From the Departament de Biologia Cellular, Institut
d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),
Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain
and the
Institute for Medical Biochemistry and Molecular
Biology, Department of Molecular Cell Biology and the
Department of Internal Medicine, University Hospital
Eppendorf, 20246 Hamburg, Germany
Received for publication, February 24, 2003, and in revised form, March 7, 2003
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ABSTRACT |
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High density lipoprotein (HDL) stimulates
multiple signaling pathways. HDL-induced activation of the
mitogen-activated protein kinase (MAPK) pathway can be mediated by
protein kinase C (PKC) and/or pertussis toxin-sensitive G-proteins.
Although HDL-induced activation of MAPK involves Raf-1, Mek, and
Erk1/2, the upstream contribution of
p21ras (Ras) on the activation of Raf-1
and MAPK remains elusive. Here we examine the effect of HDL on Ras
activity and demonstrate that HDL induces PKC-independent activation of
Ras that is completely blocked by pertussis toxin, thus implicating
heterotrimeric G-proteins. In addition, the HDL-induced activation of
Ras is inhibited by a neutralizing antibody against scavenger receptor
type BI. We conclude that the binding of HDL to scavenger
receptor type BI activates Ras in a PKC-independent manner with
subsequent induction of the MAPK signaling cascade.
In peripheral cells many beneficial effects of high density
lipoproteins (HDL)1 on the
removal of cellular cholesterol are elicited by signal transduction pathways in which HDL receptors at the cell surface are
believed to transmit the signal to intracellular signaling proteins (1,
2). This concept of HDL receptor-mediated signaling was recently
supported by the identification of a PDZ-containing adaptor protein (3)
interacting with the cytoplasmic domain of the intensively studied HDL
receptor scavenger receptor type BI (SR-BI). More importantly the
SR-BI-dependent and HDL-mediated activation of endothelial
nitric-oxide synthase has been demonstrated (4, 5).
Several laboratories have examined the plethora of signaling responses
generated by the interaction of HDL with cells. The diversity of
HDL-mediated cellular responses can in part be explained by the
heterogeneity in the content of the particles (lipids, apolipoproteins,
and enzymes) as well as by the different HDL receptors possibly
involved. HDL triggers a variety of intracellular signaling events,
including activation of phosphatidylinositol- and
phosphatidylcholine-specific phospholipases C and D (PI-PLC, PC-PLC,
and PC-PLD), protein kinase C (PKC), mitogen-activated protein kinase
(MAPK), tyrosine kinase, and heterotrimeric G-proteins (6, 7) but also
production of cyclic AMP (cAMP), nitric oxide (NO), and ceramide (4)
and intracellular Ca2+ release. Both lipid and protein
components of HDL have been implicated in the activation of different
classes of cellular phospholipases and the mobilization of
intracellular calcium but also in the stimulation of mitogenesis in
vascular smooth muscle cells. In respect to the HDL-induced activation
of the MAPK pathway it was demonstrated that
G-protein-dependent signaling proceeds phosphorylation of
Raf-1 and Mek-1 (8-10). Indeed it was recently shown that
sphingosylphosphorylcholine and lysosulfatide in HDL3
particles interact with receptors of the endothelial differentiation
gene family. This leads to dual activation of signaling through
heterotrimeric Gi-proteins that in turn activates PI-PLC.
This G-protein-dependent activation of PI-PLC is inhibited
by pertussis toxin (PTX). Activation of PI-PLC results in the immediate
production of inositol 1,4,5-trisphosphate and diacylglycerol
with the subsequent activation of PKC.
It is believed that PKC plays a pivotal role regulating the signaling
cascade for the HDL-induced phosphorylation of the Raf-1/Mek/MAPK pathway (8, 11). However, a number of observations have demonstrated that HDL-induced activation of the MAPK pathway does not completely depend on PKC signaling. First, in fibroblasts down-regulation or
inhibition of PKC only partially (40-50%) blocks HDL-induced MAPK
activation (8). Second, in smooth muscle cells HDL-mediated activation
of MAPK requires a PKC-independent but PTX-sensitive pathway,
indicating the involvement of G-proteins (9). However, the potential
contribution of p21ras (Ras), which is one of
the best studied activators of the Raf-1/MAPK pathway, remains unclear,
and to date there is no evidence that the interaction of HDL with cell
surface receptors increases Ras activity leading to MAPK phosphorylation.
Reagents and Antibodies--
Nutrient mixture Ham's F-12,
glutathione, TPA, and PDGF were from Sigma (Madrid, Spain). Pertussis
toxin was from List Biological Laboratories Inc. Fetal calf serum was
purchased from Biological Industries. Peroxidase-labeled antibodies and
SDS-PAGE molecular weight markers were from Bio-Rad. Monoclonal
anti-pan-Ras was purchased from Oncogene Sciences. Polyclonal
anti-P-Mek and anti-MAPK (P-MAPK) were purchased from Cell Signaling.
Antibodies against phospho-MARCKS were from Cell Signaling, and PKC Cell Culture--
CHO cells were grown in Ham's F-12
supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in 5% CO2. For the measurement of
Ras, Raf, and MAPK activity (see below) cells were preincubated overnight in the presence or absence of TPA (500 nM), PTX
(0.1 µg/ml), or both.
Preparation of HDL and ApoA-I--
High density lipoproteins
(HDL3, density 1.125-1.21 g/ml) were isolated from the
plasma of normolipidemic volunteers by sequential density gradient
ultracentrifugation as described previously (12, 13). After preparation
HDL was stored in KBr at 4 °C and dialyzed extensively against
phosphate-buffered saline before use. ApoA-I was isolated from HDL
preparations as described previously (12).
Measurement of Ras Activation--
The capacity of Ras-GTP to
bind to RBD (Ras-binding domain of Raf-1) was used to analyze the
amount of active Ras (15). Cells (2 × 106) were
incubated for 3 min with HDL (40 µg/ml), purified apoA-I (15 µg/ml), PDGF (10 ng/ml), or TPA (500 nM). In some
experiments binding of HDL to SR-BI was inhibited by preincubation of
cells with anti-SR-BI as described previously (14). Cells were
harvested in lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 5 mM
MgCl2, 1% (v/v) Triton X-100, 5 mM NaF, 10%
(v/v) glycerol, 0.5% (v/v) 2-mercaptoethanol, 0.1 mM
Na3VO4, and protease inhibitors). After
centrifugation at 10,000 × g the protein concentration
of the cleared cell lysate was determined (16). Then cellular proteins (600 µg) were incubated for 2 h at 4 °C with
glutathione-Sepharose 4B beads precoupled with GST-RBD. Beads were
washed four times in lysis buffer, and bound proteins were solubilized
with Laemmli loading buffer and electrophoresed on 12.5%
SDS-polyacrylamide gels (17). Proteins were transferred and
immunoblotted using the anti-pan-Ras antibody.
Raf-1 Kinase Activity Assays--
To measure Raf-1 activity,
kinase assays following immunoprecipitation were performed essentially
as described previously (18). Cells (2 × 106) were
harvested on ice in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1% (v/v)
Triton X-100, 5 mM NaF, 0.1 mM
Na3VO4, and protease inhibitors and cleared as
described above. Supernatants (equalized for protein concentration)
were immunoprecipitated for 2 h at 4 °C with 2 µg of anti-Raf
precoupled with 20 µl of protein G-Sepharose (Pierce).
Immunoprecipitates were washed three times in buffer A (30 mM Tris, 0.1 mM EDTA, 0.3%
To identify a possible role of Ras in HDL signaling we analyzed
the HDL-induced activation of the MAPK pathway in CHO cells. Ras
activity was measured by GST-RBD pull-down experiments with cell
lysates from CHO cells incubated from 3 to 20 min with HDL3 (40 µg/ml). Similar to the PDGF-induced activation of Ras (data not
shown), incubation with HDL3 resulted in a strong induction of Ras activity in CHO cells. Production of Ras-GTP peaked after 3 min
of exposure to HDL and returned to basal levels at later time points
(Fig. 1a). Kinetics
of growth factor- and TPA-induced Ras-GTP production are
characterized by a peak of Ras activity immediately after addition of
activating agents (19). These findings demonstrate that HDL-induced
activation of Ras follows kinetics similar to those for receptor
tyrosine kinase (epidermal growth factor receptor)- or PKC
(TPA)-mediated stimulation of Ras in CHO cells (20). Similar to results
described in vascular smooth muscle cells (9), incubation of CHO cells
with HDL resulted in the downstream activation of the Raf-1/MAPK
pathway as shown by the immediate increase (70 ± 10%) of Raf-1
activity after 3 min of HDL exposure (Fig. 1b) and the
HDL-induced phosphorylation of Mek and Erk1/2 (Fig. 1, c and
d).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and c-Raf-1 were from BD Biosciences. Rabbit antiserum against the
extracellular domain of mSR-BI (KKB-1) was kindly provided by Karen
Kosarzky (14).
-mercaptoethanol, 10% glycerol, 0.1% (v/v) Triton X-100, 5 mM NaF, 0.2 mM Na3VO4) with decreasing amounts of NaCl (1 M, 0.1 M,
and salt-free) and incubated for 30 min at 30 °C in 20 µl of Mek
buffer (salt-free buffer A plus 10 mM MgCl2,
0.8 mM ATP, 6.5 µg/ml GST-Mek, and 100 µg/ml GST-Erk2).
The reaction was terminated by the addition of 20 µl of ice-cold stop
buffer (salt- and glycerol-free buffer A containing 6 mM
EDTA). Following centrifugation, 6 µl of the supernatant was
incubated for 15 min at 30 °C with 24 µl of MBP buffer (salt- and
glycerol-free buffer A containing 10 mM MgCl2, 0.1 mM ATP, 2.5 µl [32P]ATP, 0.5 µg/µl
myelin basic protein, and 0.16 µg/µl bovine serum albumin), and
then aliquots of 24 µl were loaded onto P81 sheets, washed three
times (20 min each) in 75 mM orthophosphoric acid, and counted.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effect of HDL on Ras and the MAPK
signaling pathway. a, CHO cells were incubated with
HDL3 (40 µg/ml) for the indicated time periods. Lysates
were subjected to RBD pull-down to analyze the amount of Ras-GTP.
b, Histogram showing the mean ± S.D. of Raf-1 activity
(18) from lysates of cells incubated with HDL3 for 0-30
min. c and d, HDL-induced activation of Mek and
Erk1/2 by Western blot analysis with phosphospecific antibodies for Mek
(P-Mek) and Erk (P-Erk1/2). Ras activity and
P-Mek were also analyzed in CHO cells incubated with free apoA-I (15 µg/ml) (a and c). An anti-pan-Ras antibody was
used to control for similar amounts of total Ras in the lysates.
To determine whether the major apolipoprotein of HDL3, apoA-I, is responsible for Ras activation, we compared HDL3 and purified lipid-free apoA-I (21). In contrast to the immediate increase of Ras-GTP levels in HDL3-incubated cells, addition of purified apoA-I to the culture medium had no effect on Ras-GTP or the P-Mek levels as compared with the control at any time point analyzed (Fig. 1, a and c). These findings indicate that lipid-free apoA-I does not activate Ras-GTP production.
Since HDL-induced activation of PKC could be responsible not only for
the downstream induction of the Raf-1/Mek/MAPK pathway but also the
activation of Ras, we studied the involvement of PKC in the activation
of Ras after HDL stimulation. Cells were preincubated overnight in the
presence or absence of TPA, a potent PKC inhibitor (8, 12). Inhibition
of PKC was confirmed by Western blot analysis of PKC (Fig.
2) and reduced phosphorylation of a PKC
substate, P-MARCKS (22) (data not shown). HDL-induced production of
Ras-GTP was clearly detectable even after down-regulation of PKC,
indicating that HDL-induced activation of Ras in CHO cells occurs
mainly via a PKC-independent pathway (Fig. 2).
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Nofer and co-workers (9) recently reported that HDL-induced activation
of the MAPK pathway involves PTX-sensitive G-proteins. To test the
possible involvement of G-proteins in HDL-mediated activation of Ras
and Erk1/2, RBD assays were performed with lysates from
PTX-preincubated CHO cells. Strong inhibition of HDL-induced activation
of Ras-GTP production and the MAPK pathway as shown by the reduced
phosphorylation of Mek and Erk1/2 proteins was observed upon
pretreatment with PTX (Fig. 3). Taken
together these findings indicate that HDL-mediated activation of Ras
requires G-protein-mediated signaling in CHO cells.
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SR-BI, which binds HDL particles with higher affinity than lipid-free apoA-I (23, 28), could be a candidate for HDL signaling. First, SR-BI is located in caveolae (24-27) and is responsible for HDL-mediated activation of endothelial nitric-oxide synthase (4, 5). Second and similar to the results described above, Yuhanna et al. (5) reported that only binding of native HDL to SR-BI is associated with induction of NO production, whereas binding of apoA-I had no effect, indicating that binding of HDL particles to SR-BI can promote signaling events. In addition, in CHO cells the incubation of HDL (50 µg/ml) does not lead to changes in intracellular Ca2+ levels in agreement with Smart and co-workers (4) (data not shown).
Thus to identify a possible role for SR-BI in HDL-mediated Ras
activation, CHO cells were preincubated with an antibody against the
extracellular domain of SR-BI that inhibits HDL binding to SR-BI (14).
Cells were then incubated with HDL3, and lysates were prepared
to determine Ras-GTP levels in RBD assays (Fig. 4). In these experiments we observed a
strong reduction of HDL-induced activation of Ras and reduced amounts
of phosphorylated Erk1/2 in CHO cells preincubated with the inhibitory
SR-BI antibody (Fig. 4a). These findings suggest that
interaction of HDL and SR-BI plays a crucial role in the activation of
Ras in CHO cells.
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It was previously shown that, in vascular smooth muscle cells treated with HDL, the inhibition of PKC did not interfere with the Raf-1 or the MAPK activity (9). However, it has been shown in fibroblasts that PKC may contribute in some extent in the HDL-mediated stimulation of MAPK (8). The demonstration in the present study that HDL activates Ras and the complexity and cross-talk of signal transmission prompted us to determine the possible involvement of PKC in the overall stimulation of Ras. Here we clearly demonstrate that when PKC was completely depleted, by the action of TPA, no changes in the amount of Ras-GTP (Ras activity) (Fig. 2) or in the P-Erk1/2 could be observed (Fig. 4b). Therefore, as a major step in this direction, we report here for the first time that HDL activates Ras through SR-BI in a PKC-independent manner.
To understand the HDL-mediated activation of the MAPK pathway, it is
essential to identify the key signal-transducing proteins at the cell
surface and/or the endosomal compartment that are activated by the
interaction of HDL with receptors. Mapping the specific location where
HDL-mediated activation of downstream molecules occurs will have
important implications in the understanding of the development of
cardiovascular disease.
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ACKNOWLEDGEMENTS |
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We thank Cristina López (Universitat de Barcelona) for generously providing GST-RBD protein and Prof. Ulrike Beisiegel for helpful discussions relating to this project.
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FOOTNOTES |
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* This work was supported by Ministerio de Ciencia y Tecnología Grant PM99-0166, Acciones Integradas Grant HA2002-0055, and grants from the Generalitat de Catalunya BE2002, the Deutsche Forschungsgemeinschaft, and the Studienstiftung des deutschen Volkes (to M. F. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Present address: Centre for Immunology, St. Vincent's Hospital, University of New South Wales, Sydney 2010, Australia.
** Recipient of an IDIBAPS fellowship.
§§ To whom correspondence should be addressed: Dept. de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain. Tel.: 34-93-402-1908; Fax: 34-93-402-1907; E-mail: enrich@medicina.ub.es.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.C300085200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; MAPK, mitogen-activated protein kinase; SR-BI, scavenger receptor type BI; PTX, pertussis toxin; CHO, Chinese hamster ovary; TPA, 12-O-tetradecanoylphorbol-13-acetate; GST, glutathione S-transferase; RBD, Ras-binding domain; MARCKS, myristoylated alanine-rich C kinase substrate; PI, phosphatidylinositol; PC, phosphatidylcholine; PL, phospholipase; Mek, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Erk, extracellular signal-regulated kinase; PDGF, platelet-derived growth factor; P-, phospho-.
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