Activation of Epithelial Growth Factor Receptor Pathway
by Unsaturated Fatty Acids
Nathalie Vacaresse,* Isabelle Lajoie-Mazenc,* Nathalie Augé, Isabelle Suc, Marie-Françoise Frisach,
Robert Salvayre, Anne Nègre-Salvayre
Abstract—Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in
pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for
atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the
epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids
triggered a time- and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs]
were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was
used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation
derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators.
OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through
coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR- deficient B82L and EGFR- transduced B82LK1 cell lines).
OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR,
thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and
Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is
sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor
for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition
may influence vascular wall biology. (Circ Res. 1999;85:892-899.)
Key Words: fatty acid n growth factor n mitogen-activated protein kinase n EGF receptor
N
on esterified fatty acids (NEFAs) are physiologically
liberated during postprandial lipolysis and are chronically elevated in plasma in various diseases (eg, obesity,
mellitus diabetes, ketoacidosis, hypertension), known risk
factors for vascular diseases and atherosclerosis.1,2 Saturated
fatty acids (FAs) are thought to be atherogenic, whereas
unsaturated FAs (UFAs), such as oleic acid (OA) and n-3
polyunsaturated FA (PUFA), are considered to be antiatherogenic,3 despite recent contradictory reports.4 The antiatherogenic effect of UFAs may result in part from their lowering
effect on LDL cholesterol,5,6 from their “anti-inflammatory”
effect on vascular cells (eg, UFAs inhibit the expression of
endothelial proinflammatory proteins7), and from their antithrombotic effects (for n-3 PUFA).8 The mechanisms of
action of NEFAs are only in part understood and are complex
because NEFAs are involved at various stages of cell biology—namely, membrane structure, cell metabolism, energy
production, and cell signaling.9,10 UFAs are able to modulate
the activity of various intracellular signaling pathways mediated by calcium, protein kinase C (PKC), mitogen-activated
protein kinases (MAPKs), and epithelial growth factor receptor (EGFR).9 –17
EGFR, now considered to be a critical crossroad of
multiple receptor pathways,18 is potentially implicated in
the regulation of cell migration, proliferation, or differentiation and may be involved in atherogenesis.19 EGFR is a
170- kDa transmembrane receptor tyrosine kinase that is
shared by several growth factors, such as EGF, heparinbinding EGF, tumor necrosis factor-a, amphiregulin, and
betacellulin.20,21 Moreover, EGFR activation is modulated
by various non specific factors, such as UV irradiation,22
H2O2,23 oxidized lipoproteins,24 UFAs, and their oxidation
derivatives.16,25,26
Ligand binding induces EGFR dimerization, stimulation of
its intrinsic tyrosine kinase, and autophosphorylation of its
own tyrosine residues. 20,21 Phosphotyrosines of the
C-terminal domain of EGFR are binding sites for SH2
domains of adaptors or enzymatic proteins, including phospholipase Cg1, GTPase-activating protein of p21ras (rasGAP), SHP2, p85 subunit of phosphatidylinositol 3-kinase,
Received July 12, 1999; accepted August 31, 1999.
*Both authors contributed equally to this study.
From INSERM U-466 and Department of Biochemistry, IFR-31, CHU Rangueil, Toulouse, France.
Correspondence Dr A. Negre-Salvayre, Biochimie INSERM U-466, CHU Rangueil, avenue Jean Poulhès, 31403 Toulouse cedex 4, France. E-mail
salvayre@rangueil.inserm.fr or anesalv@rangueil.inserm.fr
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
892
Vacaresse et al
SHC, Nck, c-cbl, and GRB2-Sos.27,28 The activation of
GRB2-Sos complex may in turn activate p21ras and the
kinase cascade leading to MAPK activation.28 MAPK or
extracellular-regulated kinase (ERK) (p44/ERK1 and p42/
ERK2) can also be activated through receptors for growth
factors, hormones or cytokines, or G protein-coupled receptors or in response to stress.29,30
The aim of this study was to investigate whether FAs are
able to activate EGFRs and whether they are active per se or
only after metabolic activation (eg, after the generation of
oxidized or other bioactive derivatives).
Our data show that (1) in intact living cells, UFAs
induce EGFR autophosphorylation and activation, and
subsequent MAPK activation; (2) UFA activity is related
to the degree of unsaturation but is, at least in part,
independent of FA metabolism; (3) in vitro, UFAs elicit
autophosphorylation of the immunopurified EGFR, thus
suggesting that EGFR may be considered a primary target
of NEFAs; and (4) EGFR acts a sensor for amphiphiles (or
for membrane fluidity changes).
Materials and Methods
Chemicals
FCS and culture reagents were obtained from GIBCO. [125I]EGF
(150 mCi/mg) was from New England Nuclear Research Products.
E nhanced chemiluminescence reagent (ECL Kit), nitrocellulose,
and autoradiography films were from Amersham Corp. FAs,
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC), and
general reagents were obtained from Sigma Chemical Co. Recombinant EGF and antibodies against EGFR (monoclonal SC101 and rabbit polyclonal), GRB2, SHP2, ERK1/ERK2 (MK12),
and SHC were from Santa Cruz/Peprotech/Tebu. Activated
MAPK was from Promega. Phosphotyrosine (4G10) was from
UBI/Euromedex.
Cell Culture and Cell Extracts
Human endothelial ECV-304 cells (American Type Culture Collection) were grown in RPMI-1640 containing 10% FCS. The murine
B82L parental fibroblasts (EGFR deficient) and B82LK1 cells
(transduced with wild- type EGFR),31,32 a generous gift from Dr M.
Weber (Charlottesville, Va), and SrcK2 cells, C3H-10T1/2 fibroblasts overexpressing kinase defective c-src (clone 430c-src),33 a
generous gift from Dr S.J. Parsons (Charlottesville, Va), were grown
as described by Wright et al32 and Wilson et al,33 respectively.
Before experiments, cells were starved overnight in 0.5% FCScontaining medium.
Immunoprecipitation, Western Blotting,
and Dimerization
After incubation, cells were scraped off, pelleted, and solubilized in
RIPA buffer for total extracts or in solubilizing buffer for immunoprecipitates, as previously used.24 Immunoprecipitation was performed with anti-phosphotyrosine or anti-EGFR antibodies, and
immune complexes, recovered on protein G/Sepharose, were solubilized in Laemmli’s buffer. Westerns blots were performed as
previously described.24
Dimerization studies were performed according to Van der Vliet et
al.34 After agonist treatment, cells were incubated with 10 mmol/L
EDAC for 40 minutes at 37°C, immunoprecipitated by (monoclonal)
anti-EGFR, resolved by SDS-PAGE (5% polyacrylamide), and
probed with (polyclonal) anti-EGFR.
[125I] EGF Binding Assays
Competition between OA and [125I]EGF binding was performed
according to Marikovsky et al.35 Cells were incubated with [125I]EGF
EGFR Activation by Fatty Acids
893
(70 000 cpm/mL, 30 pmol/L) and with or without 50 mmol/L OA and
then washed in PBS containing 0.5% BSA, and the cell-associated
radioactivity was determined (Minaxi; Packard). Nonspecific binding was determined on the basis of excess unlabeled EGF (10
nmol/L).
In Vitro EGFR Autophosphorylation and Tyrosine
Kinase Activity
EGFR autophosphorylation and EGFR kinase, assayed on immunoprecipitated EGFR with the use of poly-GluTyr and [g-33P]ATP as
substrates, were evaluated as described by Suc et al.24
MAPK Activity
MAPK activity of cells stimulated by OA was determined through
myelin basic protein (MBP) phosphorylation with [g-33P]ATP (0.2
mCi/assay), as previously described.36 Proteins were determined
according to the bicinchoninic method. Statistical analysis was
performed with the use of the Student t test.
Results
OA and PUFA Induce Tyrosine Phosphorylation
and Dimerization of EGFR and Recruitment of
SH2-Containing Proteins
Human ECV-304 endothelial cells were used as a model
because (1) endothelium is in direct contact with circulating
UFAs and (2) this cell line expresses EGFR.
The incubation of ECV-304 cells with OA induced
tyrosine phosphorylation of a 170-kDa membrane protein
(Figure 1) that was identified as EGFR through immunoprecipitation and immunoblotting. The OA-induced EGFR
phosphorylation began rapidly (2 minutes) and was sustained for $45 minutes (Figure 1A). EGFR phosphorylation increased progressively with OA concentration, apparently without saturation up to 100 mmol/L (Figure 1B).
In the presence of BSA (50 mmol/L), the OA-induced
EGFR phosphorylation was clearly visible with a molar
ratio (OA/BSA) of 1:1 and was more intense when the ratio
was .2 (Figure 1C). This suggests that the activity may
result from unbound OA.
EGFR- phosphorylating activity of FAs was dependent on
chain length and unsaturation. Short chain FAs were inactive,
and PUFAs were more effective (C20:5, or eicosapentaenoic
acid, and C22:6, or docosahexaenoic acid, were the most
effective) (Figure 1, D and E). In the present study, we used preferentially OA (although less potent than PUFA) because (1) OA
is the major circulating UFA and (2) we sought to understand
whether UFAs may be active per se or only through their
oxidation derivatives (eg, 13(S)-hydroperoxyoctadecadienoic
and epoxyeicosatrienoic acids, which are able to activate
EGFR).25,26 OA is only slightly oxidizable and does not generate
these oxidized lipids.
The EGFR- phosphorylating activity by 50 mmol/L OA or
arachidonic acid (AA) was grossly equivalent to 50 and 200
pmol/L EGF, respectively (Figure 1F). When OA was removed from the culture medium (cells were later incubated in
delipidated medium), EGFR autophosphorylation persisted
for 15 to 20 minutes after washout (data not shown). Similar
to EGF, OA and AA were also able to induce EGFR
dimerization, concomitant with autophosphorylation of the
receptor (Figure 2A). As shown in Figure 2B, OA was
effective in activating the EGFR pathway, as assessed by the
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November 12, 1999
Figure 1. OA and PUFA induce EGFR activation in human endothelial ECV-304 cells. Cells were starved for 16 hours in
RPMI-1640 containing 0.5% FCS and were incubated with FA
or EGF, under indicated conditions. Cell lysates were analyzed
by Western blotting (SDS-PAGE on 10% polyacrylamide gels)
and probed with anti-phosphotyrosine (P-tyr) or anti-EGFR. A,
Time course of EGFR tyrosine phosphorylation in cells incubated with OA (50 mmol/L) or EGF (10 nmol/L, used as positive
control) for indicated time (minutes). B and C, Dose-response of
EGFR tyrosine phosphorylation in cells incubated for 15 minutes
with OA (concentrations expressed as mmol/L) without (B) or
with (C) BSA (50 mmol/L). EGF (10 nmol/L) as positive control. D
and E, Influence of chain length, unsaturation, or both of FA on
EGFR tyrosine phosphorylation induced by FA (50 mmol/L, 15
minutes). (PUFAs: 18:1, oleic; 18:2, linoleic; 20:4, arachidonic;
20:5, n-3 eicosapentaenoic; 22:6, n-3 docosahexaenoic acids).
F, Comparison of EGFR tyrosine phosphorylation induced by
OA or AA (50 mmol/L each) and increasing concentrations of
EGF (as pmol/L).
recruitment of SH2-containing proteins SHP-2, SHC, and
GRB2.
OA-Induced EGFR Activation Elicits
MAPK Activation
The OA-induced activation of the EGFR signaling pathway
was associated with MAPK activation, as shown by tyrosine
phosphorylation of p42/p44 MAPK (Figure 3A) and activation of MBP kinase activity of immunoprecipitated MAPK
(Figure 3B). To investigate whether EGFR and MAPK
activations were causally related or independent events,
similar experiments were performed on genetically engineered B82L-derived cell lines expressing or not expressing
Figure 2. Dimerization of EGFR and recruitment of substrates
to EGFR activated by FAs. Cells, treated as described in legend
to Figure 1, were immunoprecipitated and analyzed by Western
blotting probed with indicated antibodies. A, Dimerization of
EGFR in murine (EGFR-transduced) fibroblasts B82K1 cells,
either unstimulated (Co) or stimulated for 15 minutes by OA or
AA (50 mmol/L), Tween-20 (TW, 100 ng/mL), or EGF (10 pmol/L
or 10 nmol/L). Then cells were incubated with EDAC, immunoprecipitated by (monoclonal) anti-EGFR, resolved by SDS-PAGE
(5% polyacrylamide gels), and probed with (polyclonal) antiEGFR. B, Coimmunoprecipitation of EGFR and of SH2containing proteins in ECV-304 endothelial cells incubated with
OA (50 mmol/L) or EGF (10 nmol/L) for 15 minutes. Immunoprecipitation was performed using monoclonal anti-EGFR under
previously described conditions,24 and immune complexes were
recovered, solubilized, and resolved by SDS-PAGE (10% polyacrylamide gels), blotted by indicated antibodies.
EGFR (parental B82L cells were EGFR deficient and transduced B82LK1 cells overexpressed EGFR)31,32 (Figure 3C).
As shown in Figure 3, D and E, OA (50 mmol/L, 15 minutes)
induced no significant MAPK activation in cells lacking
EGFR (parental B82L cells), whereas it elicited both EGFR
autophosphorylation and MAPK activation in B82LK1 cells
(expressing EGFR). Conversely, OA-induced MAPK activation was inhibited when EGFR autophosphorylation was
inhibited with genistein (Figure 4). These events were not or
were slightly influenced by the PKC inhibitor bisindolylmaleimide or phorbol-12-myristate-13-acetate–mediated down
regulation of PKC (Figure 4). Taken together, these data
suggest that the moderate OA-induced activation of the
EGFR signaling pathway is effective for the inducement of
MAPK activation (whereas classic PKC is apparently
dispensable).
The next experiments were designed to understand the
mechanism of the OA-induced EGFR. It was hypothesized
that OA may (1) trigger an autocrine secretion of EGF or
(2) interact (more or less directly) with and activate EGFR.
EGFR Autophosphorylation and Activation by OA
Is Independent of Any Autocrine Effect
In our experimental system, a role for autocrine secretion
of EGF or other diffusible mediators very likely will be
Vacaresse et al
EGFR Activation by Fatty Acids
895
Figure 4. Effect of tyrosine kinase and PK C inhibitors on EGFR
phosphorylation and MAPK activation induced by OA. After preincubation with genistein (Gen) (10 mmol/L, 1 hour), bisindolylmaleimide (BIM) (10 mmol/L, 1 hour), or phorbol-12-myristate13-acetate (200 nmol/L, 24 hours), ECV-304 endothelial cells
were incubated with OA (50 mmol/L) for an additional 15 minutes. Co indicates untreated control; EGF, positive control (10
nmol/L EGF, 15 minutes). Western blotting was performed with
antibodies raised against phosphotyrosine (P-tyr), EGFR, activated (act) MAPK, and total (tot) MAPK.
OA Triggers In Vitro EGFR Phosphorylation and
EGFR Kinase Activation
Because immunopurified EGFR can be activated in vitro by
EGF and by nonspecific “agonists,” such as oxidized lipids
and hydroxynonenal (4-HNE),24 we investigated whether OA
was also able to stimulate in vitro autophosphorylation of
immunopurified EGFR.
Figure 3. OA-induced EGFR activation leads to MAPK activation. A and B, ECV-304 endothelial cells, untreated (Co) or stimulated by OA (50 mmol/L, 15 minutes) or EGF (10 nmol/L, 15
minutes), were immunoprecipitated by anti-phosphotyrosine
(P-tyr) antibody. Immunoprecipitates were immunoblotted by
anti-EGFR or anti-ERK1/ERK2 antibodies (tot MAPK) (A) or used
for assaying MAPK activity (by MBP phosphorylation). C through
E, OA- induced MAPK activation is dependent on EGFR activation (and expression) in B82L (EGFR deficient) and B82LK1
(EGFR-transduced) fibroblasts. Cells were incubated for 15 minutes without (Co) or with 50 mmol/L OA or 10 nmol/L EGF. C,
Cells were immunoprecipitated by anti-EGFR antibody, and
Western blots were probed with anti-phosphotyrosine or antiEGFR antibodies. D, Cells lysated were resolved by Western
blots and probed by anti-activated MAPK (act MAPK) or tot
MAPK (anti-ERK1/ERK2 antibodies). E, Cells lysates were used
for MAPK determination (MBP phosphorylation), as in B.
*P,0.01 compared with control.
excluded because OA-induced activations of EGFR and
MAPK were not inhibited by anti-EGF antibody (in
contrast to that elicited by added exogenous EGF) (Figure
5, A and B) and because the transfer of preconditioned
medium (from cells pretreated with OA) triggered neither
EGFR phosphorylation nor MAPK activation (Figure 5C).
This conclusion (no requirement of autocrine EGF) was
consistent with the very rapid response (2 minutes) (Figure
1) and was confirmed by the lack of inhibition with
cycloheximide and with phenylmethylsulfonyl fluoride
(inhibitors of proteins synthesis and of pro-EGF processing, respectively)37 (data not shown).
Figure 5. Activation of EGFR and MAPK by OA is independent
of any autocrine effect. A and B, B82LK1 (EGFR-transduced)
fibroblasts were stimulated by OA (50 mmol/L, 10 minutes) or
EGF (1 nmol/L, 5 minutes) in presence or absence of 10 mmol/L
neutralizing anti-EGF antibody. A, Western blot probed by antiphosphotyrosine and anti-activated (act) MAPK antibodies. B,
EGFR kinase activity of EGFR immunoprecipitates determined
by poly-GluTyr phosphorylation. Values are mean6SEM of 3
experiments. *P,0.01. C, Transfer of preconditioned medium.
After preincubation without (Co) or with OA (50 mmol/L) or EGF
(10 nmol/L) for 15 minutes, B82LK1 cells were washed once in
PBS and incubated in fresh medium (MEM containing 0.5%
FCS) for an additional 15 minutes (preconditioned medium). This
preconditioned medium was transferred onto unstimulated
(reporter) B82LK1 cells for a 15- minute incubation. Then,
extracts from reporter cells were used for Western blotting and
probed with anti-phosphotyrosine and anti-activated (act) MAPK
antibodies (EGF-treated cells as positive control).
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Circulation Research
November 12, 1999
Figure 6. OA triggers in vitro tyrosine phosphorylation and activation of immunopurified EGFR. EGFR, immunopurified from
unstimulated B82LK1 (EGFR transduced) fibroblasts, was incubated in vitro without (Co) or with OA (50 mmol/L) or with EGF
(1 nmol/L) for 10 minutes. A, Assay was performed in phosphorylation buffer24 and Western blot was probed with antiphosphotyrosine (P-tyr) or anti-EGFR antibodies. B, EGFR
kinase activity determined by poly-GluTyr phosphorylation, as in
Figure 5B. Values are mean6SEM of 3 experiments. *P,0.01
compared with control.
As shown in Figure 6, OA (50 mmol/L) incubated in vitro
with EGFR (immunopurified from B82LK1 cells) induced
both EGFR tyrosine phosphorylation (Figure 6A) and activation of EGFR intrinsic tyrosine kinase (Figure 6B).
These data suggest that OA may interact with EGFR,
thereby activating it. This led us to investigate 2 possible
mechanisms of the interaction between OA and EGFR:
(1) through specific interaction at the binding site of EGF and
(2) through nonspecific interaction involving the amphiphilic
properties of OA.
Study of Mechanism of EGFR Activation by OA:
Analogy With Mild Surfactants
As shown in Figure 7, [125I]EGF (30 pmol/L) binding was not
altered by OA (50 mmol/L) (these EGF and OA concentration
induced a grossly similar EGFR autophosphorylation). This
suggests that OA does not interfere with the EGF-binding site
of EGFR.
Figure 7. OA does not compete with [125I]EGF binding to EGFR.
Cells were incubated with a tracer amount of [125I]EGF for variable periods of time (up to 60 minutes) in absence (F) or presence of OA (50 mmol/L), added to incubation medium either simultaneously ( f ) or 30 minutes before [125I]EGF (Œ).
Figure 8. EGFR activation by surfactants Tween-20 and Triton X-100. A and B, In vitro activation of EGFR by surfactants. A, Immunopurified EGFR (from B82LK1 cells, prepared
in absence of Triton X-100) was incubated for 20 minutes in
phosphorylation buffer without or with Triton X-100 (TX, 10
ng/mL), Tween-20 (TW, 100 ng/mL), OA (30 mmol/L), or EGF
(1 nmol/L). Then, Western blots were probed with antiphosphotyrosine (P-tyr) and anti-EGFR antibodies. B, Same
as in A but in presence of [33g]ATP. After spotting onto phosphocellulose membrane, EGFR radioactivity was determined.
*P,0.01 compared with control. C and D, ECV-304 cells
were incubated for 15 minutes in the presence of increasing
concentrations of Triton X-100 or Tween-20, and cell extracts
were immunoblotted with anti-phosphotyrosine (P-tyr) or antiEGFR antibodies.
Because OA is an amphiphilic compound with mild
surfactant properties (under the study conditions), we
examined whether other mild surfactants were also able to
activate EGFR. The two mild surfactants Triton X-100 and
Tween-20 were able to induce in vitro tyrosine phosphorylation of immunopurified EGFR (Figure 8, A and B), thus
suggesting that EGFR interaction with amphiphilic compounds induces activation of its intrinsic tyrosine kinase
(probably by eliciting conformation changes). Similar to
UFAs, these mild surfactants were also able to trigger
EGFR activation in situ (under non lytic conditions, as
assessed through trypan blue exclusion) (Figure 8, C and
D). These data suggest that EGFR may act as a sensor for
amphiphilic compounds, namely UFAs, under physiological conditions (and other mild surfactants under experimental conditions).
Discussion
AA and oxidized PUFAs induce EGFR activation,17,25,26 but
the mechanism of action is largely unknown, except for
Vacaresse et al
oxidized PUFAs, which inhibit protein tyrosine phosphatases
(PTPases) and thereby increase tyrosine phosphorylation of
cell proteins.25 In the present work, we investigated whether
other major UFAs were able to activate EGFR and sought to
identify their mechanism of action.
Because FAs are liberated and transported in the blood
flow, they are in contact with vascular wall cells and may
alter their physiology. This led us to use an endothelial cell
line (ECV-304) that exhibits a stable phenotype, does not
require exogenous growth factors, and expresses sufficient
EGFR to perform Western blotting. Similar results were
obtained with vascular smooth muscle cells and other cell
types when EGFR was expressed (these data are not reported
here to avoid redundant data).
The data reported here suggest that (1) EGFR is a primary
target for UFAs, (2) UFAs activate the EGFR signaling
pathway, (3) this UFA activity is correlated to their unsaturation degree and does not require FA oxidation, and (4) EGFR
may act as a sensor of amphiphiles and of membrane fluidity
changes. This sensitivity of EGFR to its microenvironment is
not a general property of all membrane receptor tyrosine
kinases, because OA triggered no significant activation of
platelet-derived growth factor and insulin receptors (data not
shown).
The OA-induced EGFR autophosphorylation is moderate
but is effective in activation of the EGFR signaling pathway
(ie, recruitment of SH2-containing substrates) and MAPK. In
genetically engineered B82L cells, EGFR expression is necessary for OA-induced MAPK activation (no MAPK activation in EGFR- deficient cells B82L), but PKC activation is
not required (because OA-induced MAPK activation is not
inhibited by PKC inhibitors). This is consistent with the
conclusions of Casabiell et al,38 but it cannot be excluded that
the OA-induced PKC activation may be effective in other cell
types.12–14
To investigate the molecular mechanism of the OAinduced EGFR activation, several mechanistic hypotheses
were considered: (1) autocrine secretion of EGF (or other
mediators able to activate EGFR), (2) oxidative stress, lipid
oxidation, or both, which in turn may induce EGFR activation, and (3) direct EGFR activation (with EGFR being a
primary target).
An autocrine secretion of EGF was not involved in the
OA-induced EGFR activation because OA-induced EGFR
autophosphorylation is very rapid (2 minutes), is not
blocked by cycloheximide (thus excluding de novo synthesis of EGF), is not inhibited by phenylmethylsulfonyl
fluoride or leupeptin (two inhibitors of pro-EGF processing)37 (data not shown), is not blocked by anti-EGF
antibody, and is not induced by the transfer of preconditioned medium.
Oxidative stress (induced by UV-C irradiation22 or
H2O223) and oxidized lipids16,25 or 4-HNE24 may activate
EGFR either directly24 or indirectly through PTPase inhibition.39 The short-term OA-induced EGFR activation is
probably not mediated through reactive oxygen species
(ROS) generation or PTPase inhibition because (1) OA did
not generate intracellular ROS, (2) antioxidants (probucol,
tocopherol, trolox) did not inhibit the short-term OA-
EGFR Activation by Fatty Acids
897
induced EGFR autophosphorylation, (3) OA-induced
EGFR autophosphorylation occurs in vitro on immunopurified EGFR independent of any cellular generation of
ROS, and (4) all of the in vitro assays on immunopurified
EGFR contained Na3VO4, an inhibitor of PTPase (thus
excluding a role for active PTPase in vitro). Moreover,
OA-induced EGFR activation is probably not mediated via
oxidation products of OA because OA is relatively resistant to autoxidation,40 is not a substrate for lipoxygenases,
and did not induce the formation of 4-HNE/protein adducts. However, it is not excluded that the relatively
sustained phase of EGFR activation (30 to 45 minutes)
may involve ROS generation, because EGFR activation
induces H2O2 generation, which plays a role in EGFR
pathway activation.41
Chen et al26 recently reported that epoxyeicosatrienoic acid
activates Src kinase (SrcK), which initiates a tyrosine kinase
cascade (involving EGFR). Although OA is poorly oxidizable
and cannot lead to epoxyeicosatrienoic acid formation, we
investigated whether SrcK may be involved with the use of
SrcK1 and SrcK2 (overexpressing a negative dominant
SrcK2) cell lines.33 OA induced EGFR autophosphorylation
in both SrcK1 and SrcK2 cells, but the basal and OAstimulated EGFR activations were higher in SrcK1 (data not
shown). This suggests that c-src is not strictly required for the
OA-induced EGFR activation, which is in agreement with the
in vitro OA-induced activation of immunopurified EGFR.
Therefore, non oxidized OA may directly activate EGFR, via
a mechanism different from that of epoxyeicosatrienoic
acid,34 but it is not excluded that in vivo, EGFR activation
induced by OA may be potentiated by c-src.42
Finally, in vitro experiments suggest that OA interacts
(probably directly) with an EGFR domain (different from the
EGF binding site), thereby activating it. Because of its
amphiphilic properties, OA may interact with hydrophobic
domains, such as with the transmembrane domain either
directly (in vitro) or after insertion in membrane lipid bilayer,
where it elicits changes in the membrane fluidity.43 This
hypothetical mechanism was supported by EGFR activation
induced by mild detergents (Tween-20 and Triton X-100),
which is in agreement with the results of Igarashi et al.44
Amphiphiles may alter the membrane fluidity, thereby inducing conformational changes and activation of EGFR. This
hypothesis is consistent with the data of Miloso et al,45 who
report that point mutation in the EGFR transmembrane
domain induce (probably through conformational change) a
mild constitutive activation of EGFR. Finally, the reported
data suggest that EGFR may act as a sensor for amphiphiles
and membrane fluidity changes. In vivo, it cannot be excluded that OA may also modulate the activity of an EGFR
ligand.
From a pathophysiological point of view, a local NEFA
concentration effective in the activation of EGFR may be
reached acutely during intravascular lipolysis of chylomicrons at the endothelial surface; during triglyceride lipolysis of adipocytes occurring during fasting, ketoacidosis,
or other conditions associated with increased lipolysis; or
during phospholipolysis by phospholipases during inflammation. Because the level of UFA (or the ratio of unsat-
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Circulation Research
November 12, 1999
urated to saturated FA) is largely dependent on diet
composition, the reported data point out a new nutritional
mechanism that regulates EGFR activity and subsequent
cell functions. The EGFR pathway may have have interplay with the other NEFA-activated signaling pathways,9 –17 which participate in the regulation of major
intracellular events, such as cell proliferation, migration
and adhesion, gene expression, glucose transport, and
cellular metabolism. EGFR plays a role in wound healing
and may be involved in repair processes, remodeling, and
fibrosis of the vascular wall in response to injury in normal
and atherosclerotic areas (where EGFR is highly expressed
in proliferating cells).46 This may help to stabilize the
plaque and may therefore in part account for the rather
favorable effect of OA intake. The effects of PUFA are
more complex because they are oxidizable and also lead to
eicosanoid formation, which exhibits various potent effects
on the vascular wall and the hemostasis equilibrium.
In conclusion, the reported data provide novel insight into
the mechanism of cis-UFAs as mediators triggering cell
signaling via EGFR, which apparently is a novel primary
target of UFAs, acts as a sensor for amphilic agents, and may
participate in vascular wall biology regulation.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Acknowledgments
This work was supported by INSERM, University Toulouse-3, and
European Community (Biomed-2 BMH4-CT98-3191). Drs
Vacaresse and Lajoie-Mazenc were recipients of fellowships from
the Association de Recherche contre le Cancer, Ligue contre le
Cancer, and Association Française contre les Myopathies). We thank
Dr M. Weber for the B82LK1 cells and Dr S.J. Parsons for the SrcK2
cells and for fruitful discussions.
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