by Grant 08.4/0031.2/2000 (to E.M.) from the Comunidad de Madrid, Spain.

Abbreviations: ERK, Extracellular signal-regulated MAPK; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate-1; LDL, low density lipoproteins; ODN, oligodeoxinucleotides; PI3 kinase, phosphatidylinositol 3-kinase; SFM, serum-free DMEM; VLDL, very low density lipoproteins; VSMC, vascular smooth muscle cells.

Received June 6, 2001.

Accepted for publication July 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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