Activation of EGF receptor by oxidized LDL

^a INSERM U-466 and Biochemistry Department, Institut Louis Bugnard, CHU Rangueil, Toulouse, France
^b Institute of Medical Biochemistry, Karl-Franzen Universität Graz, Graz, Austria

	ABSTRACT

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Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL exhibit a wide variety of biological effects resulting from their ability to interfere with intracellular signaling. The cellular targets and primary signaling events of oxLDL are unknown. We report that oxLDL elicit, in intact cells, tyrosine phosphorylation of the epithelial growth factor receptor (EGFR) and activation of its signaling pathway. This activation triggered by oxLDL was associated with derivatization of reactive amino groups of EGFR and was mimicked by 4-hydroxynonenal (4-HNE, a major lipid peroxidation product of oxLDL). Immunopurified EGFR was derivatized and activated in vitro by oxLDL lipid extracts and 4-HNE, thus indicating that 1) EGFR may be a primary target of oxidized lipids and 2) EGFR derivatization may be associated with activation. The reported data suggest that EGFR acts as a sensor for oxidized lipids. We therefore propose a novel concept of the mechanism by which oxidized lipids (contained in oxLDL or more generally produced during oxidative stress) are able to activate receptor tyrosine kinase and subsequent signaling pathways, resulting finally in a gain of function.—Suc, I., Meilhac, O., Lajoie-Mazenc, I., Vandaele, J., Jürgens, G., Salvayre, R., Nègre-Salvayre, A. Activation of EGF receptor by oxidized LDL. FASEB J. 12, 665–671 (1998)

Key Words: signaling • 4-hydroxynonenal • tyrosine phosphorylation

	INTRODUCTION

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LOW DENSITY LIPOPROTEINS (LDL) play a major physiological role in delivering cholesterol to peripheral cells and are involved in the genesis of atherosclerosis and subsequent cardiovascular diseases that constitute the most prevalent cause of mortality in Western countries (1). Oxidized LDL (oxLDL) are present in the arterial wall (2) and are thought to play a central role in atherogenesis (3). LDL oxidation is a progressive process leading to the formation of mildly oxidized LDL (defined by low content of lipid peroxidation derivatives) and, later, to extensively oxidized LDL (characterized by high levels of lipid peroxidation derivatives and severe apoB alterations) (4). OxLDL exhibit a wide variety of biological effects potentially involved in atherogenesis, such as altera~tions of 1) lipid metabolism (leading to foam cell formation), 2) gene expression (of adhesion molecules, heat shock proteins, cytokines, growth factors, coagulation proteins), 3) cell migration, motility, and contractility, 4) cell viability, 5) local immune response, and 6) vasomotor tone (4–6).

The biological responses triggered by oxLDL are associated with lipid peroxidation derivatives (reviewed in ref 7). These bioactive molecules carried by oxLDL may be regarded as `cellular saboteurs' (7) because they are able to induce various pathogenic intracellular signals leading to cellular dysfunction. OxLDL have been shown to interfere with various signaling pathways involving calcium (8), trimeric G-proteins and cAMP (9), phospholipase D (10), protein kinase C (11), ceramide (12), and MAP kinase cascade (13). To date, the primary molecular targets and the mechanisms of their activation by oxidized lipids remain largely unknown.

We investigated the possibility that oxLDL may trigger directly intracellular signaling in cultured vascular cells. Preliminary experiments in our laboratory have shown that oxLDL induced early tyrosine phosphorylation of proteins and activation of membrane-bound tyrosine kinases. One of the tyrosine-phosphorylated proteins was a 170 kDa membrane-bound protein, possibly the epithelial growth factor receptor (EGFR) (14–17).

The EGFR is a transmembrane receptor tyrosine kinase shared by several growth factors such as EGF, heparin binding EGF (HB-EGF), TGF-, amphiregulin, and betacellulin. EGFR is implicated in various biological processes such as cell proliferation or differentiation, and may be involved in the genesis or progression of atherosclerosis and a number of human malignancies.

EGFR activation is associated with the stimulation of its intrinsic tyrosine kinase, with autophosphorylation of its own tyrosine residues, and with phosphorylation of intracellular substrate proteins. Phosphotyrosines of the COOH-terminal domain of the EGFR may bind to SH2 domains of enzymatic or adaptor proteins, including phospholipase C-1 (18), GTPase-activating protein of p21ras (18, 19), syp phosphotyrosine phosphatase (or SH2-containing phosphotyrosine phosphatase: SH-PTP1D) (20, 21), p85 subunit of phosphatidylinositol 3-kinase (PI3K) (22, 23), shc (24), Grb2-Sos (25), and nck (26–28).

We report that oxLDL elicit, in intact cells, tyrosine phosphorylation and activation of the EGFR. EGFR activation by oxLDL was associated with derivatization of reactive amino groups of EGFR and was mimicked by lipid extracts from oxLDL and by 4-hydroxynonenal (4-HNE, a major lipid peroxidation product). Immunopurified EGFR was derivatized and activated in vitro by oxLDL lipid extracts and 4-HNE, thus indicating that 1) EGFR may be a primary target of oxidized lipids, and 2) EGFR activation may result from derivatization by 4-HNE. These reported data suggest that EGFR acts as a sensor for oxidized lipids and led us to propose a novel concept about the mechanism of activation of signaling pathways (gain of function) by oxidized lipids.

	MATERIALS and METHODS

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Chemicals
[-³³P]ATP was from ICN Biomedicals (Orsay, France), 4-HNE was from Tebu-Biomol (Le Perray en Yvelines, France), anti-EGFR antibodies were from Santa Cruz (Tebu, Le Perray-en-Yvelines, France), anti-phosphotyrosine protein antibody (anti-PTyr 4-G10, UBI) was from UBI (Euromedex, Souffelweyersheim, France), RPMI-1640 (containing Glutamax), penicillin, streptomycin, and fetal calf serum were from Gibco (Cergy-Pontoise, France), and acrylamide-4x/bisacrylamide-2x solution was from Bioprobe (Montreuil, France); other chemicals were from Merck (Darmstadt, Germany), Sigma (St. Louis, Mo.), or Prolabo (Paris, France).

Cell culture
The human endothelial cell line (CRL-1998 EC) was from American Type Culture Collection (Rockville, Md.) and bovine aortic smooth muscle cells (GM 08133A) were from the NIA Aging Cell Repository (Camden, N.J.). Cells were routinely grown in RPMI 1640 medium (Life Technologies-Gibco) containing 10% fetal calf serum (Biowhittaker, Gagny, France) and antibiotics, as described (29). All passages were made at a splicing ratio of 1:4. Twenty-four hours before LDL or 4-HNE incorporation, the standard medium was changed and replaced by a serum-free medium.

LDL isolation and oxidation
LDL from human pooled sera were isolated by sequential centrifugation and oxidized by two different methods: UV-C ir~radiation in the presence of 5 µM CuSO4 or cell-mediated oxidation (one night of incubation with nonconfluent CRL-1998 EC, as described in ref 29). The level of LDL oxidation was monitored by the formation of thiobarbituric reactive substances (TBARS), according to Yagi (30). The 4-HNE content of LDL was determined by high-performance liquid chromatography (HPLC), under the conditions of Esterbauer et al. (31). Briefly, 2 mg of native or oxLDL was extracted by the Folch procedure in the presence or absence of 1 µg of pure 4-HNE (Biomol-TEBU) as internal standard. Lipid extracts were dissolved in acetonitrile and analyzed by HPLC, using a Beckman Gold System equipped with a C18 column (250 x 4.6 mm from Bischoff Chromatography; elution with acetonitrile/water 50:50, v/v, 1 ml/min; detection at 220 nm). Under standard conditions, oxLDL used here contained 4–6 nmol TBARS, 6–10 nmol 4-HNE/mg apoB, and no major alteration of the apoB moiety (29).

Immunoprecipitation and Western blot analysis
After stimulation of subconfluent CRL 1998-EC monolayers by oxLDL or 4-HNE under the conditions indicated in the text, cells were washed in phosphate-buffered saline (PBS) containing 20 mM sodium fluoride, 20 mM sodium pyrophosphate, 1 mM orthovanadate, and 5 mM EDTA. Cells were then lysed with solubilizing buffer (50 mM Tris pH 7.4, 250 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 160 mM sodium fluoride, 2.5 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 2 µM pepstatin A, 10 µg/ml aprotinin, 1% triton X-100) for 30 min, on ice. Fifty micrograms of protein cell extracts (determined by the bicinchoninic acid method) were resolved by electrophoresis in a 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel, transferred onto nitrocellulose membrane (Hybond-C, Amersham), and probed with anti-PTyr or anti-EGFR antibodies. Bound proteins were detected by an ECL detection system (Amersham), using a peroxidase-coupled secondary antibody. EGFR was immunoprecipitated by incubating cell extracts (2–4 mg cell protein) with anti-EGFR overnight at 4°C. Anti-EGFR immunoprecipitates were recovered on protein G-sepharose (2 h incubation at 4°C), eluted by boiling in SDS-containing buffer, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, as indicated above (32, 33).

Derivatization of EGFR-free amino groups
The free amino group content was evaluated on EGFR immunoprecipitates obtained from CRL 1998-EC previously incubated without (control) and with EGF (10 nM for 20 min), native or oxLDL (200 µg apoB/ml for 3 h), or with pure 4-HNE (Biomol Res. Lab.) (100 nM for 3 h). Free amino groups were labeled with [³H]succinimidyl propionate (Amersham, 99.0 Ci/mmol) (10 µCi in borate buffer 0.5 M, pH 8.5, 15 min in an ice bath), an amine-reactive probe (34). The immunoprecipitates were washed three times in borate buffer, boiled in SDS-containing buffer, and EGFRs were resolved by SDS-PAGE. The 170 kDa bands were recovered and the radioactivity was determined by liquid scintillation counting.

Alternatively, EGFR was immunoprecipitated from unstimulated CRL 1998-EC and incubated for 10 min without (control) or with EGF, lipid extracts from native or oxLDL, or pure 4-HNE. After washing in borate buffer, the immunoprecipitates were labeled by [³H]succinimidyl propionate, resolved by SDS-PAGE, and the radioactivity was counted.

Detection of 4-HNE adducts was performed by using polyclonal antibodies anti-4-HNE-protein (K5-4412) (35) on an immunoblot of immunoprecipitated EGFR.

EGFR autophosphorylation and tyrosine kinase activity
EGFR was immunoprecipitated from CRL-1998 EC that was either unstimulated or preincubated with the different agents EGF, 4-HNE, and native or oxLDL lipid extracts (37°C, 15 min). EGFR autophosphorylation was evaluated by incubating the immunoprecipitates with 20 µM ATP containing 5 µCi of [-³³P]ATP (3000 Ci/mmol, Isotopchim) in phosphorylation buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MnCl₂, 10 mM MgCl₂, 10 µM NaVO₄, 0.2 % Triton X-100). After incubation (15 min at 37°C), the reaction was stopped by spotting an aliquot of the mixture on phosphocellulose membranes (Life Technologies) and the radioactivity was counted (32). EGFR tyrosine kinase activity was evaluated under the same conditions by phosphorylation of poly Glu-Tyr (33).

Binding experiments
The ability of oxLDL to compete with the binding and uptake of [¹²⁵I]EGF to CRL 1998-EC was determined according to Marikovsky et al. (36). Briefly, CRL 1998-EC (plated in 6-multiwell culture plates) were incubated with tracer amounts of [¹²⁵I]EGF (70.000 cpm/ml) (NEN) alone or in the presence of 200 µg/ml oxLDL, up to 60 min. After washing the cells twice in PBS containing 0.5% bovine serum albumin and once in PBS alone, the cell-associated radioactivity was counted (Minaxi gamma Packard Counter).

	RESULTS

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Oxidized LDL induce tyrosine phosphorylation of EGFR in cultured cells
Incubation of AG-08133A smooth muscle cells with oxLDL (200 µg apoB/ml) elicited a relatively early tyrosine phosphorylation of cell proteins. As shown in Fig. 1a, a 170 kDa protein labeled in response to oxLDL was intensely tyrosine phosphorylated in response to EGF, thus suggesting a possible identity with the EGFR. The identity of this 170 kDa protein (EGFR) was confirmed by immunoprecipitation and Western blot analysis ( Fig. 1).

View larger version (59K): [in this window] [in a new window]   Figure 1. Oxidized LDL induce the tyrosine phosphorylation of EGFR in vascular cultured cells. a) Time course of tyrosine phosphorylation induced by oxLDL (200 µg apoB/ml) in bovine smooth muscle cells. b) Time course of EGFR phosphorylation by oxLDL in bovine smooth muscle cells: subconfluent GM-08133A (NIGM, Camden, N.J.) preincubated for 24 h in serum-free RPMI 1640 were stimulated for variable periods of time with 200 µg apoB/ml of oxLDL or with 10 nM EGF for 20 min and lysed in solubilizing buffer. The cell lysates were analyzed by SDS-PAGE (7.5% gels) and transferred to nitrocellulose membranes. Immunoblots were probed with antiphosphotyrosine (4G10) and anti-EGFR antibodies and then developed by enhanced chemiluminescence. c) Time course of EGFR phosphorylation by oxLDL in human endothelial cells (CRL-1998, ATCC) under the same conditions as in panel b. d) EGFR phosphorylation of CRL-1998 EC pulsed for 3 h with increasing concentrations of oxLDL. e) Study of EGFR phosphorylation of CRL-1998 EC pulsed for 3 or 5 h with 200 µg apoB/ml of native LDL or LDL oxidized by two different techniques: by UV/copper or by cells, as previously described (29). f) Coimmunoprecipitation of EGFR with associated SH2 proteins in CRL-1998 EC pulsed with oxLDL for 1, 3, and 5 h. EGFR immunoprecipitates were probed with antiphosphotyrosine and anti-phospholipase C, an anti-PI-3 kinase 85 kDa subunit, and anti-SH-PTP2 antibodies.

Tyrosine phosphorylation of EGFR, induced by oxLDL, was time and dose dependent ( Fig. 1b–d). The EGFR tyrosine phosphorylation induced by oxLDL was sustained for several hours ( Fig. 1b). EGFR tyrosine phosphorylation was also dependent on oxLDL concentration in the culture medium, the maximal phosphorylation being observed with oxLDL concentrations higher than 100 µg apoB/ml ( Fig. 1d). We further examined whether EGFR tyrosine phosphorylation was dependent on the conditions of LDL oxidation. As shown in Fig. 1e, cell oxLDL were also able to induce EGFR tyrosine phosphorylation. In contrast, native (i.e., nonoxidized) LDL either were not active or were only poorly active.

Effective EGFR activation is associated with autophosphorylation and specific interaction with SH2-containing proteins such as phospholipase C1, SH-PTP2/syp, PI3K, GRB-2, and SHC, thereby inducing the signaling cascade of EGFR. We therefore investigated whether oxLDL-dependent phosphorylation of EGFR was also able to induce the recruitment and activation of such specific SH2-containing proteins. As shown by Fig. 1f, in CRL-1998 EC treated with oxLDL, EGFR coimmunoprecipitated with at least three target tyrosine-phosphorylated proteins: SH-PTP2/syp (69 kDa), PI-3 kinase p85 subunit (85 kDa), and phospholipase C (145 kDa). Moreover, concomitant with phospholipase C phosphorylation, PtdIns were hydrolyzed, as assessed by inositolphosphate and diacylglycerol release (data not shown). All these data strongly suggest that oxLDL are able to trigger sustained EGFR phosphorylation as well as activation of the downstream signaling pathway (15–17).

Phosphorylation of EGFR triggered by oxLDL is independent of any autocrine effect
As oxLDL have been shown to modulate gene expression of growth factors of the EGF family—namely, HB-EGF (37, 38)—we examined the possibility that oxLDL-induced EGFR tyrosine phosphorylation (observed under the experimental conditions used here) may result from an autocrine secretion of EGF, HB-EGF, or other members of the EGF family. Addition of a neutralizing anti-EGF antibody to the culture medium simultaneously with oxLDL did not inhibit the EGFR tyrosine phosphorylation triggered by oxLDL, whereas this neutralizing anti-EGF antibody was effective in inhibiting EGFR tyrosine phosphorylation triggered by EGF ( Fig. 2a). To investigate whether any EGF-like or other autocrine mediator may be involved in this EGFR tyrosine phosphorylation, the effect of a preconditioned medium was tested on reporter cells. CRL-1998 EC were `pulsed' for 1, 3, and 5 h with oxidized LDL (200 µg apoB/ml), and after washing were `chased' for 1 h in oxLDL-free basic medium. This preconditioned chase medium was then transferred to unstimulated `reporter' CRL-1998 EC. Under these experimental conditions, the preconditioned medium induced no significant tyrosine phosphorylation of EGFR ( Fig. 2b), which suggests that, in our experimental model system, EGFR tyrosine phosphorylation is probably independent of any autocrine mediator.

View larger version (58K): [in this window] [in a new window]   Figure 2. Phosphorylation of EGFR triggered by oxLDL is independent of any autocrine effect. a) Effect of a neutralizing anti-EGF antibody on the EGFR phosphorylation triggered by oxLDL. Left: CRL-1998 EC were stimulated by oxLDL (200 µg/ml) for 3 and 5 h in the absence or presence of 10 µM of neutralizing anti-EGF antibody. Right: controls were incubated for 20 min with 1 nM EGF in the presence or absence of anti-EGF. Immunoblots from lysates were made with anti-phosphotyrosine or anti-EGFR antibodies. b) Transfer of preconditioned medium. CRL-1998 EC were pulsed for 1, 3, or 5 h with 200 µg apoB/ml oxLDL. This medium was then discarded and cells were `chased' in fresh (oxLDL-free) medium for 1 h. This preconditioned medium was transferred to unstimulated (reporter) CRL-1998 EC for 30 min, and EGFR phosphorylation was examined as above. In controls, CRL-1998 EC were incubated for 30 min in the absence (C1) or presence (C2) of oxLDL. c) Lack of influence of oxLDL on the association (binding + uptake) of 125I-EGF to CRL-1998 EC. Cells were incubated with a tracer amount of 125I-EGF (70 000 cpm/ml) for variable periods of time (up to 60 min) in the absence or presence of 200 µg/ml oxLDL. Inset: competition between unlabeled EGF and 125I-EGF at 37°C for 30 min.

Finally, to examine the possibility that oxLDL may be associated with any EGF-like activity or may bind the EGFR, binding of radiolabeled [¹²⁵I]EGF to CRL-1998 EC was evaluated in the presence or absence of oxLDL. As shown in Fig. 2c, [¹²⁵I]EGF binding was independent of the presence of oxLDL. Moreover, experiments of coincubation of cells with oxLDL and EGF showed that oxLDL did not inhibit the tyrosine phosphorylation of EGFR induced by EGF (data not shown). These data strongly suggest that the mechanism of oxLDL-induced EGFR tyrosine phosphory~lation is probably independent of any EGF-like activity associated with oxLDL.

OxLDL and 4-HNE induce derivatization of EGFR-free amino groups
The biological activity of oxLDL is associated mainly with their content in lipid peroxidation products (7), some being highly reactive compounds able to bind and modify cellular proteins. For instance, 4-HNE, one of the major aldehydes formed during LDL oxidation, is able to react with primary amines of basic amino acids and with histidine, thus generating HNE protein adducts (39, 40). We hypothesized that similar derivatization of basic amino acids of EGFR by 4-HNE may occur during treatment of cells with oxLDL. The content of free reactive amino groups (determined using [³H]succinimidyl propionate, an amine reactive probe) has been evaluated on EGFR immunoprecipitates from intact CRL-1998 EC treated with EGF, native LDL, oxLDL, or 4-HNE. The EGFR-free reactive amino group content was significantly reduced in cells treated with oxLDL or 4-HNE, but not in cells treated with EGF and native LDL ( Fig. 3A).

View larger version (45K): [in this window] [in a new window]   Figure 3. OxLDL and 4-HNE induce the derivatization of EGFR-free amino groups. A) In situ, CRL-1998 EC were treated without (control) and with EGF (10 nM for 15 min), native LDL or oxLDL (200 µg apoB/ml for 3 h), or pure 4-HNE (100 nM for 3 h). Free amino groups of EGFR immunoprecipitates were labeled with [3H]succinimidyl propionate, as indicated in Methods. Then EGFR was resolved by SDS-PAGE and the radioactivity of the 170 kDa bands was counted. B) In vitro, EGFR purified by immunoprecipitation from unstimulated CRL-1998 EC was incubated for 10 min without (control) or with EGF (10 nM), lipid extracts from native LDL or oxLDL (containing 0.6 and 9 nM 4-HNE, respectively), or pure 4-HNE (10 nM). After washing, immunoprecipitates were labeled by [3H]succinimidyl propionate, resolved by SDS-PAGE, and the radioactivity was counted as described above. C) Detection of 4-HNE adducts by polyclonal antibodies anti-4-HNE-protein (K5-4412) on immunoblots of immunoprecipitated EGFR from CRL-1998 EC incubated under the same conditions as in panel A.

This led us to investigate whether lipid peroxidation products and 4-HNE were able to react directly with EGFR. In vitro incubation of EGFR (purified by immunoprecipitation) with lipid extracts from oxLDL or 4-HNE resulted in a decrease of the free reactive amino group content of EGFR, in contrast to EGF and native LDL ( Fig. 3B). These data strongly suggest that lipid peroxidation compounds contained in oxLDL are able to derivatize free reactive amino groups of EGFR in intact cells. This assumption was supported by direct detection of 4-HNE adducts on a Western blot of the immunoprecipitated EGFR by an anti-4-HNE protein antibody ( Fig. 3C).

4-HNE is able to trigger EGFR phosphorylation and activation
We further investigated whether 4-HNE, in addition to the formation of EGFR adducts, was able to induce EGFR tyrosine phosphorylation similarly to oxLDL. Incubation of intact CRL-1998 EC with 4-HNE (100 nM) induced a time-dependent EGFR tyrosine phosphorylation ( Fig. 4a). More strikingly, as shown by Western blotting ( Fig. 4b), in vitro incubation of immunoprecipitated EGFR with oxLDL lipid extract or 4-HNE induced a significant EGFR tyrosine phosphorylation (similar to that induced by EGF). Similarly, lipid extracts from oxLDL or 4-HNE induced both in vitro EGFR autophosphorylation (as shown by ³³P incorporation, Fig. 4c) and stimulation of the activity of the EGFR tyrosine kinase ( Fig. 4d). These data strongly suggest that derivatization of reactive amino groups of EGFR by 4-HNE was associated with activation of the intrinsic tyrosine kinase activity of EGFR and subsequent EGFR tyrosine phosphorylation.

View larger version (40K): [in this window] [in a new window]   Figure 4. Triggering of EGFR phosphorylation and activation by 4-HNE. a) CRL-1998 EC were incubated for various times with 100 nM 4-HNE; cell lysates were used for Western blotting of EGFR. b–d) In vitro autophosphorylation (b, c) and tyrosine kinase activation (d) of EGFR induced by EGF, 4-HNE, and oxLDL lipid extracts. EGFR purified by immunoprecipitation from unstimulated CRL-1998 EC was incubated for 10 min without (control) or with EGF (10 nM), with lipid extracts from native LDL or oxLDL (containing 0.6 and 9 nM 4-HNE, respectively), or with pure 4-HNE (10 and 100 nM). b) EGFR tyrosine phosphorylation was performed for 15 min in phosphorylation buffer as indicated in Methods. Then, SDS-PAGE and immunoblots were performed as described above. c) Same experiments as in panel b, in the presence of 5 µCi/assay of [-33P]ATP (3000 Ci/mmol, Isotopchim). After spotting on phosphocellulose membranes, the radioactivity was counted. d) EGFR tyrosine kinase activity was evaluated under the same conditions, but in the presence of poly Glu-Tyr.

	DISCUSSION

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Collectively, our results indicate that oxLDL induce EGFR tyrosine phosphorylation and effective activation of the EGFR signaling pathway, as shown by the association of SH2-containing proteins to tyrosine-phosphorylated EGFR. EGFR activation began at nontoxic concentrations of oxLDL (50 and 100 µg apoB/ml) or 4-HNE (10 and 100 nM). Activation of the EGFR signaling pathway may be involved in a broad range of biological effects of oxLDL.

The novelty of our results lies in the identification of 1) EGFR as a primary target of oxidized lipids, 2) a new mechanism of EGFR activation by lipid peroxidation products, and 3) 4-HNE as a possible mediator of EGFR activation. EGFR activation was associated with derivatization of EGFR amino groups in intact living cells as well as in vitro. This is consistent with previous reports demonstrating that oxidized lipids, particularly 4-HNE, are able to react with amino groups of lysine and histidine and with sulfhydryl groups of proteins or peptides, leading to the generation of stable HNE protein adducts with cellular proteins during oxidative stress (39, 40).

Various nonspecific factors, such as Salmonella (41), UV-C radiation (42–44), and H₂O₂ (45) are able to induce EGFR autophosphorylation independent of EGF binding. But EGFR autophosphorylation induced by UV-C is a transient event (42), whereas that induced by oxidized lipids is sustained for several hours, thus suggesting that different mechanisms probably are involved. H₂O₂ and UV radiations, via generation of reactive oxygen species (44), act presumably through the inhibition of phosphotyrosine phosphatases (PTPases), which in turn may enhance EGFR phosphorylation. In vitro experiments using immunopurified EGFR suggest a direct mechanism involving derivatization of EGFR by oxidized lipids and subsequent autophosphorylation and activation of the receptor (in these experiments, PTPases were not involved because they were inhibited by NaVO₄ in assays as well as in controls). In intact cells, however, it is not excluded that oxidized lipids may inhibit PTPases, thereby enhancing EGFR autophosphorylation in addition to the direct activation subsequent to derivatization.

The reported data support several relatively new concepts: 1) oxLDL act as local autoparacrine mediators (formed mainly in the vascular wall and acting on neighboring cells); 2) EGFR is a novel cellular target of oxLDL in addition to those previously reported (8–13), but to our knowledge this is the first primary target identified to date; 3) derivatization of EGFR by oxidized lipids elicits directly EGFR activation (i.e., is associated with a gain of function) in contrast to the general concept that protein modification by lipid peroxidation products results generally in a loss of function and cytotoxicity (39–40); and 4) oxidized lipids and 4-HNE therefore share several properties with signaling mediators, since they are short-lived (31), effective activators of signaling pathways and are active at low concentrations.

Finally, the poor specificity of protein derivatization by oxidized lipids allows us to predict that they may alter the function of various intracellular effectors and induce a broad spectrum of biological effects.

	ACKNOWLEDGMENTS

 
The authors thank Dr. F. Clemente for fruitful discussions, Dr. J. P. Jaffrézou for reading the manuscript, Mr. J. P. Basile, J. P. Estève, C. Mora, and J. C. Thiers for technical assistance, and the SNCF Laboratory for providing serum. This work was supported by grants from INSERM, MESR, University Toulouse-3, Conseil Régional Midi-Pyrénées, Fondation pour la Recherche Médicale, AFM to U-466 and from Austrian Research Council, and special research center "Biomembranes" project F-00710 to G.J.

	FOOTNOTES

 
¹ Correspondence: Biochimie et INSERM U-466 - IFR-31-CHU Rangueil, 1, avenue Jean Poulhès, 31403 Toulouse Cedex 4, France. E-mail: anne.negre-salvayre{at}rangueil.inserm.fr or salvayre{at}rangueil.inserm.fr

² Abbreviations: oxLDL, oxidized low density lipoproteins; 4-HNE, 4-hydroxynonenal, EGFR, epithelial growth factor receptor; HB-EGF, heparin binding EGF; HPLC, high-performance liquid chromatography; PI3K, phosphatidylinositol 3-kinase; TBARS, thiobarbituric reactive substances; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PTPases, phosphotyrosine phosphatases; SH-PTP1D, SH2-containing phosphotyrosine phosphatase.

Received for publication December 17, 1997. Accepted for publication January 21, 1998.

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