Oxidized LDL-Induced Smooth Muscle Cell Proliferation Involves the EGF Receptor/PI-3 Kinase/Akt and the Sphingolipid Signaling Pathways
Objective— Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL.
Methods and Results— Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutant showed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominant-negative Δp85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of N,N-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in Δp85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway.
Conclusions— SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL.
- oxidized low-density lipoprotein
- phosphoinositide 3-kinase
- epithelial growth factor receptor
Smooth muscle cell (SMC) proliferation plays a critical role in the formation of the fibrous cap and plaque stability, whereas toxic events are potentially involved in necrotic core formation, endothelial cell injury, and plaque rupture.1,2⇓ Oxidized low-density lipoproteins (oxLDLs) exert various biological effects potentially involved in atherosclerosis development, such as chemotaxis, cell proliferation, or cytotoxicity.3–7⇓⇓⇓⇓ OxLDL-induced SMC proliferation involves the activation of the sphingomyelin-ceramide pathway that results in the generation of the mitogenic mediator sphingosine-1-phosphate.3,8⇓ On the other hand, oxLDLs elicit activation of tyrosine kinase receptors such as epithelial growth factor receptor (EGFR),9 which could play a role in cell proliferation. Recent studies provided evidence that phosphatidylinositol 3-kinase (PI-3K) is implicated in macrophage proliferation induced by oxLDL.5 PI-3K is activated by growth factors and peptide hormones10–12⇓⇓ and regulates multiple cellular processes required for cell survival and proliferation.13 A cross-talk between the sphingomyelin-ceramide and PI-3K pathways has been reported, but the data are relatively conflicting, because the exogenous permeant C2-ceramide and endogenous ceramide have been reported to activate the PI-3K pathway,14,15⇓ whereas under stress conditions, ceramide has been shown to inhibit PI-3K and suppress its survival signaling.16,17⇓ Recently, Burow et al17 claimed that cross-talk between PI-3K and sphingomyelinase pathways is a mechanism for cell survival/death decisions. To date, the mechanisms involved in PI-3K activation by oxLDL have not been identified. This study aims to clarify the hypothetical cross-talk between sphingomyelin/ceramide, EGFR, and PI-3K/Akt signaling pathways activated by oxLDL and their respective role in the balance between mitogenic and cytotoxic effects.
We report here the following findings in rabbit SMCs: (1) the oxLDL-induced activation of PI-3K/Akt pathway results mainly from EGFR activation and acts as an antiapoptotic pathway; (2) the sphingomyelin/ceramide/sphingosine-1-phosphate pathway does not cross-talk with the EGFR/PI-3K pathway but is involved in mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK) 1/2 activation; and (3) both signaling pathways act in cooperation during SMC proliferation induced by oxLDL.
[3H]Thymidine (5 Ci/mmol) was purchased from Amersham, horseradish peroxidase conjugated rabbit anti-sheep IgG from Chemicon, and [γ-33P]ATP (5 Ci/mmol) from Isotopchim. Sheep anti-Akt-1, mouse anti-PY-protein (4G10), or anti-p85 PI-3K subunit antibodies were purchased from Euromedex, anti–phospho-Akt from New England BioLabs, monoclonal PY20, rabbit anti-ERK1 from Santa Cruz Biotechnologies, and rabbit anti-active MAPK and anti-hemagglutinin (anti-HA) were from Promega. SYTO-13 was from Molecular Probes, and other reagents and chemicals were from Sigma. Human LDLs were isolated from pooled sera and oxidized by UV-C irradiation under the previously defined conditions.3
Rabbit femoral artery SMCs were grown under previously described conditions.3 The plasmids pRC (empty vector) and pRC-Δp85 (expressing a dominant-negative mutant form of p85)18 were kindly provided by Dr Len Stephens (Babraham Institute, London, UK). pCMV5 (empty vector) and pCMV5 Akt-KD (expressing a dominant-negative kinase dead form of Akt tagged with hemagglutinin HA epitope19) were provided by Dr Brian Hemmings (Friedrich Miescher Institute, Basel, Switzerland). SMCs were transfected using Lipofectamine (Life Technologies, Inc) according to the manufacturer’s instructions. Stably transfected cells, pRC SMCs, and Δp85 SMCs and pCMV SMC and Akt-KD SMC were selected in the presence of 0.4 mg/mL geneticin.
DNA Synthesis, Viability, and Toxicity
DNA synthesis was evaluated by [3H]thymidine incorporation,3 and toxicity was determined by the MTT (dimethylthiazolyldiphenyltetrazolium bromide) test.20 Necrosis and apoptosis were evaluated by fluorescence microscopy using SYTO-13 (0.6 μmol/L) and propidium iodide (15 μmol/L) as described.21
Determination of Akt and PI-3K Activities and Sphingolipids
Akt activity was determined after immunoprecipitation according to Zhou et al,22 using myelin basic protein (100 μg/sample) and 1 μCi [γ-33P] ATP (5 Ci/mmol) in HEPES buffer (20 mmol/L, pH 7.2) containing 10 mmol/L MgCl2, 10 mmol/L MnCl2, and 1 mmol/L DTT).
PI-3K activity was determined on phosphotyrosine protein immunoprecipitates according to Burgering et al,23 using 20 μg phosphatidylinositol in a 30-mmol/L HEPES buffer containing 200 μmol/L adenosine and 1 μCi [γ-33P] ATP/40 μmol/L ATP/30 mmol/L MgCl2 per assay. [33P]phosphoinositides were localized by autoradiography, scraped off, and counted by liquid scintillation. Sphingomyelin and sphingosine-1-phosphate were determined as reported.3,24⇓
Western Blot Experiments
After incubation with (or without) oxLDL, cells were washed with cold PBS, lysed, and subjected to SDS-PAGE, as previously described.9 Alternatively, EGFR was immunoprecipitated (1.5 mg cell protein) and analyzed by Western blot with anti-phosphotyrosine, anti-EGFR, and anti-p85 antibodies, as described.9
Total cellular RNA was isolated from SMCs using the guanidinium isothiocyanate–derived method RNABle. Membrane hybridization was carried out using a probe obtained by digestion of the Δp85 cDNA by BglII-ApaI digest. The probe was radiolabeled by random priming with [α-32P]dCTP using the oligolabeling kit (MBI Fermentas, Lithuania) and purified using the QIAquick Nucleotide removal kit (Qiagen, France). RNA was also probed with a similarly labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. Membranes were revealed by autoradiography (Kodak Biomax light at −80°C).
Statistical analysis was performed using analysis of variance (one-way ANOVA, Tukey test, SigmaStat software).
EGFR, PI-3K, and Sphingomyelin/Ceramide Pathways Are Involved in oxLDL-Induced SMC Proliferation
The oxLDL-induced DNA synthesis is evaluated as [3H]thymidine incorporation above the basal level (100%), which results from the mitogenic effect of low FCS (0.5%). This minimal serum concentration cannot be reduced, because the level of apoptosis of cultured SMCs increased rapidly in serum-free medium. Under optimal conditions, the sphingomyelin/ceramide pathway inhibitors D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) (20 μmol/L) and N,N-dimethyl-sphingosine (DMS) (2 μmol/L) inhibited by 83% and 100% the oxLDL-induced sphingosine-1-phosphate formation activation (online Table 1, available at http://atvb.ahajournals.org), in agreement with our previous report,24 and inhibited the oxLDL-induced DNA synthesis (Figure 1A, blacks bars). Because D-MAPP and DMS are not toxic per se and are only slightly toxic in the presence of oxLDL (Figures 1A through 1C), it was suggested that these inhibitors block DNA synthesis through inhibition of mitogenic signaling24 (and see below). Inhibitors of PI-3K (wortmannin and LY294002) or EGFR (AG1478) also inhibited the oxLDL-induced mitogenic effect (Figure 1A), but these inhibitors are nontoxic when used alone (Figures 1A and 1B, white bars), rendered oxLDL toxic (Figure 1B), and induced apoptosis (Figures 1B through 1D). In contrast, pertussis toxin (PTox), which has been implicated in the mitogenic effect of oxLDL,25 had no effect under our experimental conditions. This led us to study the relationship between the sphingolipid and PI-3K pathways and to investigate their respective role in oxLDL-induced SMC proliferation.
OxLDLs Trigger PI-3K Activation Through EGFR Activation
OxLDL (100 μg/mL) triggered EGFR tyrosine phosphorylation, sustained for at least 5 hours and maximal at 30 minutes (Figure 2A). The maximal activation was equivalent to that induced by 0.5 nmol/L EGF (data not shown). Coimmunoprecipitation experiments showed the recruitment of the p85 regulatory subunit of PI-3K to the phosphorylated EGFR (Figure 2B).26 Recruitment of p85 to EGFR was associated with a concomitant activation of PI-3K enzymatic activity, which was inhibited by the PI-3K inhibitor LY294002 (Figure 2C). As expected, the EGFR inhibitor AG1478 inhibited efficiently both the oxLDL- or EGF-induced EGFR phosphorylation, p85 recruitment to EGFR, and PI-3K activation. Pertussis Toxin, PD098059, and GF 109203X, inhibitors of Gi, MEK, and protein kinase C, respectively, had no effect. The reported data suggest that the oxLDL-induced PI-3K activation is subsequent to EGFR activation (EGFR/PI-3K pathway).
OxLDL-Activated PI-3K and Sphingomyelin/ Ceramide Pathways Do Not Cross-Talk
We investigated whether a relationship exists between EGFR/PI-3K and sphingomyelin/ceramide pathways activated by oxLDL. In Δp85 SMC, genetically engineered SMCs overexpressing a dominant-negative form of the p85 subunit, oxLDL, failed to activate PI-3K (Figure 3A) but triggered sphingomyelin hydrolysis (Figure 3B). In control cells, the PI-3K inhibitor LY294002, which strongly inhibited PI-3K activation (Figure 2E), did not inhibit sphingomyelin hydrolysis (Figure 3B) triggered by oxLDL. Conversely, mitogenic concentration24 of the short-chain permeant C2-ceramide did not trigger any activation of PI-3K (Figure 3C). Consistently, the sphingosine kinase inhibitor DMS (2 μmol/L) inhibited both DNA synthesis activation (see online Table 1, available at http://atvb.ahajournals.org) and sphingosine-1-phosphate generation but not PI-3K activation triggered by oxLDL (Figure 3D).
All these data suggest that PI-3K activation by oxLDL is not involved in sphingomyelin/ceramide pathway activation, the sphingomyelin/ceramide pathway is not implicated in PI3K activation, and oxLDLs activate independently EGFR/PI-3K and the sphingomyelin/ceramide pathways that do not cross-talk under the used conditions. This led us to investigate the precise role of each signaling pathway in the oxLDL-induced proliferation of SMCs.
The Sphingomyelin/Ceramide Pathway But Not PI-3K Is Involved in oxLDL-Induced ERK1/2 Activation
Because the mitogenic effect of oxLDL is mediated through ERK1/2 activation,3 we investigated the respective roles of PI-3K and sphingomyelin/ceramide pathways in MAPK activation. D-MAPP and DMS strongly inhibited MAPK activation induced by oxLDL, whereas PI-3K inhibitors wortmannin and LY294002 were inactive (Figure 3E). These data suggest that activation of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway and MAPK is the main mitogenic signal triggered by oxLDL (Figures 1A and 3⇑E) and is independent of the PI-3K pathway.
OxLDLs Induce Akt/PKB Activation and Survival Through PI-3K Activation
Because PI-3K inhibitors render oxLDL toxic to SMC (independently of any MAPK inhibition) (Figures 1C and 3⇑E), we hypothesized that PI-3K may activate a survival pathway (MAPK independent). This led us to investigate the possible involvement of the serine-threonine kinase Akt/PKB, a downstream antiapoptotic mediator of PI-3K. Akt was phosphorylated and activated in response to oxLDL. The time course of Akt phosphorylation, the inhibitory effect of LY294002, and the lack of Akt activation in Δp85-transfected cells are consistent with the hypothesis that the oxLDL-induced Akt activation is mediated through PI-3K activation (see online Figure I, available at http://atvb.ahajournals.org).
The crucial role of PI-3K/Akt to prevent the toxicity of moderate concentrations of oxLDL was clearly shown by the use of PI-3K inhibitors wortmannin and LY294002 and Δp85 SMC and Akt-KD SMC (expressing a dominant-negative form of inactive Akt), because the inhibition of PI-3K or Akt (see online Figure II, available at http://atvb.ahajournals.org) induced a dramatic decrease of cell proliferation and viability concomitant with a rise of apoptosis. Together, these data suggest that Akt/PKB is activated through PI-3K and is involved in SMC survival in response to oxLDL.
SMC proliferation in intimal area is a main feature of atherosclerotic lesions.1,2⇓ OxLDLs are thought to play a role in this process because they activate various signaling pathways leading to activation of MAPK and transcription factors involved in proliferation and survival.6 We recently reported that oxLDLs trigger ceramide generation, concomitant to sphingosine-1-phosphate formation,24 followed by activation and nuclear translocation of MAPK and cell proliferation.3 On the other hand, we also reported that EGFR is activated by oxLDL independently of any autocrine EGF secretion.9,27⇓ Martens et al5 reported that PI-3K is involved in macrophage proliferation triggered by oxLDL. The precise role of these signaling pathways, their interrelationship, and their respective involvement in SMC proliferation elicited by oxLDL are only partly understood.
We report here that the oxLDL-induced SMC proliferation requires the activation of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which triggers ERK1/2 activation and subsequent DNA synthesis, and the EGFR/PI-3K/Akt pathway, which is required for cell survival (see online Figure III, available at http://atvb.ahajournals.org).
In our model system, oxLDL-induced activation of the sphingomyelin/ceramide pathway is independent of PI-3K activation, as shown by the use of inhibitors and dominant-negative mutants. Conversely, the sphingomyelin/ceramide pathway does not contribute to PI-3K activation, as shown by the lack of effect of permeant C2-ceramide and of inhibitors of the sphingomyelin/ceramide metabolism. This was unexpected and suggests that these two signaling pathways are activated concomitantly by oxLDL but do not cross-talk, in contrast to the data obtained in other experimental systems in which cross inhibition between PI-3K/Akt and sphingomyelin/ceramide pathways has been observed.17,28⇓ But it may be noted that in these latter systems, ceramide generally triggers apoptosis, whereas the oxLDL-induced activation of the sphingomyelin/ceramide pathway is not involved in apoptosis29 but plays a role in SMC proliferation,3 after conversion of ceramide into sphingosine-1-phosphate,24 in agreement with the general view of Spiegel and Merrill.30 Finally, the oxLDL-induced EGFR activation is not required for sphingosine kinase activation (data not shown).31
Moreover, we did not observe any cross-talk between the oxLDL-induced activation of PI-3K and MAPK. These data are in contrast with those of Rakhit et al,32 who reported that PI-3K is involved in MAPK activation triggered by exogenous sphingosine-1-phosphate and Edg-1 but are in agreement with earlier studies with H2O2 that did not implicate PI-3K in MAPK activation but rather in survival.33,34⇓
The mechanism of the oxLDL-induced EGFR activation involves two successive phases, an early one (0.5 to 1 hour) resulting from EGFR derivatization by 4-hydroxynonenal issued from oxLDL9 and a second phase (3 to 5 hours) mediated by reactive oxygen species generated in cells treated by oxLDL.27 The experiments reported here (coimmunoprecipitation, effect of inhibitors, and dominant-negative mutants) support a role for EGFR in the PI-3K and Akt activation induced by oxLDL. This is consistent with previous reports indicating that reactive oxygen species induced by various agonists may activate Akt and enhance cell survival.34,35⇓ Moreover, our results with inhibitors of EGFR/PI-3K/Akt, which enhance the toxic effect of oxLDL, are also consistent with the observation that EGFR inhibition by blocking antibodies or specific inhibitors enhances the toxic effect of several cellular stresses, such as UV irradiation or H2O2 treatment.34,36⇓
This study provides evidence that oxLDL activates the EGFR/PI-3K/Akt pathway, which is required to prevent oxLDL-mediated apoptosis. The PI-3K dependence of Akt activation was demonstrated by the lack of Akt activation in Δp85-expressing SMCs (which did not proliferate and underwent apoptosis in the presence of oxLDL) and by the use of the PI-3K inhibitor LY294002, which prevented Akt activation by oxLDL in SMCs. Moreover, the major role of Akt in promoting cell survival and protection against oxLDL-mediated apoptosis was clearly assessed on SMCs transfected with a kinase-dead form of Akt, which is much more susceptible to the toxic effect of oxLDL than untransfected cells. These data are in agreement with the protective role of PI-3K/Akt against the oxLDL-induced apoptosis of macrophages37 and endothelial cells38 and with the general role of Akt in cell survival.39
Finally, it may be noted that some apparent conflicting data, oxLDL being able to promote cell proliferation or cell death, result from the dose-dependent dual effect of oxLDL that promotes proliferation at low and cell death at higher concentrations.40,41⇓ Moreover, the susceptibility of cultured cells may depend on the cell type, endothelial cells being generally more susceptible to oxLDL-induced toxicity than SMCs29 (and unpublished results).
In conclusion, the reported data suggest that oxLDL-induced SMC proliferation requires the concomitant activation of the EGFR/PI-3K/Akt pathway, which promotes cell survival and resistance against the toxic effect of oxLDL, and the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which evokes ERK1/2 activation and DNA synthesis.
The financial support of INSERM, Université Paul Sabatier, and Fondation pour la Recherche Médicale is acknowledged. The authors wish to thank Drs L. Stephens and B. Hemmings for providing the plasmids, Dr S. Parthasarathy for RFA SMC, J. Dumoulin, C. Mora, M.F. Frisach, and J.C. Thiers for technical assistance and Dr V. Gallet (SNCF laboratory, Toulouse) for providing human sera.
Received August 21, 2002; revision accepted September 30, 2002.
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