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(Circulation Research. 2003;92:1115.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Phosphatidylinositol 3-Kinase and Calcium-Activated Transcription Pathways Are Required for VLDL-Induced Smooth Muscle Cell Proliferation

Larissa Lipskaia, Marie-Luce Pourci, Claudine Deloménie, Laurent Combettes, Dominique Goudounèche, Jean-Louis Paul, Thierry Capiod, Anne-Marie Lompré

From INSERM U446 (L.L., C.D., A.-M.L.), Laboratoire de Biochimie Appliquée (M.-L.P., D.G., J.-L.P.) and IFR-75: Institut de signalisation et innovation thérapeutique, Faculté de Pharmacie, Châtenay-Malabry, France; and INSERM U442, IFR: Signalisation cellulaire (L.C., T.C.), Faculté des Sciences Bâtiment 443, Orsay, France.

Correspondence to Anne-Marie Lompré, INSERM U446/IFR-75, Institut de signalisation et innovation thérapeutique, Faculté de Pharmacie, Tour D4, 5 rue JB Clément, 92296 Châtenay-Malabry, France. E-mail anne-marie.lompre{at}egm.u-psud.fr

	Abstract

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Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca²⁺ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca²⁺ release was inhibited and the IP3-sensitive Ca²⁺ release induced by ATP (10 µmol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3ß in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca²⁺-induced Ca²⁺ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.

Key Words: vascular disease • smooth muscle • calcium signaling • transcription factors • lipoproteins

	Introduction

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Hypertriglyceridemia is considered an independent risk factor of atherosclerosis and coronary disease,^1,2 but the mechanisms of atherogenicity are not clearly defined. An increasing body of evidence supports multiple roles for very low-density lipoproteins (VLDLs), the main carriers of triglycerides, in the development of atherosclerosis and cardiovascular diseases. The VLDLs may have an indirect role because of the strong relationship between elevated triglycerides and other atherogenic metabolic changes, including decreased levels of high-density lipoprotein (HDL) and a predominance of atherogenic small and dense low-density lipoprotein (LDL) particles. They may also have a direct influence on atherosclerosis by altering physiological functions of arterial wall cells.^2,3 A potential relationship between hypertriglyceridemia and disease progression has been suggested in studies showing that atherogenic lipoproteins induce smooth muscle cell (SMC) proliferation, a phenomenon of primary importance in atherosclerosis, restenosis after balloon injury, or neointimal occlusion of grafts. How hypertriglyceridemia alters vascular cells toward the atherosclerotic phenotype is still under intense investigation. Previous studies showed that VLDL stimulated SMC DNA replication and growth by themselves^4,5 via activation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1) and ERK2.⁵ ßVLDL, a cholesteryl ester-rich atherogenic lipoprotein, alone was unable to inspire quiescent cells into S phase but activated ERK1/ERK2 via transactivation of the epidermal growth factor receptor.⁶ Furthermore, recent clinical trials have provided evidence that calcium channel blockers can inhibit progression of atherosclerotic disease.^7,8

Ca²⁺ is an essential regulator of the cell cycle and the Ca²⁺ response amplitude and duration control gene expression in various cell types.⁹ Proliferation of SMCs is accompanied by modifications in calcium homeostasis and expression of genes encoding Ca²⁺ transporting proteins. Indeed, decreased L-type and enhanced T-type Ca²⁺ channel activity^10–12 as well as enhanced capacitative Ca²⁺ entry¹³ and upregulation of the TRP1 channels¹⁴ were described in proliferating SMCs. We also demonstrated that the ryanodine receptor (RyR) and the sarcoplasmic reticulum Ca²⁺-ATPase, SERCA 2a, were lost in proliferating cells.¹⁵ Similar changes were observed under chronic exposure of SMCs to minimally oxidized LDL.^16,17

Two main Ca²⁺-regulated transcription factors were described: c-AMP responsive element binding protein (CREB) and nuclear factor of activated T-cells (NFAT). Transient Ca²⁺ influx through the L-type voltage-activated channels is particularly effective at activating CREB via Ca²⁺/CaM-dependent phosphorylation by Ca²⁺/calmodulin kinase or MAPK.^18–20 By contrast, a sustained increase in cytosolic Ca²⁺, obtained for example by PDGF activation of IP3R, is necessary to dephosphorylate NFAT by calcineurin, a Ca²⁺/calmodulin-dependent phosphatase, and induce its translocation into the nucleus.^21,22 GSK-3ß is a negative regulator of this signaling pathway because it phosphorylates NFAT and induces its export from the nucleus. Inactivation of GSK-3ß by phosphorylation on Ser9 by protein kinase B (also called Akt) is necessary to assure NFAT-dependent transcription. Akt itself is activated by the phosphatidylinositol 3-kinase (PI3K). Very recently, by using VLDL receptor-deficient mice, Beffert at al²³ demonstrated that in cultured embryonic neurons, the VLDL receptor is coupled to the PI3K-dependent signaling cascade.

We designed experiments to determine whether VLDL could induce proliferation of isolated rat aortic SMCs in culture or could alter Ca²⁺ homeostasis and the activation of Ca²⁺-regulated transcription factors to induce a new gene expression program characteristic of the proliferating SMC. We analyzed the pathways involved in these processes.

	Materials and Methods

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Preparation and Characterization of VLDLs
Total plasma was recovered from normotriglyceridemic donors free from infectious disease, with prior consent and following the local ethic committee guidelines. VLDLs (density (d) <1.006 g/mL) and HDL (1.063 <d <1.210 g/mL) were prepared by sequential ultracentrifugation.²⁴ For detailed characterization, please refer to the expanded Materials and Methods section, available in the online data supplement at http://www.circresaha.org.

Isolation and Culture of SMCs: Analysis of Cell Proliferation
Rat aortic SMCs were isolated from the media of the thoracic aorta from male Wistar rats as described.¹⁵ All experiments were performed on primary culture. After dissociation, cells were allowed to adhere for 24 hours in DMEM supplemented with 1% penicillin-streptomycin-amphotericin mixture (ICN) and 20% FCS and then maintained for 24 to 36 hours in 0.1% FCS medium before beginning the experiments. For the experiment, the medium was replaced by DMEM containing either 0.1% lipoprotein-deprived FCS (LPDS) with or without purified VLDL or 10% FCS. The concentration of VLDL vehicle (PBS) was kept constant in all compared cultures. Various components were added together with the VLDL fraction to analyze their influence on proliferation; LY294002 (No. 9901, Cell Signaling), diltiazem, ryanodine, cyclosporin A (CsA), and rapamycin were from Sigma.

Proliferation of SMCs was measured by using the colorimetric-based CellTiter96 Cell Proliferation Assay kit (Promega) according to manufacturer instructions.

Reverse Transcriptase-Polymerase Chain Reaction for NFAT Isoforms
Total RNA was prepared from aorta or cultured SMCs by using the RNAxis Reagent (Genaxis) according to the manufacturer’s protocol. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described.²¹ Amplified PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining.

Western Blot Analysis and Immunofluorescence
Total cell lysates were prepared according to standard protocol (Upstate Biotechnology). Cytosolic and nuclear fractions were obtained by hypotonic lysis as described.²⁵

The primary antibodies were anti-phosphorylated CREB (a-pCREB) and a-CREB from Upstate Biotechnology (Nos. 06-519 and 06-863); a-NFATc1 and a-NFATc3 from Santa Cruz Biotechnology, Inc (K-18 and M-75); a-pAkt, a-Akt, a-pGSK-3ß, a-pPTEN, a-pPDK1, and a-FKHR from Cell Signaling (No. 9916) and a-p53.²⁶ Proteins were visualized using the enhanced chemiluminescence detection system (ECL+, Amersham).

For immunofluorescence, proteins were visualized by using either secondary antibodies directly conjugated to Texas Red or the biotin/streptavidin-Texas Red conjugated amplification method (Amersham). Antibodies to RyR and to SERCA 2a and 2b were previously described.^27,28 Anti-PCNA (proliferating nuclear antigen) was from Dako-France. Nuclei were stained with Hoechst (Sigma). Images were collected with a Zeiss LSM-510 confocal scanning laser microscope as described online in the expanded Materials and Methods section.

[Ca²⁺]_i Measurements
Directly after isolation from the aorta, cells were plated on glass coverslips coated with collagen and treated as for the other experiments. Cells were loaded with 4 µmol/L Fura 2 (Molecular Probes) and continuously superfused with control or test solutions at 37°C using a PTR 200 perfusion temperature regulator (ALA Scientific Instruments). The control solution contained (in mmol/L) NaCl 116, KCl 5.6, CaCl₂ 1.8, MgCl₂ 1.2, NaHCO₃ 5, NaH₂PO₄ 1, and HEPES 20, pH 7.3. Caffeine (10 mmol/L), ATP (10 µmol/L), or 2,5-di-(t-butyl)-1,4-benzo-hydroquinone (tBHQ, 50 µmol/L) was used in the test solution. Fluorescence images were collected every 2 seconds by a Sensicam QE CCD camera (PCO Computer Optics GmbH), digitized, and integrated in real time by an image processor (Metafluor). For details, please refer to the expanded Materials and Methods section available online.

Statistical Analysis
All quantitative data are presented as mean of at least 3 independent experiments±SEM. Data were analyzed by using the SigmaStat Statistical Software Package, version 2.0 (Jandel Scientific). One-way ANOVA or Kruskal-Wallis ANOVA on ranks followed by the Dunn’s or Tukey method was performed for multiple comparison of values. Statistical comparison of 2 values was done by an unpaired Student’s t test. P<0.05 was considered to be statistically significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
VLDLs Stimulate Proliferation of SMCs in a Dose- and Time-Dependent Manner
Exposure of rat aortic SMCs to VLDL significantly promoted cell proliferation in a concentration-dependent (Figure 1A) and time-dependent (Figure 1B) manner. The maximal response was observed with 0.15 g protein/L (1 mmol/L triglycerides). The number of cells in VLDL-treated culture was significantly increased after 24 hours, and saturation was reached at 48 hours. Proliferation was increased by 2.5-fold compared with control cells cultured in 0.1% LPDS. Similar levels of proliferation were obtained with VLDL (0.15 g/L) and 10% FCS. HDL isolated from the same plasma never enhanced proliferation, even at high dose.

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of VLDL on proliferation of SMCs. A, SMCs were treated for 48 hours either with 0.1% LPDS in the presence of indicated concentrations of VLDL or HDL or with 10% FCS. B, SMCs were treated for 5 to 96 hours with VLDL (0.15 g/L proteins=1 mmol/L triglycerides). Each point represents the mean of 3 to 6 independent experiments. For each time point or each lipoprotein concentration, the number of cells was normalized to the number of cells in 0.1% LPDS with PBS as vehicle. Values were plotted as the percentage of change from control. *P<0.05 vs control.

The cells in 0.1% FCS expressed SERCA 2a, SERCA 2b, and the RyR. PCNA was not detected in the nucleus, attesting for a quiescent/differentiated phenotype (online Figure 1). In the presence of VLDL for 2 or 3 days, PCNA was present in the nucleus, indicating that cells were proliferating. SERCA 2a and the RyR were absent, whereas SERCA 2b was still expressed, as already described in dedifferentiated SMCs.¹⁵

VLDLs Alter [Ca²⁺]_i Transient
We tested the hypothesis that VLDLs could alter Ca²⁺ release through the RyR or the IP3R by analyzing the caffeine- or ATP-induced Ca²⁺ transients, respectively. In control conditions, SMCs were sensitive to both caffeine and ATP (Figure 2A, top). Addition of VLDL together with caffeine (or ATP, not shown) did not alter the Ca²⁺ signal (Figure 2A, middle), indicating that VLDL did not trap the agonists, rendering them unavailable to the cells. However, after preincubation with VLDL for 4 hours, most SMCs did not respond to 10 mmol/L caffeine, but 10 µmol/L ATP induced an increase in [Ca²⁺]_i (Figure 2A, bottom). The number of caffeine-sensitive SMCs was down to 25% (14 of 55 cells) in the presence of VLDL compared with 71% (15 of 21 cells) in the control condition. By contrast, the number of ATP-sensitive cells remained significantly the same in the presence (89%, 49 of 55 cells) or absence (90%, 19 of 21 cells) of VLDL. In the presence of VLDL for 24 hours, no cells were sensitive to caffeine (0 of 19 cells,) while 15 of 19 cells (79%) responded to ATP. The rate of cytosolic Ca²⁺ clearance after ATP application was clearly affected by VLDL treatment (Figure 2B). Similar results were obtained when using 50 µmol/L tBHQ, a SERCA blocker. The duration of the ATP- and tBHQ-evoked responses was estimated as the duration of the [Ca²⁺]_i transient at half amplitude. Durations of the ATP-evoked Ca²⁺ responses were significantly (P<0.001) increased to 40±5 seconds (n=10) from 18±1 seconds (n=12), and those of tBHQ increased to 157±2 seconds (n=6) from 80±5 seconds (n=7) in the presence of VLDL for 4 hours compared with control conditions.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of VLDL on caffeine- and ATP-evoked Ca2+ transients. A, Top, Caffeine-induced (10 mmol/L) or ATP-induced (10 µmol/L) increase in cytosolic [Ca2+] in control conditions. Middle, Caffeine-induced Ca2+ release in cells perfused with VLDL (1 mmol/triglycerides) (middle). Bottom, Caffeine- or ATP-induced Ca2+ release in cells pretreated for 4 hours with VLDL (1 mmol/L triglycerides). B, ATP-evoked Ca2+ transient in control conditions and in presence of VLDL (1 mmol/L triglycerides) for 4 hours. When applying the agonists, cells were perfused with an external solution containing no calcium and 100 µmol/L EGTA. C, Ca2+ influx generated by KCl depolarization in the presence of VLDL (1 mmol/L triglycerides) (top) is inhibited by diltiazem (60 µmol/L) (bottom).

Depolarization-induced Ca²⁺ influx, measured in the presence of 60 mmol/L KCl, was not inhibited by VLDL (Figure 2C). The KCl-induced Ca²⁺ influx was inhibited by diltiazem, an inhibitor of the L-type voltage-dependent Ca²⁺ channels, even in the presence of VLDL. Altogether, these data indicate that the agonists remain active in the presence of VLDL and that VLDLs have little effect on the L-type Ca²⁺ channels and affect preferentially the Ca²⁺-induced Ca²⁺ release mechanism.

CREB Phosphorylation Is Decreased in VLDL-Treated SMCs
Most of the nuclei of control SMCs (0.1% LPDS) and of cells incubated with VLDL for 4 hours were stained with anti-pCREB (Figure 3A). However, the number of pCREB-positive nuclei was dramatically decreased after 24 hours with VLDL. The level of CREB phosphorylation was quantitated by Western blotting using anti-pCREB and a-CREB sequentially after stripping (Figure 3B). The level of CREB was similar in all samples. The ratio of pCREB/CREB was decreased >50% in the presence of VLDL (1 mmol/L triglycerides) for 24 or 48 hours or 10% FCS for 48 hours compared with control conditions (0.1% LPDS) or VLDL for 4 hours. Because pCREB regulates expression of the tumor suppressor gene, p53,²⁹ we analyzed the level of p53 after VLDL treatment (Figure 3C). No variation was observed after 4 hours with VLDL, but after 48 hours p53 expression was lowered.

View larger version (36K): [in this window] [in a new window]   Figure 3. CREB phosphorylation is decreased in VLDL-treated SMCs. A, Immunofluorescence with a-pCREB in control conditions and after exposure to VLDL (1 mmol/L triglycerides) for 4 or 24 hours. Nuclei are visualized with Hoescht. Bar=10 µm. B, Western blot analysis of pCREB levels in control conditions and after exposure to VLDL (1 mmol/L triglycerides) for 4, 24, or 48 hours or to 10% FCS for 48 hours. After exposure, the membrane was stripped and probed with a-CREB. The films were scanned, and values of pCREB in each sample were normalized to the level of CREB. The results in the bar graph are presented as percent of control value and represent the average of 2 independent experiments. The bars refer to the lanes in the Western blot above. C, Western blot analysis of p53 expression in control condition or after exposure to VLDL for 4 or 48 hours.

VLDLs Activate the NFAT Pathway
The NFAT isoform expressed in rat aortic SMCs was determined by RT-PCR (Figure 4A). Only NFATc3 (NFAT4) was detected in aorta free of endothelium and adventitia as well as in SMCs maintained in presence of 0.1% LPDS for 24 hours. NFATc4 (NFAT3) transcript was induced by exposure to VLDL or 10% FCS for 72 hours, and trace amounts of NFATp (NFAT2) were induced in cells exposed to 10% FCS for 72 hours. Because NFAT is transcriptionally active only when it is in the nucleus, we analyzed the localization of NFAT protein by immunofluorescence (Figure 4B) and by Western blot of nuclear extracts (Figure 4C). Both the specific a-NFATc3 antibody and an a-NFAT antibody that recognized all NFAT isoforms (data not shown) were used for immunofluorescence with the same results. In control cells (Figure 4Ba), NFAT was located in the cytosol, whereas NFAT staining was more abundant in the nuclei after exposure to VLDL for 24 hours (Figure 4Bb). In cells exposed to VLDL for 24 hours in the presence of the calcineurin inhibitor CsA (5 µmol/L), NFAT was arrested in the cytosol (Figure 4Bc), as expected. In the presence of LY294002 (10 µmol/L), an inhibitor of PI3K, NFAT was mainly in the cytosol (Figure 4Bd), suggesting that VLDL activated the PI3K pathway.

View larger version (53K): [in this window] [in a new window]   Figure 4. VLDLs activate the NFAT signaling pathway. A, RT-PCR analysis of NFAT mRNA isoforms in quiescent and proliferating SMCs. NFATc1 (NFATc), 1; NFATc2 (NFATp), 2; NFAT C4 (NFAT3), 3; NFAT C3 (NFAT4), 4. B, Immunofluorescence with a-NFATc3 in control conditions (a) and after 24 hours exposure to VLDL (1 mmol/L triglycerides) alone (b) or in presence of 5 µmol/L cyclosporin A (c) or 10 µmol/L LY294002 (d). Bar=10 µm. C, Western blot analysis of the presence of NFAT in nuclear extracts from SMCs in control conditions (0.1% LPDS) or treated for 4 and 24 hours with VLDL (1 mmol/L triglycerides). Cells were also cultured for 24 hours with VLDL plus cyclosporin A (5 µmol/L) and with 10% FCS.

Western blot analysis of total extracts from freshly isolated cells using the common a-NFAT antibody (not shown) revealed only 1 NFAT protein (110 to 115 kDa) corresponding to NFATc3. In nuclear extracts, the amount of NFATc3 was increased after exposure to VLDL for 24 hours or after exposure to 10% FCS for 24 hours (Figure 4C). CsA blocked NFAT transport into the nucleus. Thus, VLDLs activate NFAT translocation in a calcineurin-dependent and PI3 kinase-dependent way.

Pharmacological Analysis of the Calcium Signaling Pathways Involved in VLDL-Induced CML Proliferation
To determine the importance of CREB and NFAT pathways on VLDL-induced proliferation, we tested the effect of various drugs known to act on these pathways (Figure 5). Ca²⁺ influx through L-type channels was shown to be essential in CREB phosphorylation,¹⁸ but diltiazem (still active in presence of VLDL, Figure 2C) had no effect on VLDL-induced proliferation. Ryanodine did not influence VLDL-induced proliferation, in agreement with the data in Figure 2, showing that the caffeine-induced Ca²⁺ release was already inhibited by VLDL. Rapamycin, a drug known to block proliferation by acting on mTOR (mammalian target of rapamycin), completely blocked VLDL-induced proliferation at picomolar concentrations. Cyclosporin A (5 µmol/L) and LY294002 (10 µmol/L) prevented VLDL-induced proliferation at doses that also prevented NFAT translocation. These latter data suggest that there is a relationship between NFAT translocation and proliferation and that activation of the PI3 kinase is involved in both processes.

View larger version (43K): [in this window] [in a new window]   Figure 5. Pharmacological analysis of the calcium signaling pathways of VLDL-induced proliferation. SMCs were cultured for 36 hours in the presence of 0.1% LPDS (vehicle) and VLDL (1 mmol/L triglycerides) alone (control) or with several drugs at indicated concentrations. Rya indicates ryanodine; Dil, diltiazem; LY, LY294002; and Rap, rapamycin. Data are expressed as percent of the cell number in control wells. The results represent the average of at least 3 independent experiments performed in pentaplicate. *P=0.05; **P<0.005; and ***P<0.0001 vs control.

VLDLs Activate the PI3K Signaling Cascade
Thus, we analyzed the effect of VLDL (1 mmol/L triglycerides) on the PI3K signaling cascade by Western blot (Figure 6). A 30-minute exposure to VLDL induced phosphorylation of Akt on Thr308 and Ser473, and phosphorylation was maintained for up to 24 hours. The level of total Akt was stable. We also tested phosphorylation of 2 of the targets of Akt, GSK3-ß on Ser9 and FKHR (a forkhead transcription factor) on Ser256. Both were phosphorylated within 30 minutes after application of VLDL. Phosphorylation of FKHR was transient, whereas that of GSK3-ß was additionally increased after 24 hours. Phosphorylation was abolished or markedly decreased by LY294002 (10 µmol/L), indicating that PI3K is involved in this process. The levels of phospho-PTEN (Ser380) and of phospho-PDK1 (Ser241), as expected, were insensitive to LY294002. These results demonstrate that VLDLs activate the Akt pathway through activation of the PI3 kinase.

View larger version (59K): [in this window] [in a new window]   Figure 6. VLDLs activate the PI3K-dependent signaling cascade. Western blot analysis of total extracts from SMCs in control conditions (0.1% LPDS) or treated for 30 minutes, 4 hours, and 24 hours with VLDL (1 mmol/L triglycerides) alone or in the presence of LY294002 (10 µmol/L). The membranes were probed with a-pAkt (Thr308), a-pAkt (Ser473), a-pGSK-3ß (Ser9), or a- p-FKHR (Ser256). Then they were stripped and probed with a-Akt, a-pPTEN (Ser380), and a-pPDK (Ser241), confirming equivalent protein loading.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Results of the present study provide evidence that VLDLs induce proliferation of rat aortic smooth muscle cells by activating the PI3 kinase/Akt pathway leading to NFAT nuclear translocation. Furthermore, VLDLs inhibit the ryanodine-sensitive store and CREB phosphorylation, additionally contributing to induce a proliferating phenotype.

LDLs, either native or mildly oxidized, were clearly shown to be mitogenic for vascular SMCs,^6,30 but the effect of VLDL, a triglyceride-rich lipoprotein, was more controversial. In agreement with our results, VLDLs were shown to promote proliferation of human or rat arterial SMCs by themselves.^4,5 However, ßVLDL, a cholesteryl ester-rich atherogenic lipoprotein, did not induce proliferation of quiescent smooth muscle cells alone but increased the proliferative rate of actively dividing cells and exacerbated the effect of epidermal growth factor.⁶ Yet it was not clear how lipoproteins, and especially VLDL, exert their proliferating effect.

In the present study, we demonstrate that VLDLs induce rapid (30-minute) phosphorylation of Akt and of its downstream targets GSK-3ß and FKHR. Phosphorylation of FKHR regulates its nuclear translocation and promotes cell survival. These phosphorylations, being blocked by LY294002, are attributable to activation of the PI3K. This results in agreement with very recent data showing that in cultured embryonic neurons, the VLDL receptors are coupled to the PI3K-dependent signaling cascade. Thus, we propose that VLDLs induce proliferation of vascular SMCs by acting via the VLDL receptors and by a consecutive alteration in intracellular signaling pathways, resulting in inhibition of the CREB pathway and activation of NFAT (Figure 7).

View larger version (74K): [in this window] [in a new window]   Figure 7. Schematic representation of VLDL-induced signaling pathway in vascular SMCs. VLDLR indicates VLDL receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homologue; PDK1/2-3, phosphoinositide-dependent protein kinase-1/2; Akt/PKB, protein kinase B; GSK-3ß, glycogen synthase kinase-3ß; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; IP3R, IP3 receptor; SOC, store-operated calcium channel; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; Dil, diltiazem, and Rya, ryanodine.

VLDLs induce a calcineurin-dependent, PI3K-dependent translocation of NFATc3 to the nucleus, which coincides with proliferation, because both phenomenon are inhibited by CsA and LY294002. Furthermore, VLDLs induce phosphorylation of GSK-3ß and thus inhibit its activity, which is to phosphorylate and export NFAT from the nucleus. Both nuclear translocation and inhibition of export will favor NFAT activity. Our results indicate that NFATc3 is present in aortic SMCs as in cerebral artery^21,31 and that NFATc4 is induced in proliferating cells only. This is consistent with the fact that mice lacking NFATc3/c4 genes died with defects in vascular wall assembly.³² Other studies on passaged SMCs reported the presence of NFATc1 and NFATc2.³³

Rapamycin very effectively prevented VLDL-induced proliferation, in agreement with previous data showing that it inhibits proliferation of vascular SMCs in vitro and in vivo,^34,35 but rapamycin did not prevent VLDL-induced NFAT translocation (data not shown). The complex rapamycin/FKPB12 directly arrests cell cycle progression by acting on mTOR (mammalian target of rapamycin).³⁶

Activation of calcineurin requires a sustained increase in Ca²⁺. In our hands, VLDL did not increase [Ca²⁺]_i immediately when applied by the overflow technique,³⁷ at the opposite of data reporting a maximal elevation in [Ca²⁺]_i in a few seconds by all lipoproteins.⁵ However, incubation with VLDL for several hours resulted in prolongation of Ca²⁺ release from the ATP-sensitive pool because of a slower rate of clearance of Ca²⁺. Activation of phospholipase C by PIP3 and production of IP3 will also favor the increase in cytosolic Ca²⁺ either directly by activation of the IP3R or indirectly via the capacitative Ca²⁺ current (store-operated channel). Interestingly, the VLDL-induced blockade of the ryanodine-sensitive Ca²⁺ pool observed on the short-term resulted in long-term alteration of protein expression. Indeed, after 2 to 3 days of VLDL treatment, SERCA2a and RyR were lost (online Figure 1).

In the presence of VLDL, CREB is dephosphorylated. Phosphorylation of CREB was shown to be coupled to Ca²⁺ entry through the voltage-gated L-type channels,¹⁸ and the present results show that pCREB is associated with a quiescent/differentiated phenotype of SMC. CREB dephosphorylation is probably the result of VLDL-induced inhibition of the Ca²⁺-induced Ca²⁺ release mechanism and is associated with SMC proliferation. Diltiazem and ryanodine have no effect on VLDL-induced proliferation, because the Ca²⁺-induced Ca²⁺ release mechanism is already blocked. Others have shown that p-CREB recruits p53, the tumor suppressor gene.²⁹ Interestingly, a decrease in expression of p53 was observed with VLDL. The role of p53 in regulating proliferation in atherosclerosis or restenosis after angioplasty is highly controversial,³⁸ but loss of p53 accelerates neointimal lesions of vein bypass grafts.³⁹ Furthermore, apoE/p53 double-knockout mice had a significant increase in cell proliferation and atherosclerotic lesions induced by hyperlipidemia.⁴⁰

The exact nature of the alteration in Ca²⁺ movement is not clear but seems to be indirect, because several hours were needed to observe an effect of VLDL on Ca²⁺ signaling. VLDLs are lipoproteins enriched in triglycerides and relatively poor in phospholipids, and this may alter the composition of cellular membranes. Alterations in the lipid composition of biological membranes, including sarcoplasmic reticulum (SR), have been shown to modulate the function of integral proteins and especially of SERCA.^41,42 Alternatively, activation of protein kinase C (PKC), a second substrate of PDK1, may also account for this effect, because PKC activation decreases SR Ca²⁺ transport⁴³ and reduces the storage capacity of the sarcoplasmic reticulum Ca²⁺ pool.⁴⁴ Furthermore, PKC can inhibit spontaneous Ca²⁺ release in SMCs by lowering the Ca²⁺ sensitivity of RyR channels^45,46 or inhibition of Ca²⁺ influx through L-type channels.⁴⁷

In conclusion, we have shown that VLDLs induce proliferation of SMCs by activating the PI3 kinase cascade, resulting in alteration of intracellular calcium homeostasis and in NFAT translocation into the nucleus. VLDLs also inhibit the ryanodine-sensitive store and CREB phosphorylation, additionally favoring proliferation.

	Acknowledgments

 
L.L. is supported by a postdoctoral fellowship from Association Française contre les Myopathies. The authors thank V. Nicolas, service d’imagerie IFR-75, for assistance in elaborating the microscopy data and N. Moatti for critical reading of the manuscript.

Received January 15, 2003; revision received April 16, 2003; accepted April 17, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Forrester JS. Triglycerides: risk factor or fellow traveler? Curr Opin Cardiol. 2001; 16: 261–264.[CrossRef][Medline] [Order article via Infotrieve]

2. Malloy MJ, Kane JP. A risk factor for atherosclerosis: triglyceride-rich lipoproteins. Adv Intern Med. 2001; 47: 111–136.[Medline] [Order article via Infotrieve]

3. Ginsberg HN. Hypertriglyceridemia: new insights and new approaches to pharmacologic therapy. Am J Cardiol. 2001; 87: 1174–1180.[CrossRef][Medline] [Order article via Infotrieve]

4. Bjorkerud S, Bjorkerud B. Lipoproteins are major and primary mitogens and growth promoters for human arterial smooth muscle cells and lung fibroblasts in vitro. Arterioscler Thromb. 1994; 14: 288–298.[Abstract/Free Full Text]

5. Sachinidis A, Kettenhofen R, Seewald S, Gouni-Berthold I, Schmitz U, Seul C, Ko Y, Vetter H. Evidence that lipoproteins are carriers of bioactive factors. Arterioscler Thromb Vasc Biol. 1999; 19: 2412–2421.[Abstract/Free Full Text]

6. Zhao D, Letterman J, Schreiber BM. ß-Migrating very low density lipoprotein (ßVLDL) 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 ßVLDL plus EGF-induced cell proliferation. J Biol Chem. 2001; 276: 30579–30588.[Abstract/Free Full Text]

7. Zanchetti A, Bond MG, Hennig M, Neiss A, Mancia G, Dal Palu C, Hansson L, Magnani B, Rahn KH, Reid JL, Rodicio J, Safar M, Eckes L, Rizzini P. Calcium antagonist lacidipine slows down progression of asymptomatic carotid atherosclerosis: principal results of the European Lacidipine Study on Atherosclerosis (ELSA), a randomized, double-blind, long-term trial. Circulation. 2002; 106: 2422–2427.[Abstract/Free Full Text]

8. Tulenko TN, Sumner AE, Chen M, Huang Y, Laury-Kleintop L, Ferdinand FD. The smooth muscle cell membrane during atherogenesis: a potential target for amlodipine in atheroprotection. Am Heart J. 2001; 141: S1–S11.[CrossRef][Medline] [Order article via Infotrieve]

9. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca²⁺ response amplitude and duration. Nature. 1997; 386: 855–858.[CrossRef][Medline] [Order article via Infotrieve]

10. Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Haller H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J. 1998; 12: 593–601.[Abstract/Free Full Text]

11. Kuga T, Kobayashi S, Hirakawa Y, Kanaide H, Takeshita A. Cell cycle-dependent expression of L- and T-type Ca²⁺ currents in rat aortic smooth muscle cells in primary culture. Circ Res. 1996; 79: 14–19.[Abstract/Free Full Text]

12. Richard S, Neveu D, Carnac G, Bodin P, Travo P, Nargeot J. Differential expression of voltage-gated Ca²⁺-currents in cultivated aortic myocytes. Biochim Biophys Acta. 1992; 1160: 95–104.[CrossRef][Medline] [Order article via Infotrieve]

13. Golovina VA. Cell proliferation is associated with enhanced capacitative Ca²⁺ entry in human arterial myocytes. Am J Physiol. 1999; 277: C343–C349.[Medline] [Order article via Infotrieve]

14. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, Yuan JX. Upregulated TRP and enhanced capacitative Ca²⁺ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol. 2001; 280: H746–H755.[Abstract/Free Full Text]

15. Vallot O, Combettes L, Jourdon P, Inamo J, Marty I, Claret M, Lompre AM. Intracellular Ca²⁺ handling in vascular smooth muscle cells is affected by proliferation. Arterioscler Thromb Vasc Biol. 2000; 20: 1225–1235.[Abstract/Free Full Text]

16. Massaeli H, Austria JA, Pierce GN. Lesions in ryanodine channels in smooth muscle cells exposed to oxidized low density lipoprotein. Arterioscler Thromb Vasc Biol. 2000; 20: 328–334.[Abstract/Free Full Text]

17. Massaeli H, Austria JA, Pierce GN. Chronic exposure of smooth muscle cells to minimally oxidized LDL results in depressed inositol 1,4,5-trisphosphate receptor density and Ca²⁺ transients. Circ Res. 1999; 85: 515–523.[Abstract/Free Full Text]

18. Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science. 2001; 294: 333–339.[Abstract/Free Full Text]

19. Cartin L, Lounsbury KM, Nelson MT. Coupling of Ca²⁺ to CREB activation and gene expression in intact cerebral arteries from mouse: roles of ryanodine receptors and voltage- dependent Ca²⁺ channels. Circ Res. 2000; 86: 760–767.[Abstract/Free Full Text]

20. Hardingham GE, Chawla S, Johnson CM, Bading H. Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature. 1997; 385: 260–265.[CrossRef][Medline] [Order article via Infotrieve]

21. Stevenson AS, Gomez MF, Hill-Eubanks DC, Nelson MT. NFAT4 movement in native smooth muscle: a role for differential Ca²⁺ signaling. J Biol Chem. 2001; 276: 15018–15024.[Abstract/Free Full Text]

22. Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997; 15: 707–747.[CrossRef][Medline] [Order article via Infotrieve]

23. Beffert U, Morfini G, Bock HH, Reyna H, Brady ST, Herz J. Reelin-mediated signaling locally regulates PKB/Akt and GSK-3ß. J Biol Chem. 2002; 277: 49958–49964.[Abstract/Free Full Text]

24. Alaupovic P, Lee DM, McConathy WJ. Studies on the composition and structure of plasma lipoproteins: distribution of lipoprotein families in major density classes of normal human plasma lipoproteins. Biochim Biophys Acta. 1972; 260: 689–707.[Medline] [Order article via Infotrieve]

25. Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T. Glycogen synthase kinase-3ß is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 2000; 151: 117–130.[Abstract/Free Full Text]

26. Gurney EG, Harrison RO, Fenno J. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct subclasses of large T antigen and for similarities among nonviral T antigens. J Virol. 1980; 34: 752–763.[Abstract/Free Full Text]

27. Marty I, Robert M, Villaz M, De Jongh K, Lai Y, Catterall WA, Ronjat M. Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle. Proc Natl Acad Sci U S A. 1994; 91: 2270–2274.[Abstract/Free Full Text]

28. Eggermont JA, Wuytack F, Verbist J, Casteels R. Expression of endoplasmic-reticulum Ca²⁺-pump isoforms and of phospholamban in pig smooth-muscle tissues. Biochem J. 1990; 271: 649–653.[Medline] [Order article via Infotrieve]

29. Arnould T, Vankoningsloo S, Renard P, Houbion A, Ninane N, Demazy C, Remacle J, Raes M. CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation. EMBO J. 2002; 21: 53–63.[CrossRef][Medline] [Order article via Infotrieve]

30. Auge N, Escargueil-Blanc I, Lajoie-Mazenc I, Suc I, Andrieu-Abadie N, Pieraggi MT, Chatelut M, Thiers JC, Jaffrezou JP, Laurent G, Levade T, Negre-Salvayre A, Salvayre R. Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein. J Biol Chem. 1998; 273: 12893–12900.[Abstract/Free Full Text]

31. Gomez MF, Stevenson AS, Bonev AD, Hill-Eubanks DC, Nelson MT. Opposing actions of inositol 1,4,5-trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle. J Biol Chem. 2002; 277: 37756–37764.[Abstract/Free Full Text]

32. Graef IA, Chen F, Chen L, Kuo A, Crabtree GR. Signals transduced by Ca²⁺/calcineurin and NFATc3/c4 pattern the developing vasculature. Cell. 2001; 105: 863–875.[CrossRef][Medline] [Order article via Infotrieve]

33. Yellaturu CR, Ghosh SK, Rao RK, Jennings LK, Hassid A, Rao GN. A potential role for nuclear factor of activated T-cells in receptor tyrosine kinase and G-protein-coupled receptor agonist-induced cell proliferation. Biochem J. 2002; 368: 183–190.[CrossRef][Medline] [Order article via Infotrieve]

34. Marx SO, Marks AR. Bench to bedside: the development of rapamycin and its application to stent restenosis. Circulation. 2001; 104: 852–855.[Free Full Text]

35. Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Hayashi E, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, Molnar F, Falotico R. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med. 2002; 346: 1773–1780.[Abstract/Free Full Text]

36. Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med. 2002; 8: 1249–1256.[CrossRef][Medline] [Order article via Infotrieve]

37. Tong J, Du GG, Chen SR, MacLennan DH. HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca²⁺ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine. Biochem J. 1999; 343(pt 1): 39–44.[Medline] [Order article via Infotrieve]

38. Scott S, O’Sullivan M, Hafizi S, Shapiro LM, Bennett MR. Human vascular smooth muscle cells from restenosis or in-stent stenosis sites demonstrate enhanced responses to p53: implications for brachytherapy and drug treatment for restenosis. Circ Res. 2002; 90: 398–404.[Abstract/Free Full Text]

39. Mayr U, Mayr M, Li C, Wernig F, Dietrich H, Hu Y, Xu Q. Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice. Circ Res. 2002; 90: 197–204.[Abstract/Free Full Text]

40. Guevara NV, Kim HS, Antonova EI, Chan L. The absence of p53 accelerates atherosclerosis by increasing cell proliferation in vivo. Nat Med. 1999; 5: 335–339.[CrossRef][Medline] [Order article via Infotrieve]

41. Madden TD, Chapman D, Quinn PJ. Cholesterol modulates activity of calcium-dependent ATPase of the sarcoplasmic reticulum. Nature. 1979; 279: 538–541.[CrossRef][Medline] [Order article via Infotrieve]

42. Hunter GW, Negash S, Squier TC. Phosphatidylethanolamine modulates Ca-ATPase function and dynamics. Biochemistry. 1999; 38: 1356–1364.[CrossRef][Medline] [Order article via Infotrieve]

43. Rogers TB, Gaa ST, Massey C, Dosemeci A. Protein kinase C inhibits Ca²⁺ accumulation in cardiac sarcoplasmic reticulum. J Biol Chem. 1990; 265: 4302–4308.[Abstract/Free Full Text]

44. Pedrosa Ribeiro CM, McKay RR, Hosoki E, Bird GS, Putney JW Jr. Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells. Cell Calcium. 2000; 27: 175–185.[CrossRef][Medline] [Order article via Infotrieve]

45. Bonev AD, Jaggar JH, Rubart M, Nelson MT. Activators of protein kinase C decrease Ca²⁺ spark frequency in smooth muscle cells from cerebral arteries. Am J Physiol. 1997; 273: C2090–C2095.[Medline] [Order article via Infotrieve]

46. Jaggar JH, Nelson MT. Differential regulation of Ca²⁺ sparks and Ca²⁺ waves by UTP in rat cerebral artery smooth muscle cells. Am J Physiol Cell Physiol. 2000; 279: C1528–C1539.[Abstract/Free Full Text]

47. Lacerda AE, Rampe D, Brown AM. Effects of protein kinase C activators on cardiac Ca²⁺ channels. Nature. 1988; 335: 249–251.[CrossRef][Medline] [Order article via Infotrieve]

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