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J Biol Chem, Vol. 273, Issue 52, 35153-35160, December 25, 1998
From the § Dipartimento di Medicina Sperimentale,
Università di L'Aquila, Via Vetoio Coppito 2, L'Aquila 67100, Italy, In this report the molecular mechanism(s)
involved in the rapid and selective endocytosis of cell surface
glycoprotein CD4 induced by exogenous monosialoganglioside GM3 in human
peripheral blood lymphocytes have been investigated. Inhibition of the
GM3-induced CD4 down-modulation was observed in the presence of
specific protein kinase C (PKC) inhibitors. Scanning confocal
microscopy revealed the translocation and clustering on the cell
surface of PKC isozymes Gangliosides, sialic acid-containing glycosphingolipids, are
ubiquitous constituents of cell membranes and have been implicated in a
variety of biological events occurring at the cell surface (1).
Cellular gangliosides have been shown to function as receptors for
several different molecules and for bacterial toxins (2) and are
involved in the binding or release of transmitters (3), regulation of
cell cycle (4), cell differentiation (5), and oncogenic transformation
(6). Gangliosides show cell type- and cell differentiation-specific
expression patterns. In T lymphocytes, monosialoganglioside
GM31 represents the main
ganglioside constituent of the plasma membrane (72% of total
gangliosides), whereas monosialoganglioside GM1 is not detectable
(7). In a previous investigation, we identified GM3-enriched domains on
lymphocyte plasma membrane associated with the integral membrane
glycoprotein CD4 and its noncovalently linked Src family tyrosine
kinase p56lck, suggesting a functional role for GM3-enriched
domains in cell-cell interactions, antigen recognition, cell
activation and signal transduction (8).
Exogenous gangliosides rapidly and significantly bind to biological
membranes, modulating the expression and/or the activity of specific
surface receptors. Indeed, GM3 is able to down-regulate fibroblast
growth factor and epidermal growth factor receptor activities, through
the inhibition of tyrosine-kinase activity (9, 10); GM1 specifically
inhibits platelet-derived growth factor receptor activity and insulin
receptor-associated kinase activity (11) and enhances the action of
nerve growth factor by binding to Trk, the tyrosine kinase-type
receptor for nerve growth factor (12). Furthermore, exogenous GM1 or
binding and cross-linking of endogenous GM1 with B subunit of cholera
toxin induces early tyrosine phosphorylation of phospholipase C In human T lymphocytes, exogenous GM1 induced a selective
dose-dependent down-modulation of CD4 molecules on the
plasma membrane (17), reducing in vitro HIV-1 infectivity
(18). CD4 down-regulation on T cell plasma membrane is a well known
molecular event triggered by different stimuli, which, through
activation of PKC, induce the phosphorylation of serine residues in the
cytoplasmic tail of CD4 molecule and, consequently, the dissociation of
the CD4·p56lck complex (19). The most effective triggers for
CD4 down-regulation are PKC-activating phorbol esters (20). The
GM1-induced CD4 down-modulation, although involving dissociation of
CD4·p56lck complex, has been reported to be independent of
both CD4 serine phosphorylation and PKC activation (21). We previously
reported that also addition of exogenous GM3 induces
down-modulation of CD4 molecules in human T lymphocytes, with a CD4
redistribution on the cell surface, and clustering and internalization
via endocytic pits and vesicles, followed by intracellular degradation
(22). Interestingly, the CD4 endocytosis due to GM3 was more rapid
(5-15 min) than that reported for GM1 (2 h) (23). Therefore, the
specific molecular mechanism(s) triggered by GM3 await(s) further
investigation. This work has been undertaken to characterize the early
biochemical pathways upstream to the GM3-induced CD4 endocytosis in
human T lymphocytes. We presented evidence of PKC involvement, CD4
phosphorylation, and CD4·p56lck dissociation. The possible
mechanisms underlying these events have been also analyzed.
Cells--
Human peripheral blood lymphocytes (PBLs) were
isolated from fresh heparinized blood by Lymphoprep (Nycomed AS Pharma
Diagnostic Div., Oslo, Norway) density-gradient centrifugation and
washed three times in phosphate-buffered saline (PBS), pH 7.4. PBLs
were then incubated for 30 min at 4 °C with anti-CD4 monoclonal
antibody DAKO T4 (Dakopatts, Glostrup, Denmark). CD4+ cells
were separated using Dynabeads M-450 coated with affinity-purified sheep anti-mouse IgG covalently bound to the surface (Dynal, Oslo, Norway). CD4+ lymphocytes were about 80%, as detected by
cytofluorimetric analysis.
Flow Cytometry Analysis of GM3-treated Cells--
Cells (1 × 106 in 1 ml of PBS) were incubated in the presence of 50 nM staurosporine (Calbiochem, La Jolla, CA) (24), 50 µM bisindolylmaleimide GF109203X (Boehringer Mannheim,
Milano, Italy) (25), or 10 µM rottlerin (Calbiochem)
(26). After a 15-min incubation time, 50 µg/ml GM3 (kindly provided
by Fidia Research Laboratories, Abano Terme, Padova, Italy) or 50 µg/ml GM3-derived 3'-sialyllactose (BioCarb Chem., Lund, Sweden) or GM3-derived lactosyl-ceramide (Matreya, Inc., Pleasant Gap, PA) or
ceramide (Sigma), were added for 30 min at 37 °C. Alternatively, cells were incubated with 50 µg/ml GM1 or GT1b (Sigma) for 2 h, since only after this incubation time have been shown to induce a
complete CD4 endocytosis (21, 23). In parallel experiments, cells were
alternatively incubated in the presence of 10 µM PI-PLC inhibitor U73122
(1-(6-(17
Cells either untreated or treated with the different gangliosides in
the presence or in the absence of PKC inhibitors were stained directly
using fluorescein-conjugated monoclonal antibody OKT4 (Ortho
Diagnostic, Raritan, NJ), which recognizes the juxtamembrane epitope of
CD4, or mAb DAKO T4 (Dakopatts), which recognizes the distal epitope of
the NH2-terminal domain. After washing with PBS, cells were
fixed in 2% formaldehyde in PBS. Green fluorescence intensity was
analyzed on an EPICS profile flow cytometer (Coulter Electronics,
Hialeah, FL). Vital cells were gated on the bases of forward angle
light scatter and 90° light scatter parameters.
GM3 Incorporation and Extraction--
PBLs were incubated with
staurosporine, bisindolylmaleimide GF109203X or rottlerin, and then
with GM3, at 50 µg/ml/106 cells, as reported above. After
washing with PBS, containing 10% FCS and 0.1% trypsin to remove the
gangliosides not inserted in the membrane bilayer (30), lymphocytic
gangliosides were extracted according to the method of Svennerholm and
Fredman (31), with minor modifications. Briefly, untreated and treated
cells were extracted twice in chloroform:methanol:water (4:8:3, by
volume) and subjected to Folch partition by the addition of water to
give a final chloroform:methanol:water ratio of 1:2:1.4. The upper phase, containing polar glycosphingolipids, was desalted and purified of low molecular weight contaminants using 3-ml Supelclean LC-18 tubes
(Supelco Inc., Bellefonte, PA), according to the method of Williams and
McCluer (32).
The eluted glycosphingolipids were dried down and separated by high
performance thin layer chromatography (HPTLC), using Silica gel 60 HPTLC plates (Merck, Darmstadt, Germany), preactivated at 100 °C for
30 min. Chromatography was performed in chloroform:methanol:0.25% aqueous KCl (5:4:1, by volume).
Ganglioside standards GM3, GM1, GD1a, GD1b, and GT1b (Sigma) were
included in every HPTLC plate. Plates were air-dried and gangliosides
visualized with resorcinol, which specifically stains sialic
acid-containing glycosphingolipids (33).
Scanning Confocal Microscopy--
CD4+ lymphocytes
(1 × 106 in 1 ml of PBS) were incubated with 50 µg/ml GM3 for 5 min at 37 °C. GM3-treated and untreated cells were
then fixed with acetone/methanol (1/1; v/v) for 10 min at 4 °C.
Cells were soaked in balanced salt solution (Sigma) for 30 min at
25 °C and incubated for 20 min at 25 °C in the blocking buffer of
2% BSA in PBS containing 5% glycerol and 0.2% Tween 20. Cells were
then labeled with anti-PKC- Labeling of Cells with [32P]Orthophosphate and
Immunoprecipitation of CD4--
Cells were washed twice with
phosphate-free medium (150 mM NaCl, 5 mM
MgCl2, 5 mM KCl, 2 mM glutamine,
and 1.8 mM glucose in 10 mM Tris acetate, pH
7.4) incubated for 1 h (i.e. starved) and equilibrated
with 1 mCi of [32P]orthophosphate for another 1 h;
phosphorylation was then induced by adding GM3 for 5 and 15 min. Cells
were washed twice with ice-cold PBS, incubated at 4 °C for 10 min in
RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 10 mg/ml phenylmethylsulfonyl fluoride (PMSF), 50 µg/ml
aprotinin, 100 mM sodium orthovanadate), and then disrupted
by repeated aspiration through a 21-gauge needle. Lysates were cleared
by centrifugation at 15,000 × g for 30' at 4 °C and
protein concentration determined using Bio-Rad protein assay reagent.
Cell-free lysates normalized for proteins were incubated for 4 h
at 4 °C with anti-CD4 mAb (CBT4, kindly provided by Dr. Malavasi).
The same amount of anti-CD4 mAb was used in each experiment. Then,
protein A/G plus agarose was added for an overnight incubation at
4 °C. The mixtures were centrifuged and washed three times with 0.4 ml of the RIPA buffer. The pellets resuspended in loading buffer were
resolved on 10% SDS-PAGE under reducing conditions.
Immunoprecipitation--
Cells (107/10 ml) were
incubated with GM3, 50 µg/ml for 5 or 15 min at 37 °C, in
serum-free RPMI 1640. Where indicated, the cells were treated with
GF109203X or rottlerin, as reported above, before GM3 addition,
transferred to 0.45-mm nitrocellulose paper (Schleicher & Schuell,
Dassel, Germany). Blots were blocked with 10 mM Tris, pH
8.0, 0.15 M NaCl, 0.05% Tween 20, 10% nonfat dry milk,
for 1 h at 25 °C. After repeated washes, the filter was probed
with the following antibodies: anti-phosphoserine antibody (Sigma),
anti-CD4, monoclonal anti-p56lck (Santa Cruz Biotechonology) or
anti-mouse serum under the same conditions. Bound antibodies were then
visualized with a polyclonal horseradish peroxidase-conjugated
secondary antibody diluted 1:3000, followed by incubation with the
Enhanced Chemiluminescence Western blotting detection reagent (Amersham
Corp.). Manufacturer-specified protocols were used to strip the
membrane (ECL manual, Amersham) to reprobe with each antibody.
Phosphatidylinositol-specific Phospholipase C (PI-PLC) Activity
Assay--
Cells (1 × 106) were incubated with GM3
or GM1 (50 µg/ml) for different time periods (1-20 min) at 37 °C
in RPMI 1640. Stimulation was stopped by centrifugation at 500 × g for 1 min at 4 °C. The cell pellets were resuspended in
50 mM Tris-HCl buffer, pH 8.5, containing 10 µM PMSF, 100 µM bacitracin, 1 mM benzamidine, 1 µM aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, and 5 µg/ml soybean trypsin
inhibitor. Cells were lysed by sonication with a cell sonifier
(Vibracell; Sonic & Materials Inc., Danbury, CT). Protein
concentrations were determined using a Bio-Rad protein assay. PI-PLC
activity in cell extracts was determined in vitro by its
ability to hydrolyze 14C-labeled PI vesicles to generate
diacylglycerol (DAG) and/or monoacylglycerol (MAG).
Vesicles were obtained by sonicating 25.5 mCi/mmol phosphatidylinositol
L- Phospholipase A2 (PLA2) Activity
Assay--
Cells (1 × 106) were incubated with GM3
or GM1 (50 µg/ml) for different time periods (1-20 min) at 37 °C
in RPMI 1640. Where indicated, the cells were treated with
AACOCF3 (10 µM) or PD098059 (50 µM) for 1 h, before GM3 or GM1 addition. Incubation
was stopped by centrifugation at 500 × g for 1 min at
4 °C. The cell pellets were resuspended in 50 mM
Tris-HCl buffer, pH 8.5, containing 10 µM PMSF, 100 µM bacitracin, 1 mM benzamidine, 1 µM aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin,
and 5 µg/ml soybean trypsin inhibitor. Radiolabeled PC vesicles were
prepared by sonicating the radiolabeled phospholipid
(L- PKC Involvement in GM3-induced CD4 Endocytosis--
In agreement
with our previous results (22), incubation of PBL with GM3
was followed by a significant decrease of CD4 staining on
both epitopes recognized by DAKO-T4 (Fig.
1, A and B) and
OKT4 (data not shown). In order to assess the specificity of the
GM3-induced endocytosis, we treated the cells with GM3-derived
3'-sialyllactose, a simple carbohydrate structure of GM3 lacking the
ceramide moiety (Fig. 1C), with GM3-derived
lactosylceramide, or ceramide (data not shown). In these conditions,
CD4 down-modulation did not occur. These results clearly indicate that
GM3 effect relies on the integrity of the molecule with its lipid
moiety linked to sialylated oligosaccharide. We also observed that
exogenous GT1b (data not shown) or GM1 (Fig. 1D) exerted a
complete down-modulation of CD4 surface expression after 2 h (Fig.
1D), as already reported (17, 21, 23).
In order to ascertain a possible PKC involvement in the CD4
down-modulation consequent to GM3 membrane insertion, we performed cytofluorimetric analysis of the CD4 surface expression in the presence
of different PKC inhibitors. We analyzed the GM3 effect in the presence
of staurosporine, a nonspecific kinase inhibitor (24);
bisindolylmaleimide GF109203X, a highly selective PKC inhibitor (25);
or rottlerin, which inhibits selectively the Ca2+-independent isozyme PKC-
Next, we investigated the PKC translocation from the cytosol to the
plasma membrane in GM3-treated CD4+ cells. This change of
localization pattern is generally associated with enzyme activation
(35). As shown in Fig. 2A,
after 5-15 min of treatment with GM3, translocation of PKC- GM3 Incorporation--
To exclude the possibility that the PKC
inhibitors could modify GM3 incorporation in the cell plasma membrane,
we analyzed the ganglioside pattern of lymphocytes incubated with GM3
for 30 min at 37 °C in the presence or in the absence of
staurosporine, bisindolylmaleimide GF109203X, or rottlerin. In Fig.
3 is reported the HPTLC analysis of
lymphocyte ganglioside pattern, showing the GM3 cell membrane
incorporation, either in the absence (Fig. 3, lane
d), or in the presence of bisindolylmaleimide GF109203X (Fig. 3, lane e), without modification of its
RF (relative mobility). The ganglioside pattern
of untreated lymphocytes is shown in Fig. 3 (lanes
b and c, respectively). Similar findings were
obtained with the other inhibitors under investigation (data not
shown).
CD4 Phosphorylation and Dissociation from p56lck
following GM3 Treatment--
The results suggesting a PKC involvement
on GM3-induced CD4 endocytosis led us to examine the ganglioside effect
on CD4 phosphorylation.
Human PBLs, after labeling for 2 h with
[32P]PO4, were incubated for 5 and 15 min in
the presence or in the absence of GM3 and then lysed as reported above
and immunoprecipitated with anti-CD4 mAb. The autoradiograms of the SDS
gel of these immunoprecipitates revealed that GM3 exposure induced a
significant increase of CD4 phosphorylation as early as after 5 min of
treatment (Fig. 4A).
Western blot analysis, performed using anti-phosphoserine antibody,
showed that the CD4 phoshorylation was on the serine residues (Fig.
4B). In order to better investigate the PKC involvement in
the CD4 phosphorylation, we analyzed the effects of rottlerin or GF
109203X on GM3-induced CD4 phosphorylation. PKC inhibitors were able to
totally prevent GM3-induced serine phosphorylation of CD4 molecule. In
Fig. 4C, the densitometric analysis of the experiments shown
in Fig. 4B is reported. After 5 min, GM3 induced a 5-fold
increase of CD4 serine phosphorylation level, which significantly decreased after 15 min. In the presence of PKC inhibitors, CD4 serine
phosphorylation returned to control levels (Fig. 4B). The results obtained in the presence of the specific PKC-
Since CD4 phosphorylation and endocytosis are related with the
dissociation of p56lck, it was of interest to further examine
the CD4·p56lck complex, over the time course of GM3
treatment. The results clearly indicated a p56lck dissociation
from CD4, which was evident as early as after 5 min of incubation with
GM3 (Fig. 4D). Specificity of anti-p56lck binding
was confirmed by testing an anti-mouse serum that did not cross-react
with p56lck (Fig. 4D, lane
d).
cPLA2 Activation following GM3 Treatment--
In the
attempt to characterize the early biochemical pathway(s) induced by GM3
treatment in T human lymphocytes, we carried out a series of
experiments to assess the activity of second messenger-generating enzymes, such as PI-PLC and cytosolic PLA2.
To address the possibility of GM3 inducing PI-PLC activation, an
in vitro determination of enzymatic activity using
radiolabeled PI vesicles and TLC analysis of reaction products was
performed. Whole cell extracts were prepared from either GM3-treated or
-untreated cells after different incubation times (1-20 min) and
incubated with radiolabeled PI vesicles. No DAG and/or MAG (data not
shown) were detectable by TLC analysis of PI hydrolysis products,
indicating the absence of PI-PLC activity (data not shown).
To assess the possibility of GM3 inducing cPLA2 activation,
extracts from GM3-treated or -untreated cells were assayed for the
in vitro measurement of enzymatic activity by analyzing AA release from radiolabeled AA-PC vesicles. The time course of
cPLA2 activity (Fig. 5)
revealed a rapid AA release from PC vesicles, which was maximal at 1 min and declined thereafter. The observed AA generation could be
attributed to cPLA2 activity, since AACOCF3, a
specific inhibitor of cPLA2 (27), totally inhibited PC
hydrolysis. Parallel experiments performed to compare GM3 and GM1 on
cPLA2 activation confirmed the different activity of the
two gangliosides under test (Fig. 5).
cPLA2 Involvement in GM3-induced CD4 Endocytosis and
PKC- MAP Kinase Involvement in GM3-induced CD4 Endocytosis--
Several
studies have shown a role for ERK in the phosphorylation and activation
of cPLA2 (36, 37). To investigate whether GM3-induced
cPLA2 activity was under the control of ERK, we utilized the synthetic MEK inhibitor PD 098058, which is known to specifically prevent MEK-1 activation without affecting the activity of other kinases (29). T lymphocytes stimulated with GM3 either in the presence
or in the absence of PD098058 showed that PD098058 completely inhibited
both GM3-induced CD4 down-modulation (Fig.
7A) and AA release from PC
vesicles (Fig. 7B). These results suggest that GM3 leads to
ERK activation, which in turn is relevant to GM3-induced cPLA2 stimulation.
The aim of this study was to further elucidate our previous
observation that the treatment of lymphocytes with GM3 induce CD4
redistribution, clustering, and internalization via endocytic pits and
vesicles (22). Ganglioside-induced CD4 down-modulation has also been
reported following treatment with GM1 and shown to trigger a
PKC-independent signal responsible for the dissociation of
CD4·p56lck complex (21). Primarily, we focused on the
different kinetics of the GM3-induced effects. Unlike GM1, which exerts
its effect after 2 h (23), GM3 induces CD4 internalization as
early as after 15 min (22), an observation consistent with the
hypothesis that this molecule triggers peculiar signaling pathway(s).
CD4 down-regulation on T cell plasma membrane has also been observed in
several functional conditions, such as the antigenic trigger of T-cell
receptor complex, the antibody cross-linking of the complex (38, 39),
the exposure of CD4+ T cells to phorbol myristate acetate
or phorbol dibutyrate (20). All these stimuli activate PKC isozymes
that cause phosphorylation of the serine residues within the CD4
intracellular domain and thereby lead to the dissociation of
CD4·p56lck complex. Interestingly, the abrogation of
GM3-induced CD4 endocytosis by different PKC inhibitors suggests that
the activation of this enzyme play a key role in this event. A further
support to this hypothesis derives from the analysis of cell
redistribution of PKC isozymes from cytosol to plasma membrane. This
molecular event is generally associated with specific PKC activation
(19). After GM3 treatment, the Ca2+-independent PKC
isozymes PKC- Among the possible upstream pathways triggered by exogenous GM3, the
observation of the activation of cPLA2 with AA release as
early as after 1 min suggests a role for this enzyme in GM3-induced PKC
activation and CD4 down-modulation. The hydrolysis products of
cPLA2, AA and lyso-PC, are more frequently recognized as
lipid second messengers and though to regulate a number of potential molecular targets. AA and lyso-PC are well known molecules directly responsible for the regulation of key intracellular players such as PKC
isozymes (42). Our results showing that both AA generation and PKC
translocation are blocked by AACOCF3, an inhibitor of cPLA2, associate cPLA2 and PKC activations as a
result of treatment with GM3. Moreover, it was of interest to analyze
the molecular signals triggering GM3-induced cPLA2
activation. Our findings indicate that cPLA2 activation may
be mediated by ERKs (36, 37), a downstream effector of a Ras-regulated
cyoplasmic kinase cascade, which includes RAF-1 and MAP kinase kinase
(43). Since in our cell system no activation of PI-PLC was observed,
this result seems to be in agreement with the finding that U73122, a
selective inhibitor of PI-PLC, does not affect GM3-induced CD4 down-modulation and PKC- Exogenously added GM3 may have biological effects similar to
endogenously synthesized ones. Specific changes in GSL pattern have
been observed during cell growth and differentiation, suggesting that
the regulation of their expression on the cell surface represents a
precise event. An increase in the activity of GM3 synthase in leukemia
cell lines HL-60 or CEM has been observed during differentiation induced by treatment with phorbol ester (49, 50) and the increase of
endogenous GM3 expression has been reported following lymphocyte activation with phytohemagglutinin, which activates sialyltransferase, leading to an increased GM3 biosynthesis (51). Moreover, we recently
reported a 4-fold increase of GM3 concentration at the surface of
HIV-infected lymphocytes, which appears to be consistent with the
increase of GM3 concentration observed in human normal lymphocytes
after incorporation of exogenous GM3 (22). These results suggest that
GM3 may play a role in the CD4 down-modulation occurring during HIV
infection (52, 53). It is noteworthy that gangliosides modulate a
number of immune functions (54) and the down-regulation of CD4
expression may be essential for normal T responses. Indeed, CD4 is
associated with the T cell receptor/CD3 complex during T cell
activation (55, 56) and can induce early signaling events following T
cell receptor triggering. Interestingly, the coreceptor function
mediated via CD4 depends on its association with p56lck (57),
which appears, in our experimental conditions, to be regulated by GM3 treatment.
The possibility of a functional GM3-CD4 interaction is strongly
supported by our previous observations that lymphocyte surface endogenous GM3 is present in relatively detergent-resistant
microdomains of the membrane where some proteins, including CD4 and
p56lck are highly enriched (8). In particular, CD4 molecules
are closely associated with GM3, as demonstrated by the observation that anti-CD4 co-immunoprecipitates GM3 molecules (8). These plasma
membrane microdomains of distinct proteins (Src family, PKC, and CD4)
and lipids (GM3, cholesterol) correspond to the glycosphingolipid-enriched microdomains. They may function as binding
factors for many different molecules, including complementary GSL,
antibodies, and selectins, and are involved in transducing stimulatory
and/or inhibitory cellular signals (58, 59).
Altogether, our findings describe a novel mechanism of GM3-triggered
CD4 endocytosis in T lymphocytes (summarized in Fig. 8), suggesting a role for gangliosides as
structural components of a cell membrane multimolecular signaling
complex (60, 61).
We thank Dr. Angelo Del Nero for excellent
graphic and photographic work, and Dr. Roberto Gradini and Dr.
Patrizio Sale for precious help on image acquisition with confocal
microscope. We are indebted to Dr. Cesare Montecucco for critical
reading of the manuscript.
*
This work was supported in part by grants from MURST, from
Consiglio Nazionale delle Ricerche PF ACRO, and from Associazione Italiana Ricerca sul Cancro, Italy.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.
The abbreviations used are:
PKC, protein kinase
C; PI, phosphatidylinositol; PLC, phospholipase C; PLA2, phospholipase A2; PC, phosphatidylcholine; HIV, human
immunodeficiency virus; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PBL, peripheral blood lymphocyte; AA, arachidonic acid; FITC, fluorescein isothiocyanate; mAb, monoclonal
antibody; BSA, bovine serum albumin; MAP, mitogen-activated protein; DAG, diacylglycerol; MAG, monoacylglycerol; HPTLC, high performance
thin layer chromatography; FCS, fetal calf serum; ERK, extracellular
signal-regulated kinase; MEK, MAP/ERK kinase; AACOCF3, trifluoromethylketone analog of arachidonic acid.
1
The abbreviated designations of glycolipids are
according to Svennerholm's nomenclature for ganglio series
glycopeptides (33).
A Novel Mechanism of CD4 Down-modulation Induced by
Monosialoganglioside GM3
INVOLVEMENT OF SERINE PHOSPHORYLATION AND PROTEIN KINASE C 
TRANSLOCATION*
,,,,
Dipartimento di Medicina Sperimentale e Patologia,
Università di Roma "La Sapienza," Viale Regina Elena 324, Roma 00161, Italy, and ¶ Centro di Microscopia, Università
di L'Aquila, Via Monteluco di Roio, L'Aquila 67100, Italy
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and 
(more evidently than 
and 
)
after GM3 treatment, suggesting the involvement of these isozymes in
the ganglioside-induced CD4 down-modulation. Exogenous GM3 induced
phosphorylation of CD4 molecule, which then dissociated from
p56lck, as early as after 5 min. Moreover, addition of GM3
resulted in a rapid (1 min) cytosolic phospholipase
A2 activation with consequent arachidonic acid
release, whereas no phosphatidylinositol-phospholipase C activity was
observed. Both PKC translocation and CD4 down-modulation were blocked
by the trifluoromethylketone analog of arachidonic acid, a selective
inhibitor of cytosolic phospholipase A2 and by
mitogen-activated protein kinase inhibitor PD98059. Taken together, these findings strongly suggest that GM3 may trigger a novel mechanism of modulation of the CD4 surface expression through the activation of
enzyme(s) involved in the regulation of cellular functions.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-1
(13) and increase of intracellular calcium through a
p56lck-dependent pathway (14). It has also been
reported that gangliosides may control cell growth by modulating the
activity of protein kinase C
(PKC)2 (15) or activating
mitogen-activated protein (MAP) kinase isoform ERK2 (16).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; Calbiochem) (27), 10 µM cPLA2 inhibitor
AACOCF3 (trifluoromethylketone) analog of arachidonic
acid (Biomol, Plymouth Meeting, PA) (28), or 50 µM MAP
kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone; Calbiochem) (29)
for 30 min, after which GM3 was added as reported above. Incubation
with GM3 at 37 °C did not affect the viability of the cells. At the
end of the incubation time, the cells were washed with PBS before
incubation with antibodies.
(Transduction Laboratories, Lexington,
KY), -II, -, or - mAbs (Santa Cruz Biotechnology Inc., Santa Cruz, CA), for 1 h at 4 °C. After three washes in PBS, cells were incubated with FITC-conjugated goat anti-mouse or
anti-rabbit IgG for 45 min at 4 °C. The redistribution of the PKC
isozymes was analyzed by scanning confocal microscopy. Images were
acquired through a confocal laser scanning microscope Sarastro 2000 (Molecular Dynamics, Sunnyvale, CA) adapted to a Nikon Optiphot microscope (objective PLAN-APO 60/1.4 oil) and equipped with argon ion
laser (25 milliwatts). FITC was excited at 488 nm, and laser power was
set at 1 milliwatts. Images were collected at 512 × 512 pixels
with voxel dimensions 0.08 µm (lateral), 0.49 µm (axial). After
having been processed with routines for noise filtering, serial optical
sections were assembled in Depth-Coding mode. Acquisition and
processing were carried out using Image Space software (Molecular Dynamics). In parallel experiments, untreated and GM3-treated cells
were labeled with the anti-PKC- polyclonal Abs (Santa Cruz Biotechnology) for 1 h at 4 °C. After three washes in PBS,
cells were incubated with Texas Red-conjugated goat anti-rabbit IgG (Calbiochem) for 45 min at 4 °C. After washing three times in PBS,
pH 7.4, cells were then incubated for 1 h at 4 °C with anti-CD4 mAb, followed by three washes in PBS and the addition (30 min at
4 °C) of FITC-conjugated goat anti-mouse IgG (-chain-specific) (Sigma). Cells were finally washed three times in PBS and then mounted
upside down onto a glass slide in 1× glycerol/Tris-HCl, pH 9.2, and
analyzed by scanning confocal microscopy in dual fluorescence configuration where the FITC (green) and the Texas Red (red)
fluorophores were excited at 518 nm.
-1-stearoyl-2-arachidonyl
(arachidonyl-1-1-14C), in 20 mM Tris-HCl, pH
7.4, containing fatty acid-free 0.01% BSA in an ice bath (5 min, 5 watts, and 80% output). Vesicles were resuspended at 1 µM in the reaction buffer (50 mM Tris-HCl, pH
7.0, 5 mM CaCl2, 5 mM
MgCl2, 0.01% fatty acid-free BSA). Whole cell lysate
(60-100 µg) was added to 250 µl of reaction buffer containing the
vesicles and incubated for 1 h at 37 °C, and the reaction was
stopped by the addition of 250 µl of chloroform/methanol/acetic acid
(4:2:1). Phospholipids were extracted by the method of Bligh and Dyer
(34), dried under nitrogen, resuspended in 200 µl of chloroform, and
applied in duplicate to a silica gel TLC plate (Merck, Darmstadt,
Germany), with an automatic applicator (Linomat IV; Camag, Muttenz,
Switzerland). Samples were chromatographed in petroleum ether/diethyl
ether/acetic acid (70/30/1, by volume) to separate the parent
phospholipid PI from DAG. Authentic standards were co-chromatographed
with the lipid extracts to locate the compounds of interest. The
radioactive spots were visualized by autoradiography, scraped from the
plate, and counted by liquid scintillation. PI-PLC activity was
quantitated by the release of DAG generation from PI and expressed as
picomoles of DAG produced/106 cells.
-palmitoyl-2-arachidonyl
(arachidonyl-1-14C)-phosphatidylcholine for the detection
of released arachidonic acid (AA), in 50 mM Tris-HCl
buffer, pH 8.5, in an ice bath (5 min, 5 watts, and 80% output).
Vesicles resuspended at 1 µM in the reaction buffer (50 mM Tris-HCl, pH 8.5, 5 mM CaCl2, 5 mM MgCl2, 0.01% fatty acid-free BSA) were
reacted with whole cell lysate (60-100 µg) and phospholipids
extracted as described above. Samples were chromatographed in
chloroform/methanol/acetic acid/water (100:60:16:8, by volume) to
separate the parent phospholipid from the labeled product of
PLA2 activity, i.e. AA. Authentic standards were
co-chromatographed with the lipid extracts to locate the compounds of
interest. The radioactive spots were visualized by autoradiography,
scraped from the plate, and counted by liquid scintillation.
PLA2 activity was quantitated by the release of AA from PC
and expressed as picomoles of AA produced/106 cells.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
GM3-induced CD4 down-modulation in the
presence of PKC inhibitors. Cells (1 × 106 in 1 ml of PBS) were incubated in the presence of the following PKC
inhibitors: 50 nM staurosporine, 50 µM
bisindolylmaleimide GF109203X, or 10 µM rottlerin. After
a 15-min incubation time, 50 µg/ml GM3 or GM1 was added for 30 min or
2 h at 37 °C, respectively. Untreated and treated cells were
stained directly using fluorescein-conjugated anti-CD4 mAb and analyzed
by flow cytometry. A, cells incubated in the absence of GM3;
B, cells incubated in the presence of GM3; C,
cells incubated in the presence of GM3-derived 3'-sialyllactose;
D, cells incubated in the presence of GM1; E,
cells incubated in the presence of staurosporine, and then with GM3;
F, cells incubated in the presence of bisindolylmaleimide
GF109203X, and then with GM3; G, cells incubated in the
presence of rottlerin, and then with GM3; H, cells incubated
in the presence of staurosporine, and then with GM1. Histograms
represent log fluorescence versus cell number, gated on
lymphocyte population of a side scatter/forward scatter
(SS/FS) histogram. Cell number is indicated on the
y-axis, and fluorescence intensity is represented in three
logarithmic units at the x-axis.
(26). The results shown in
Fig. 1 (E-G) revealed that, in the presence of
staurosporine, bisindolylmaleimide GF109203X, or rottlerin, the
GM3-induced CD4 down-modulation was inhibited. This finding suggests
that the GM3-specific effect is PKC-dependent. Moreover,
the inhibition by rottlerin is indicative of a PKC- involvement.
These results appear to be in contrast with the PKC-independent signal
generated by GM1 (14, 21). In fact, we also analyzed the CD4 surface
expression in GM1-treated cells in the presence of staurosporine. Also
in our hands, the inhibition of GM1-induced CD4 down-modulation did not
occur (Fig. 1H).
and
- was more evident than - and -, as revealed by scanning
confocal microscopy. In GM3-treated cells, the anti-PKC- and -
signal appeared uneven and punctate over the plasma membrane, whereas
in untreated cells PKC isozymes were mostly diffuse in the cytoplasm.
The clustered distribution of the PKC- and - suggests that the
enzymes translocate mostly in correspondence of specific membrane
domains. Dual staining of CD4 and PKC- revealed nearly complete
colocalization yellow stained portions of membrane (Fig.
2B), indicating that a large fraction of translocated
PKC- effectively colocalizes and becomes associated with CD4, as
early as after 5 min of GM3 treatment. Therefore, GM3 treatment
preferentially promotes translocation of PKC- in selective membrane
domains in which PKC- may interact with CD4.
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Fig. 2.
A, PKC redistribution after GM3
treatment as revealed by scanning confocal microscopic analysis.
CD4+ lymphocytes (1 × 106 in 1 ml of PBS), separated using Dynabeads M-450 coated with
affinity-purified sheep anti-mouse IgG covalently bound to the surface,
were incubated with 50 µg/ml GM3 for 5 min at 37 °C. GM3-treated
and untreated cells were then fixed with acetone/methanol 1/1 (v/v) for
10 min at 4 °C. Cells were labeled with anti-PKC- ,
-II, -, or - for 1 h at 4 °C. After three
washes in PBS, cells were incubated with FITC-conjugated goat
anti-mouse or anti-rabbit IgG for 45 min at 4 °C. The redistribution
of the PKC isoforms was analyzed by scanning confocal microscopy.
Lane A, untreated cells; lane B, GM3-treated
cells (50 µg/ml, 5 min, 37 °C). B, scanning confocal
microscopic analysis of PKC--CD4 association on lymphocyte surface
after GM3 treatment. GM3-treated cells were labeled with anti-PKC-
polyclonal antibody, followed by the addition of goat anti-rabbit IgG
conjugated with Texas Red. After washing with PBS, the cells were
incubated with CD4 mAb, followed by the addition of FITC-conjugated
goat anti-mouse IgG. a, cells stained with anti-CD4 mAb,
followed by the addition of FITC-conjugated goat anti-mouse IgG;
b, dual immunolabeling of anti-CD4 and anti-PKC- revealed
nearly complete colocalization of white stained portions of membrane;
c, cells stained with anti-PKC-, followed by the addition
of goat anti-rabbit IgG conjugated with Texas Red.
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Fig. 3.
GM3 incorporation by cells. HPTLC
analysis of gangliosides extracted in chloroform/methanol/water from
2 × 107 lymphocytes. Lane A,
standard gangliosides GM3, GM1, GD1a, GD1b, and GT1b; lane
B, gangliosides obtained from untreated lymphocytes;
lane C, gangliosides obtained from lymphocytes
incubated in the presence of 50 mM bisindolylmaleimide
GF109203X; lane D, gangliosides obtained from
lymphocytes incubated in the presence of 50 mg/ml GM3/106
cells for 30 min at 37 °C, and then washed with PBS/FCS (10%) and
PBS/trypsin (0.1%); lane E, gangliosides
obtained from lymphocytes incubated in the presence of 50 µM bisindolylmaleimide GF109203X and then with 50 µg/ml
GM3/106 cells for 30 min at 37 °C, washed with PBS/FCS
(10%) and PBS/trypsin (0.1%). The plate was stained with resorcinol
(ganglioside-specific stain).
View larger version (18K):
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Fig. 4.
A, GM3 induces phosphorylation of CD4 in
human PBLs. Cells were washed twice with phosphate-free medium,
incubated for 1 h (i.e. starved), equilibrated with 1 mCi of [32P]orthophosphate for another 1 h, and then
treated with GM3 for 5 min. Cell lysates were immunoprecipitated with
anti-CD4 mAb as described under "Experimental Procedures." CD4
phosphorylation was evaluated by autoradiography of the SDS gel.
Lane a, untreated cells; lane
b, GM1-treated cells (50 µg/ml, 5 min, 37 °C);
lane c, GM3-treated cells (50 µg/ml, 5 min,
37 °C). B, GM3 induces serine phosphorylation of CD4 in
human PBLs. Cells were treated for the indicated times with GM3 (50 µg/106 cells/ml). Cell lysates were immunoprecipitated
with anti-CD4 mAb as described under "Experimental Procedures." The
amount of serine-phosphorylated CD4 was evaluated by and visualized by
chemiluminescence. C, densitometric scanning analysis
of the immunoblotting with anti-phosphoserine mAb. D, GM3
induces the dissociation of p56lck from CD4 molecule. Cells
were treated for the indicated times with GM3 (50 µg/106
cells/ml). Cell lysates were immunoprecipitated with anti-CD4 mAb as
described under "Experimental Procedures." The amount of
CD4-associated p56lck was evaluated by immunoblotting using
1× anti-p56lck mAb (lanes a-c) or
anti-mouse serum (lane d) and visualized by
chemiluminescence.
inhibitor further support the selective involvement of PKC- in GM3-induced CD4
down-modulation.
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Fig. 5.
cPLA2 activation following GM3
treatment. Cells (106/ml) were treated with GM3 (50 µg/ml) for the indicated times. cPLA2 activity in cell
lysates was tested against radiolabeled arachidonyl-PC vesicles. AA
generation was analyzed by TLC and quantitated. Results are expressed
as AA release and are representative of one from three independent
experiments. Mean values of two determinations are reported. S.D. < 5% of mean values. 
, untreated; 
, GM3; 
, GM3 + AACOCF3; 
, GM1.
Translocation--
To confirm the possible direct involvement
of cPLA2 in CD4 internalization, the GM3 effect on CD4
surface expression was analyzed in the presence of AACOCF3.
Flow cytometric analysis revealed that the GM3-induced CD4
down-modulation could be completely blocked by 30 min of pretreatment
with the cPLA2 inhibitor (Fig.
6). Of note, also PKC- translocation
(Fig. 6, inset) could be totally blocked by
AACOCF3, indicating that cPLA2 is involved in
PKC activation following GM3 treatment. Parallel experiments performed
in the presence of PI-PLC inhibitor U73122 revealed that this agent did not influence the GM3-induced CD4 down-modulation (data not shown), thus confirming that PI-PLC activation is not involved in this event.
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Fig. 6.
Inhibition of CD4 down-modulation after GM3
treatment by cPLA2 inhibitor AACOCF3
(trifluoromethylketone) analog of arachidonic acid. Cells (1 × 106) were incubated in the presence of cPLA2
inhibitor AACOCF3. Cells were stained directly using
fluorescein-conjugated anti-CD4 mAb and analyzed by flow cytometry.
Cell number is indicated on the y-axis, and fluorescence
intensity is represented in three logarithmic units at the
x-axis. Inset, inhibition of PKC-
redistribution after GM3 treatment by cPLA2 inhibitor
AACOCF3 (trifluoromethylketone) analog of arachidonic acid.
CD4+ lymphocytes (1 × 106 in 1 ml of PBS)
were incubated in the presence of cPLA2 inhibitor
AACOCF3 and then with 50 µg/ml GM3 for 5 min at 37 °C.
Cells were then fixed with acetone/methanol (1/1, v/v) for 10 min at
4 °C. Cells were labeled with anti-PKC- Ab for 1 h at
4 °C. After 3 washes in PBS, cells were incubated with
FITC-conjugated goat anti-mouse IgG for 45 min at 4 °C. The
redistribution of the PKC isozymes was analyzed by scanning confocal
microscopy.
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Fig. 7.
A, inhibition of CD4 down-modulation
after GM3 treatment by MAP kinase inhibitor PD98059. Cells were stained
directly using fluorescein-conjugated anti-CD4 mAb and analyzed by flow
cytometry. Cell number is indicated on the y-axis, and
fluorescence intensity is represented in three logarithmic units at the
x-axis. B, inhibition of AA release from PC
vesicles by MAP kinase inhibitor PD98059. Cells (1 × 106/ml) were incubated with GM3 (50 µg/ml) for 1 min in
the presence or absence of PD98059 (50 µM) as described
under "Experimental Procedures" and then analyzed for
cPLA2 activity. Results, expressed as AA release (mean
values of two determinations; S.D. < 5%), are representative of one
from three experiments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and - translocate more evidently than - and
-, as shown by scanning confocal microscopy. In addition, the
inhibition of GM3-induced down-modulation of CD4 molecule by rottlerin,
a highly specific inhibitor of PKC- (26), strongly suggests the
involvement of PKC-. Moreover, we observed that, after addition of
GM3, CD4 is rapidly phosphorylated on serine residues and this effect
is significantly inhibited in the presence of rottlerin. We also
demonstrated that GM3-induced phosphorylation of CD4 molecule
correlates with its dissociation from p56lck. This is a
fundamental step, which allows the association of CD4 with uncoated and
clathrin-coated pits and subsequently the endocytosis and intracellular
degradation of the molecule (19, 22). As a whole, these findings
indicate that, unlike GM1, which triggers a PKC-independent signal
(23), GM3 may elicit CD4·p56lck dissociation via PKC-
activation and serine phosphorylation. A different effect of GM3 as
against GM1 on receptor phosphorylation has already been reported, such
as modulation of EGF receptor activity in A431 cells, induced by GM3,
but not GM1 (40). The relevant structural diversity of the glycan
moiety of the gangliosides and the different carbohydrate head group
affect the physical interactions between gangliosides and proteins and
may account for specific ganglioside effects (41). The observations
that GM3-derived 3' sialyllactose (GM3 molecule lacking the ceramide moiety) and GM3-derived ceramide are unable to induce CD4
down-modulation suggest that the the integrity of the GM3 molecule is
required in order to have this effect. Further studies are in progress to analyze the possible effect of gangliosides on different CD4 mutants
and so clarify the functional role of glycolipid-protein interactions.
translocation. Other biochemical pathways that may underline the cPLA2-dependent PKC-
activation following GM3 treatment are under investigation. Since we
observed the absence of PI-PLC activity in GM3-treated cells, the link
between cPLA2 and PKC- activation could be the induction
of PC breakdown by PC-specific phospholipases C (PC-PLC) and
phospholipase D (44, 45), leading to DAG formation without mobilizing
Ca2+. These pathways may represent an alternative route for
PKC stimulation, particularly the Ca2+-independent isotypes
such as PKC-
and - (46). On the other hand, the ability of AA
and/or lysophospholipids to directly activate the
Ca2+-independent PKC has been already reported (47,
48).
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Fig. 8.
Possible mechanisms of GM3-triggered CD4
endocytosis in T lymphocytes.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dipartimento di
Medicina Sperimentale, Università di L'Aquila, Via Vetoio,
Coppito 2, 67100 L'Aquila, Italy. Tel.: 39-0862-433683; Fax:
39-0862-433523; E-mail: pavan{at}univaq.it.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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