G-protein-stimulated Phospholipase D Activity Is Inhibited by
Lethal Toxin from Clostridium sordellii in HL-60 Cells*
Noomen Ben
El Hadj
,
Michel R.
Popoff§,
Jean-Christophe
Marvaud§,
Bernard
Payrastre¶,
Patrice
Boquet
, and
Blandine
Geny
**
From
INSERM U332, ICGM, 22 rue Méchain,
75014 Paris, § Institut Pasteur, Unité des Toxines
Microbiennes, 75724 Paris, Cedex 15, ¶ INSERM U326,
Hôpital Purpan, 31059 Toulouse Cedex, and
INSERM U452,
Faculté de Médecine de Nice,
06107 Nice, Cedex 2, France
 |
ABSTRACT |
Lethal toxin (LT) from Clostridium
sordellii has been shown in HeLa cells to glucosylate and
inactivate Ras and Rac and, hence, to disorganize the actin
cytoskeleton. In the present work, we demonstrate that LT treatment
provokes the same effects in HL-60 cells. We show that guanosine
5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD)
activity is inhibited in a time- and dose-dependent manner
after an overnight treatment with LT. A similar dose response to the
toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin
effect on actin organization seemed unlikely to account directly for
PLD inhibition as cytochalasin D and iota toxin from Clostridium
perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease
observed in phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2). Likely in a relationship with this
decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were
not able to reconstitute PLD activity in LT-treated cells permeabilized
and depleted of cytosol. Studies of phosphoinositide kinase activities
did not allow us to attribute the decrease in PtdIns(4,5)P2
to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a
major inhibition in phosphatidylinositol 3-kinase that could not
account for the inhibition of PLD activity because wortmannin, at doses
that fully inhibit phosphatidylinositol 3-kinase, had no effect on the
phospholipase activity. Among the three small G-proteins, Ras, Rac, and
RalA, inactivated by LT and involved in PLD regulation, inactivation of
Ral proteins appeared to be responsible for PLD inhibition as LT toxin
(strain 9048) unable to glucosylate Ral proteins did not modify PLD
activity. In HL-60 cells, LT treatment appeared also to modify cytosol
components in relationship with PLD inhibition as a cytosol prepared
from LT-treated cells was less efficient than one from control HL-60
cells in stimulating PLD activity. Phosphatidylinositol transfer
proteins involved in the regulation of polyphosphoinositides and
ADP-ribosylation factor, a major cytosolic PLD activator in HL-60
cells, were unchanged, whereas the level of cytosolic protein kinase
C
was decreased after LT treatment. We conclude that in HL-60 cells,
lethal toxin from C. sordellii, in inactivating small
G-proteins involved in PLD regulation, provokes major modifications at
the membrane and the cytosol levels that participate in the inhibition
of PLD activity. Although Ral appeared to play an essential role in PLD
activity, we discuss the role of other small G-proteins inactivated by
LT in the different modifications observed in HL-60 cells.
 |
INTRODUCTION |
Phospholipase D (PLD)1
hydrolyzes phosphatidylcholine, the major membrane phospholipid,
yielding choline and phosphatidic acid (PA). PA, a possible second
messenger, is fusogenic, and its increase is associated with several
important cellular modifications such as activation of protein kinases,
phospholipase C-
and cytosolic phospholipase A2
(cPLA2), Ca2+ influx, DNA synthesis, and
c-fos and c-myc transcription. In cells, PA is
rapidly converted by a PA phosphohydrolase into diacylglycerol, the
natural activator of protein kinase C (PKC), and hence, PLD activity
can lead to a long term activation of protein kinase C (see review Ref.
1).
In mammalian cells, PLD is stimulated via several membrane receptors.
Its activation can occur by different intracellular pathways. Over the
past few years, several components participating in the control of PLD
activity have been deciphered. Small G-proteins, including ARF which is
involved in membrane traffic and RhoA which participates in actin
polymerization, were demonstrated to be potent activators of PLD. Other
small G-proteins implicated in the regulation of actin polymerization,
namely Rac and Cdc42, were also shown to be PLD activators but to a
lesser extent (see review Ref. 2). PLD activation was also reported to
occur through a Ras-Ral pathway (3-6); activated RalA leads to
membrane recrutement of ARF (3, 4). Other PLD activators have been
identified, such as conventional PKC isoforms (see review Ref. 2) and
the actin-binding protein gelsolin (7). PLD is under the negative control of several proteins, one of which was demonstrated to be the
cytoskeletal protein, fodrin, an actin-binding protein (8) and two
others were identified as synaptojanin (9) and clathrin assembly
protein 3 (10). Moreover, phosphoinositides are also involved in PLD
regulation. In particular, phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2), the PtdIns-phospholipase C substrate, was
shown to be a necessary cofactor for PLD activity (11). How all the
components involved in the regulation of PLD activity interact in
resting or activated cells is not yet well understood.
Bacterial protein toxins of large molecular weight isolated from
Clostridium difficile (toxins A and B) have been shown to inactivate Rho proteins and, thus, to affect the actin cytoskeleton (12). Toxin B has Rho subfamily small G-proteins, including RhoA, Rac,
and Cdc42, as targets. It inactivates these proteins by modifying Rho
by a glucosylation on threonine 37 and Rac and Cdc42 on threonine 35 (12). This toxin was demonstrated to inhibit m3 muscarinic receptor
stimulation of PLD in human embryonic kidney (HEK) cells (13) by
decreasing the membrane level in PtdInsP2 (14).
Clostridium sordellii, the organism responsible for gas
gangrene, produces a large molecular weight toxin, lethal toxin (LT), which exhibits some similarities with toxin B (15). LT also inactivates
small G-proteins by having a glucosyltransferase activity leading to
the glucosylation of small G-proteins (Ras, Rac, RalA, and Rap).
In vivo, its major target was reported to be Ras which is
glucosylated on threonine 35 (16, 17). It has also been reported that
LT produced by some strains of C. sordellii is able to
glucosylate the Ral protein on threonine 47 in vitro and
in vivo (18). So far, LT has not been found to have an
activity on other related small G-proteins from the same superfamily
including Rho, Cdc42, Rab, and ARF (16).
As Ras, Ral, and Rac have been reported to be involved in PLD
activation, we investigated the effect of LT on PLD activity in HL-60
cells. We observed that cell treatment with LT inhibits ARF-, RhoA-,
RalA-, and Rac-stimulated PLD activities. We investigated the mechanism
by which LT inhibits the enzyme in HL-60 cells and found that it
modifies the levels of cellular polyphosphoinositide and of some
phosphoinositide kinases. We discuss the responsibility of the small
G-proteins, Rac, Ras, and RalA, inactivated by LT in the decrease of
PLD activity in toxin-treated cells.
 |
EXPERIMENTAL PROCEDURES |
Materials
RPMI 1640 and medium 199 were purchased from Life Technologies,
Inc. Fetal bovine serum was from Dutcher. Radiolabeled molecules, [methyl-3H]choline chloride (82 Ci/mmol),
[myo-2-3H]inositol (16.5 Ci/mmol), were
obtained from Amersham Pharmacia Biotech.
1-O-[alkyl1',2'-3H]lysoPAF(30-60 Ci/mmol),
L-dipalmitoyl,
[choline,methyl-3H]-(30-60 Ci/mmol),
[
-32P]ATP (5000 Ci/mmol), and EN3HANCE
spray were purchased from NEN Life Science Products. Streptolysin O was
from Murex Diagnostic Ltd. GTP
S, phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P2), protease inhibitor
mixture tablets (CompleteTM), and protease and fatty
acid-free bovine serum albumin were purchased from Roche Molecular
Biochemicals (Germany). Bio-Rex 70 cation-exchange resin was purchased
from Bio-Rad. LY 294002 was obtained from Calbiochem. Cytochalasin D,
wortmannin, FITC phalloidin, protein A-agarose beads and all other
chemical products were from Sigma. Scintillant liquid, Optiphase
"HiSafe" 3, was from EG & G. Silica gel 60 plates for TLC analysis
were obtained from Whatman. Anti-Ha-Ras (259) and anti-Rac (T-17) were
from Santa Cruz Biotechnology. Rabbit anti-rat IgG and anti-goat IgG were from Cappel. ARF antibodies were obtained after immunization of
rabbits with recombinant ARF.
Lethal toxin and iota toxin were purified to homogeneity from C. sordellii (IP82) and (9048) strains and Clostridium
perfringens E, respectively, as extensively reported elsewhere
(19, 20). Toxin B from C. difficile was prepared according
to von Eichel-Streiber et al. (21).
Recombinant ARF, RhoA, Rac1, and RalA were made in Escherichia
coli through glutathione S-transferase fusions and were
prepared by thrombin digestion to yield the 21-kDa proteins.
Methods
Cells--
HL-60 cells were cultured in suspension in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 2% L-glutamine at 37 °C in a humidified incubator
containing 5% CO2.
Glucosylation by Lethal Toxin of Small GTP-binding Proteins in
HL-60 Cells--
HL-60 cells grown in RPMI 1640 medium were treated by
LT from C. sordellii as indicated. After treatment, cells
(2 × 107) were washed twice in PBS. After
centrifugation, the cell pellets were resuspended in 0.5 ml of lysis
buffer, pH 7.4, containing 0.5% Triton X-100, 20 mM Hepes,
50 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.1 mM orthovanadate, 10 mM GDP, and protease inhibitor mixture (CompleteTM) according to instructions. Cells were
incubated on ice for 20 min, and complete lysis was achieved by three
cycles of freeze-thawing. Cell lysates were centrifuged at 400 × g to remove intact cells and nuclei, and the amount of
protein in each supernatant was determined. For in vitro
glucosylation of small GTP-binding proteins, 32 µg of cell lysate or
2 µg of purified small G-proteins was added to 3 µl of dried
UDP-[14C]glucose with 5 mg/ml LT in glucosylation buffer,
pH 7.5, made of 50 mM triethanolamine, 2 mM
MgCl2, 1 mM DTT, and 0.3 mM GDP. The reaction was carried out for 1 h at 37 °C and stopped by
adding 10 µl of sample buffer (3 times). This mixture was boiled and electrophoresed on a 15% SDS-PAGE. The gel was stained with Coomassie Blue, destained, dried, and autoradiographed.
Immunoprecipitation of Small G-proteins
In vitro glucosylation was performed on a cell lysate
containing 320 µg of proteins per assay in a final volume of 300 µl. At the end of the reaction (1 h at 37 °C), bovine serum
albumin (1 mg/ml final) and antibody anti-ras (3 µg) or anti-Rac (6 µg) were added. After 1 h incubation at 4 °C under constant
gentle shaking, 30 µl of rabbit anti-rat IgG and anti-goat IgG were
added, followed by a 1-h incubation at 4 °C. Protein A-agarose beads (25 µl) previously equilibrated in washing buffer made of
triethanolamine (5 mM), MgCl2 (2 mM), NaCl (50 mM), DTT (1 mM), and
bovine serum albumin (0.1%) were added. After incubation at 4 °C
overnight under constant shaking, beads were washed 6 times in washing
buffer before addition of 8 µl of sample buffer (3 times).
Immunoprecipitates were analyzed by SDS-PAGE and autoradiographed as
described above.
Fluorescence Experiments, Effect of LT on the Actin
Cytoskeleton
After treatment with LT, cells were fixed with 3%
paraformaldehyde for 15 min and then washed three times with PBS. Free
aldehyde groups were quenched in NH4Cl (50 mM)
in PBS containing CaCl2 and MgCl2 (1 µM) for 10 min. Cells were permeabilized for 5 min with
Triton X-100 0.3% in PBS for 5 min and then incubated with FITC-phalloidin for 30 min. After labeling, cells were washed three
times in PBS, fixed on slides covered with polylysine, and mounted in
Mowiol. All steps were carried out at room temperature. Fluorescent
images were taken with a confocal laser scanning microscope (Bio-Rad
MRC-1000) attached to a diaphot 300 microscope (Nikon). For FITC
fluorescence detection, a krypton/argon laser at 488 excitation was
used, and emission was collected with a 522-nm filter.
Determination of Phospholipase D Activity in Permeabilized
Cells and in Intact Cells
In HL-60 cells, PLD hydrolyzes preferentially
phosphatidylcholine, the major phospholipid, to give choline and
phosphatidic acid, both can reflect PLD activity but can also result
from other cellular activities. PLD possesses a specific
transphosphatidylation activity (22); in the presence of a short chain
alcohol, instead of PA, a stable phosphatidyl alcohol will be formed.
Measurement of phosphatidyl alcohol (Ptd-alcohol) is highly specific of
PLD in comparison to choline measurement. We have previously checked that the formation of both products of PC hydrolysis, Ptd-alcohol and
choline, both occurred in parallel and reflected PLD activity in
streptolysin O-permeabilized HL-60 cells (23). Thus PLD activity was
estimated by measuring choline formed in experiments made with
permeabilized cells or PtdEt formed in the presence of 1.5% ethanol in
intact cells.
Choline Measurement--
Cells were labeled at isotopic
equilibrium for 48 h in medium 199 containing 10% fetal bovine
serum in the presence of 0.5 µCi/ml [3H]choline
chloride. At the end of labeling, HL-60 cells were counted, and
viability was monitored by trypan blue exclusion. Cells were washed and
resuspended at a concentration of 107 cells/ml in a buffer
made of 137 mM NaCl, 2.7 mM KCl, 20 mM PIPES, and 0.1% protease and fatty acid-free bovine
serum albumin, pH 6.8 (PIPES buffer). Phospholipase D activity
measurement was carried out in a final volume of 100 µl at 37 °C
in the presence of MgCl2 (2 mM final
concentration), MgATP (2 mM final concentration), GTP
S
(25 µM final concentration), Ca2+
(10
5 M) buffered with EGTA (3 mM
final concentration), and streptolysin O (0.4 units/ml final
concentration) as permeabilizing agent. The enzymatic reaction was
started by addition of 50 µl of labeled cells (5 × 106 cells) and stopped after 20 min at 37 °C by addition
of 500 µl of chloroform/methanol (1:1, v/v), and then 150 µl of
water were added to extract the aqueous phase containing radiolabeled
metabolites. The aqueous phase was loaded on columns of 1 ml of Bio-Rex
70 cation-exchange resin to separate newly formed choline from
phosphorus-containing choline metabolites as described by Martin (24).
Radioactivity present in choline was quantified after addition of
scintillant liquid using a Wallac 1410 liquid scintillation counter.
Total radioactivity associated with cells was counted. PLD activity was
estimated by measuring choline formed and expressed as the percentage
of total radioactivity incorporated in cells or as indicated.
Cytosol-depleted cells were obtained after permeabilization for 10 min
at 37 °C in the presence of 0.4 units/ml streptolysin O as described
in detail elsewhere (23).
PtdEt Measurement--
HL-60 cells were washed in a buffer made
of 137 mM NaCl, 2.7 mM KCl, 20 mM
Hepes, 5.6 mM glucose, and 0.1% protease and fatty acid-free bovine serum albumin, pH 6.8, and incubated at 37 °C for
30 min in the same buffer containing 2 mCi/ml
[lsqb3H]lyso-PAF. After labeling, PLD measurement was
carried out as described above except that 1.5% ethanol was added to
the reaction mixture. After phase separation as above, the lower
organic phase was dried under vacuum and resuspended in 25 µl of
chloroform and spotted onto oxalate-impregnated TLC silica plates, and
phospholipids were separated by migration in a solvent made of
chloroform/methanol/acetic acid/water (75:45:3:1). Radioactivity
present in silica spots corresponding to PtdCho, PA, and PtdEt was
counted in 4 ml of scintillation liquid.
Phosphoinositide Measurement
Cells were labeled at isotopic equilibrium for 48 h in
medium 199 containing 10% of fetal bovine serum, previously dialyzed against PBS, in the presence of 2 µCi/ml
[myo-2-3H]inositol. After 24 h treatment
without or with toxin LT, labeled cells were washed in PIPES buffer,
and lipids were extracted by addition of
chloroform/methanol/concentrated HCl (100:200:5). Extracts were
vortexed, and phase separation was achieved by the procedure of Bligh
and Dyer (25). The organic phase was then evaporated under vacuum, and
labeled phospholipids were separated onto oxalate-impregnated TLC
silica plates using a solvent of chloroform/methanol/acetone/acetic
acid/H2O (80:26:30:24:16, by volume). Lipids were localized
by staining with iodine vapor, and identification of the different
phosphoinositides was made by autoradiography after spraying the plates
with EN3HANCE and using labeled standards. Silica spots
corresponding to phosphatidylinositol (PtdIns), phosphatidylinositol
monophosphate (PtdInsP), and phosphatidylinositol bisphosphate
(PtdInsP2) were scraped and transferred into scintillation
vials. Lipids were extracted from silica by adding 100 µl of
methanol, and radioactivity associated with each phospholipid was
counted after addition of 4 ml scintillation liquid.
Cell Loading in PtdIns(4,5)P2
HL-60 cells labeled with [3H]choline were washed
with PIPES buffer and resuspended in the same buffer containing 0.1 mM EGTA, and 100 µM phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P2) was incubated for 15 min
at 37 °C. Streptolysin O was then added, and incubation was carried
out for an additional 10 min. In unlabeled cells, the pool of
PtdInsP2, incorporated into cells, was calculated to be
increased by 30-50% after that time, using 3H-labeled
PtdIns(4,5)P2 as a tracer. After loading with
PtdIns(4,5)P2, cells, depleted of cytosol, were used for
measurement of GTP
S-stimulated PLD activity in the presence of
different small G-proteins as described above.
Determination of PtdIns 4-Kinase and PtdIns(4)P 5-Kinase
Activities
Phosphatidylinositol 4-kinase (PtdIns 4-kinase) and
phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase)
activities were performed in a final volume of 100 µl containing 40 µl of HL-60 cell lysates in the presence of 10 µl of a sonicated
solution containing PtdIns4P (1.4 mg/ml) and PS (0.6 mg/ml) as
described by Chong et al. (26). The reaction was started by
addition of 10 µl of buffer containing [
-32P]ATP
(0.1 mCi/ml) and 0.2 mM unlabeled MgATP and performed at room temperature. Incorporation of 32PO4 into
PtdIns4P and PtdIns(4,5)P2 was found to be linear for 6 min
under such conditions. Five min after addition of the radioactive substrate, the reaction was stopped with 0.6 ml methanol, 1 N HCl (1:1). Phospholipids were extracted by adding 0.5 ml
of chloroform. The organic phase was dried under vacuum, and lipids
resuspended in chloroform (40 µl) were separated onto TLC plates
according to Chong et al. (26). 32P-Labeled
phosphoinositides were visualized by autoradiography and quantified
using a molecular imager (Molecular Dynamics).
Determination of PtdIns 3-Kinase Activity
PtdIns 3-kinase activity was performed in 500 µl final volume
under conditions similar to those described above for other PtdIns
kinases except that the amount of radiolabeled ATP was equivalent to
0.25 mCi per sample. This enzyme activity was estimated by measuring
the amount of D3-phosphorylated inositol lipids present in cells using
an HPLC technique as described (27, 28). Analysis of the deacylated
lipids was performed as described (28). Radioactivity eluted from the
Partisphere SAX column (Whatman International, Maidstone, Kent, UK) was
monitored and quantified by LB 506C detector (Berthold, Munich,
Germany), using the Cerenkov effect.
HL-60 Cell Cytosol Preparation
HL-60 cells (108) were washed 3 times in isotonic
PIPES buffer, pH 6.8, and resuspended in 3 ml of hypotonic buffer made
of Tris (20 mM), MgCl2 (5 mM), EDTA
(1 mM), sucrose (250 mM), orthovanadate (0.1 mM), DTT (1 mM), and protease inhibitor mixture
(CompleteTM), pH 7.5. After 30 min incubation on ice, cells
were homogenized with a Dounce homogenizer (50 strokes) followed by
three cycles of freeze-thawing. Unbroken cells and nuclei were removed
by a 5-min centrifugation at 400 × g. Particulate
fractions were pelleted and removed from cytosol by centrifugation for
20 min at 300,000 × g in a TL100 ultracentrifuge
(Beckman). Cytosol extracts were tested for their ability to
reconstitute PLD activity in cytosol-depleted permeabilized cells.
Immunoblot Analysis
After SDS-PAGE, proteins were transferred electrophoretically
onto a nitrocellulose membrane. Blocking was achieved overnight at
4 °C in isotonic PBS/glycine (2%) with 3% non-fat dry milk. Western blot analysis was performed for 90 min at room temperature using a polyclonal anti-recombinant ARF antibody (1:500 dilution) or
anti-PKC
antibody (1:500 dilution) in the same buffer containing 10% FBS and 0.3% Tween 20. The blots were then incubated for the same
period with 125I-protein A (0.2 µCi/ml). Immunoblots were
analyzed with a PhosphorImager (Molecular Dynamics) for quantitative
evaluation of the radioactivity in bands.
Western blot analysis with anti-PITP antibodies and protein
quantification were kindly performed as described (29).
Protein Estimation--
Protein content in cytosolic fractions
was measured according to Bradford (30) using bovine albumin as a standard.
 |
RESULTS |
LT-IP82 Glucosylates Several Small G-proteins in HL-60
Cells--
Lethal toxin from C. sordellii (strain IP82) was
found to glucosylate and inactivate small G-proteins in Swiss 3T3
cells: Ras primarily and Rac and Rap to a lesser extent (16). Moreover, this strain was also found to glucosylate Ral in vitro. We
checked that LT toxin exerted the same effect on small G-proteins in
the promyelocytic HL-60 cell line. As shown in Fig.
1A, overnight treatment of
HL-60 cells with 100 ng/ml LT would glucosylate proteins that could no
longer be glucosylated in vitro as in control cells. Demonstration that Ras and Rac present in HL-60 cells were glucosylated by LT was performed by immunoprecipitation of a cell lysate after in vitro glucosylation using specific antibodies against Ras
and Rac (Fig. 1B). As controls we used recombinant Ras, Rac,
and Ral glucosylated in vitro by LT (Fig. 1, A
and B).

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Fig. 1.
In vivo glucosylation of cellular
21-kDa proteins in HL-60 cells by lethal toxin from C. sordellii and in vitro glucosylation of
Ras and Rac in this cell line. A, in
vivo glucosylation of HL-60 cell proteins by LT treatment. After
incubation overnight without or with 100 ng/ml LT, HL-60 cells were
washed, lysed, and glucosylated by LT with
UDP-[14C]glucose as described under "Experimental
Procedures." HL-60 cell lysates (100 µg) from untreated cells
(lane 1) and LT-treated cells (lane 2) were
analyzed by SDS-PAGE and autoradiography for glucosylated protein
content. 0.5 µg of purified Ras (lane 3) and 1 µg of Ral
(lane 4) proteins were glucosylated in vitro in
similar conditions in the presence of LT and used as internal controls.
B, immunoprecipitation of LT-treated and -untreated HL-60
cell lysates with anti-Ras (lane 1) or anti-Rac (lane
2) after in vitro glucosylation of proteins by
incubation with UDP-[14C]glucose. Purified Ras
(lane 3), Rac1 (lane 4), and Rac2 (lane
5) were glucosylated in vitro in the same experiment
and run as controls.
|
|
LT-IP82 Causes Cytoskeletal Effect in HL-60 Cells--
LT has been
observed to provoked morphological and cytoskeletal effects in
epithelial and fibroblastic cells. In HL-60 cells, the effect of LT
toxin was checked on cell viability and on actin polymerization.
Overnight treatment of the promyelocytic cell line, HL-60, was found to
have some effect on cell viability determined by trypan blue exclusion.
Up to 30 ng/ml LT toxin has no detectable effect on cell viability; at
100 ng/ml, cell mortality was around 10%. At 300 ng/ml LT, the
percentage of dead cells was increased to 20-30%. Thus, we tested the
effect of 100 ng/ml LT on the cytoskeleton in this cell line. As shown
in Fig. 2, overnight treatment of HL-60
cells with LT induced a neat decrease in cortical actin polymerization,
indicating the efficiency of the treatment.

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Fig. 2.
FITC-phalloidin staining of the actin
cytoskeleton in HL-60 cells after treatment with lethal toxin from
C. sordellii. After staining with
FITC-phalloidin, cells were mounted in Mowiol, and examined.
Fluorescent images were taken with a confocal laser scanning
microscope. The equatorial plane of focus was defined as the midpoint
between the top and bottom of the cell.
A, FITC-phalloidin staining of untreated HL-60 cells
cultured in RPMI. B, FITC-phalloidin staining of HL-60 cells
cultured in RPMI and treated overnight with LT (100 ng/ml).
|
|
LT Inhibits PLD Activity--
As LT glucosylates several small
G-proteins reported to be involved in PLD regulation, we then studied
the effect of LT on PLD activity. As reported in Fig.
3, LT inhibits PLD activity in a time-
and dose-dependent manner. In HL-60 cells,
GTP
S-stimulated PLD activity, measured in streptolysin
O-permeabilized HL-60 cells, was decreased by 20-30% after 6 h
of treatment with LT (100 ng/ml), and maximal inhibition (50-60% of
the control) was reached after 16 h (Fig. 3A). This PLD
activity was inhibited in a dose-dependent manner after an
overnight treatment of HL-60 cells with LT. Maximal effect that reduces
GTP
S-stimulated PLD activity by 60% was obtained with 30 ng/ml. A
slight decrease in basal PLD activity was also observed at the highest
LT dose studied (100 ng/ml) (Fig. 3B), and PMA-stimulated
PLD activity, measured in intact cells, was inhibited to the same
extent by similar doses of LT (Fig. 3C).

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Fig. 3.
Time course effect of lethal toxin on
GTP S-stimulated PLD activity and dose
dependence on GTP S- and on PMA-stimulated PLD
activity in streptolysin O-permeabilized HL-60 cells. A,
[3H]choline-labeled cells were treated for various
periods with LT (100 ng/ml). After washing, PLD activity was determined
in permeabilized cells by measuring choline formation in the presence
(diamonds) or absence (squares) of GTP S (25 µM) as described under "Experimental Procedures."
Data were expressed as the percentage of total incorporated
radioactivity and represent the means ± S.E. In the experiment
shown, the total radioactivity in cells was 150,397 ± 22,160 dpm.
This experiment was repeated three times with similar results.
B, GTP S-stimulated PLD activity was measured as in Fig.
3. [3H]Choline-labeled cells were treated overnight with
LT at the indicated concentrations, and formation of
[3H]choline was estimated in the presence
(diamonds) or absence (squares) of GTP S (25 µM) in permeabilized cells as described under
"Experimental Procedures." Data were expressed as the percentage of
total incorporated radioactivity and represent the means ± S.E.
In the experiment shown, the total radioactivity in cells was
153,287 ± 33,161 dpm. This experiment was repeated three times
with similar results. C, PMA-stimulated PLD activity was
measured in intact cells treated or not with LT and labeled with
[3H]lyso-PAF. After treatment with various concentrations
of LT, cells were labeled for 30 min, washed, and resuspended in the
washing medium containing 1.5% ethanol. After a preincubation of 10 min at 37 °C, stimulation was started by adding PMA
(10 7 M final). After 30 min, the reaction was
stopped with chloroform/methanol, and samples were analyzed for PtdEt
content. PtdEt formed in the presence (filled triangles) and
in the absence (open triangles) of PMA was estimated in
intact cells as detailed under "Experimental Procedures." Data were
expressed as the percentage of radioactivity incorporated in PtdCho and
represent the means ± S.E. In the experiment shown, the total
radioactivity in cells was 167,950 ± 10,150 dpm. This experiment
was repeated three times with similar results.
|
|
Effect of LT Treatment on Reconstitution of GTP
S-stimulated PLD
Activity with Small G-proteins in Cytosol-depleted HL-60 Cells--
We
have already shown that HL-60 cells permeabilized with streptolysin O
can be depleted of cytosol (23). Thus, cytosol-depleted cells represent
an accurate model to study whether the decrease in PLD activity,
observed after LT treatment, is related to a change occurring at the
membrane or the cytosol level. The first possibility was tested using
LT-treated and control HL-60 cells depleted of cytosol after
permeabilization. PLD activity was then stimulated in the presence of
GTP
S by different small G-proteins known to be PLD activators as
follows: ARF and RhoA which cannot be modified by LT and Rac which is a
LT target. ARF- and Rho-stimulated PLD activities were inhibited by up
to 65% after an overnight treatment of HL-60 cells with LT and
Rac-stimulated PLD activity by 33% as shown in Fig.
4, A
C, respectively. It is
noticeable that ARF and RhoA are efficient PLD activators in HL-60
cells, with ARF being a better one. Indeed, an equivalent stimulation of PLD activity was obtained with 5 and 10 µg of ARF and RhoA, respectively. In comparison, Rac is a poor PLD activator in HL-60 cells. ARF and Rac have been reported to act synergistically on PLD in
different cell lines. They were tested together, and their stimulating
effect on PLD activity was found to be additive and not synergistic in
HL-60 cells (Fig. 4D). Moreover, the effect of RalA and Ras
on PLD activity was checked. PLD stimulation obtained with 10 µg of
the recombinant protein RalA was similar to that given by the same
amount of Rac (1.6 ± 0.04% of PtdCho hydrolysis). After LT
treatment (100 ng/ml overnight), RalA-stimulated PLD activity was
inhibited by about 35%. In cytosol-depleted HL-60 cells, Ras was never
found to stimulate PLD activity in similar conditions (data not shown).
These results indicate that, in membrane of toxin-treated HL-60 cells,
a factor necessary for PLD activation is missing or decreased.

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Fig. 4.
Effect of LT on ARF-, RhoA-, and
Rac-stimulated PLD activity in HL-60 cells.
[3H]Choline-labeled cells were treated overnight with LT
at different concentrations, then washed and permeabilized for 15 min
with streptolysin O at 37 °C. Cellular cytosol was removed by
dilution, and ghost cells were pelletted by centrifugation. PLD
activity in ghost cells was measured after stimulation in the presence
of ARF (5 µg/assay) (A), RhoA (10 µg/assay)
(B), Rac (10 µg/ml) (C), and ARF + Rac (5 and
10 µg/ml, respectively) (D) in the absence (open
symbols, dotted lines) or in the presence of 25 µM
GTP S (closed symbols, filled lines). In the experiment
shown, the total radioactivity in cells was 105,537 ± 8,804 dpm.
This experiment was repeated twice with similar results.
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Relationship between LT Effect on the Cytoskeleton and PLD
Inhibition--
To study if the decrease in PLD activity provoked by
LT treatment was directly related to actin polymerization, we studied the effect of different molecules modifying the cytoskeleton on PLD
activity. As reported in Table I,
overnight treatment of cells with toxin B (15 ng/ml, overnight) from
C. difficile was able to inactivate PLD in HL-60 cells to an
even greater extent than LT; this toxin was reported to modify the
actin cytoskeleton in different cell types. However, cytochalasin D (1 µM/ml, overnight) which disrupts the cytoskeleton and
iota toxin (100 ng/ml, overnight) which depolymerizes actin (31) did
not provoke marked changes in PLD activity in cytosol-depleted cells
stimulated with ARF or RhoA. In HL-60 cells, the effect on the actin
cytoskeleton of toxin B, cytochalasin D, and iota toxin was checked;
they all provoked a clear decrease in cortical polymerized actin (data not shown). These results do not exclude an indirect link between LT
effect on the actin cytoskeleton and on GTP
S-stimulated PLD activity
such as a change in phosphoinositides required for actin polymerization
and for PLD activation.
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Table I
Effect of different molecules modifying the actin cytoskeleton on PLD
activity in HL-60 cells depleted of cytosol
HL-60 cells were labeled with [3H]choline chloride (0.5 µCi/ml) for 48 h in medium 199 and treated overnight with
different molecules before PLD activity measurement. The enzymatic
reaction was carried out in the presence or not of GTP S and small
G-proteins (ARF, RhoA). The values in the table represent PLD activity
measured in triplicate and expressed as the percentage of PtdCho
hydrolysis ± S.E. This experiment is representative of three
different ones.
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LT Treatment Decreases Polyphosphoinositides Levels; Its Effect on
Phosphatidylinositol Kinases--
As PtdIns(4,5)P2 is a
cofactor for ARF-stimulated PLD activity, we studied the effect of LT
treatment on phosphoinositide composition. As reported in Fig.
5, overnight treatment of HL-60 cells
induced modifications in polyphosphoinositide levels in the range of LT
concentrations that provoked inactivation of ARF- and RhoA-stimulated
PLD activity. PtdInsP (Fig. 5A) and PtdInsP2 (Fig. 5B) levels were both decreased by LT in a
dose-dependent manner. At 30 ng/ml, a dose shown to inhibit
maximally small G-protein-stimulated PLD activity, PtdInsP and
PtdInsP2 were decreased by about 30 and 50%, respectively.
PtdIns level was not modified regardless which concentration of LT was
used (data not shown).

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Fig. 5.
Effect of LT treatment on the levels of
polyphosphoinositides in HL-60 cells. Cells were incubated for
48 h to reach metabolic equilibrium with
[3H]inositol (0.5 µCi/ml) and then treated or not with
various amounts of LT. After overnight incubation, cells were washed,
and the amount of radioactivity associated with PtdInsP and
PtdInsP2 was determined. A represents tritiated
inositol incorporated in PtdInsP (striped bars), and
B represents tritiated inositol incorporated in
PtdInsP2 (dotted bars). Data are expressed as
the percentage of radioactivity incorporated in phosphoinositides and
represent the means ± S.E. in triplicate. This experiment is
representative of three different ones. In the experiment shown, the
total radioactivity in cells was 405,857 ± 34,403 dpm.
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Since PtdIns(4,5)P2 (PtdInsP2) is a cofactor
for PLD activity, a decrease in the level of this minor
polyphosphoinositide could be responsible for the inability of small
G-proteins such as ARF and RhoA to stimulate PLD activity fully in
LT-treated cells. Therefore, the effect of PtdInsP2
addition was tested on small G-protein-stimulated PLD activity. PLD
activity stimulated with ARF, RhoA, Rac, or RalA in the presence of
GTP
S was measured in untreated and LT-treated cells. As shown in
Fig. 6, control and LT-treated cells
loaded with PtdInsP2 responded slightly better to each
small G-protein. However, PtdInsP2 was not able to restore fully GTP
S- and small G-protein-stimulated PLD activity in
LT-treated HL-60 cells.

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Fig. 6.
Effect of PtdIns(4,5)P2
cell loading on PLD activity. HL-60 cells labeled with
[3H]choline chloride were treated overnight with LT (100 ng/ml). PLD activity was determined in cells loaded or not loaded with
PtdIns(4,5)P2. After 30 min incubation with
PtdIns(4,5)P2 (100 µM) at 37 °C, cells
were permeabilized with streptolysin O for 15 min and then washed. PLD
was measured in the absence ( ) or in the presence of GTP S (25 µM) ( ). Small G-protein-stimulated PLD activity was
achieved with ARF (5 µg/assay) ( ), RhoA (10 µg/assay) ( ), Rac
(10 µg/assay) ( ), or RalA (10 µg/assay) ( ) in the presence of
GTP S. PLD activity was expressed as the percentage ± S.E. of
PtdCho hydrolyzed in cells unloaded or loaded with
PtdIns(4,5)P2 and treated or not with LT. In the experiment
shown, the total radioactivity in cells was 160,214 ± 1,208 dpm.
Similar results were obtained on three different occasions.
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As both Rac and Ras have been reported to participate in in PtdIns4P
5-kinase and PtdIns 3-kinase regulation responsible for PLD cofactor
synthesis, the effect of LT treatment was examined on
polyphosphoinositide kinases.
PtdIns 4-kinase and PtdIns4P 5-kinase activities were measured under
conditions similar to PLD activity measurement, in the absence and in
the presence of GTP
S. As shown in Fig.
7, after overnight treatment of cells
with 100 ng/ml LT, PtdIns 4-kinase was markedly increased by about
200% compared with untreated cells (Fig. 7A), whereas
PtdIns4P 5-kinase (Fig. 7B) was not modified either with or
without GTP
S. Therefore, the decrease in PtdInsP and
PtdInsP2 levels cannot be related to a change in the
activity of the kinases involved in their synthesis.

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Fig. 7.
Effect of LT treatment on PtdIns 4-kinase and
PtdIns(4)P 5-kinase. The rate of 32P incorporation in
PtdInsP and PtdInsP2 representing PtdIns 4-kinase and
PtdIns(4)P 5-kinase activities, respectively, was measured at room
temperature over a period of 5 min in a lysate from control HL-60 cells
and from cells treated overnight with 100 ng/ml LT. The experiment was
achieved in the presence of 20 µM radioactive Mg-ATP
(specific activity 10-50 cpm/pmol) with or without GTP S (25 µM). A represents 32P incorporated
in PtdInsP in the absence (open bars) or in the presence
(hatched bars) of GTP S. B represents
32P in PtdInsP2, in control cells and in
LT-treated cells (30 ng/ml) in the absence (open bars) or in
the presence (dotted bars) of GTP S. Results were
expressed as the percentage of kinase activity in control cells in the
absence of GTP S. This experiment was repeated three times with
similar results.
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PtdIns 3-kinase activity was measured in control and LT-treated cells.
As reported in Table II, the activity of
this enzyme was profoundly inhibited after cell treatment with LT, as
estimated by the amount of the different polyphosphoinositide isoforms
phosphorylated in position 3 of inositol lipids identified by HPLC
analysis. In LT-treated cells, phosphoinositides generated by PtdIns
3-kinase represent 15-20% of those in untreated cells. Wortmannin, a
specific PtdIns 3-kinase inhibitor, decreased the enzyme activity by
95% in HL-60 cells when used at 20 nM for 10 min
(conditions known to specifically inhibit PtdIns 3 kinase
activity).
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Table II
PtdIns 3-kinase activity in HL-60 cells treated with lethal toxin from
C. sordellii
After labeling and stimulation of HL-60 cells as detailed under
"Experimental Procedures," phosphoinositides labeled in position 3 were measured by HPLC. Phosphatidylinositol 3-kinase was specifically
inhibited by treating cells for 10 min with 20 nM
wortmannin. The effect of LT (100 ng/ml) on D3-phosphoinositide
formation was measured after an overngith treatment. Results are given
in dpm, and the experiment shown is respresentive of
two.
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Relationship between LT Inactivation of Ras and PLD
Inhibition--
To check if the observed decrease in PtdIns 3-kinase
activity could account for PLD inhibition, HL-60 cells were treated for 20 min with 20 and 50 nM wortmannin. No inhibition of PLD
activity was observed (Fig. 8) whichever
the concentration used. Similar results were obtained using LY 294002, another specific PtdIns 3-kinase inhibitor (data not shown). Therefore,
it is unlikely that inhibition of PLD by LT would be related to
Ras/PtdIns3-kinase inhibition in this model.

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Fig. 8.
Effect of wortmannin a PtdIns3-kinase
inhibitor on PLD activity. [3H]Choline-labeled cells
were washed and incubated at 37 °C for 20 min in the absence or in
the presence of the indicated concentrations of wortmannin;
[3H]choline release was then measured in the absence
(open bars) and in the presence (dotted bars) of
GTP S (25 µM). PLD activity was expressed as the
percentage of total [3H]choline incorporated in cells
present in choline. In the experiment shown, the total radioactivity in
cells was 164,639 ± 5,398 dpm. This experiment was repeated three
times with similar results.
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Involvement of Ral in the Inhibition of GTP
S-stimulated PLD
Activity--
RalA, also inactivated by LT-IP82, has been reported to
take part in PLD activation. To study the role of this small G-protein on GTP
S-stimulated PLD activity, we used a different lethal toxin extracted from C. sordellii, strain 9048, that does not
possess the capacity to glucosylate Ral in vitro but is
still able to inactivate Ras and Rac (Fig.
9A). As reported in Fig.
9B, GTP
S-stimulated PLD activity in HL-60 cells was not
modified after a treatment with 1 µg/ml LT-9048, whereas it was
decreased by about 50% when cells were treated with only 0.1 µg/ml
of LT-IP82.

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Fig. 9.
Effect of lethal toxin from strain 9048 of
C. sordellii on small G-protein glucosylation in
vitro and on GTP S-stimulated PLD
activity. A, recombinant Ras, Rac, and Ral were glucosylated
in vitro, as in Fig. 1, in the presence of LT extracted from
strain 9048 of C. sordelli. B,
[3H]choline-labeled HL-60 cells were treated or not
treated overnight with LT from strains 9048 and IP-82.
[3H]Choline release was measured in the absence
(open bars) and in the presence (dotted bars) of
GTP S (25 µM) in permeabilized cells. Lane
1, not treated (control cells); lane 2, treated with
1000 ng/ml from strain 9048; and lane 3, treated with 100 ng/ml strain IP-82. PLD activity was expressed as the percentage of
total [3H]choline incorporated in cells present in
choline. In the experiment shown, the total radioactivity in cells was
145,500 ± 9,080 dpm. This experiment was repeated three times
with similar results.
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Inhibition of GTP
S-stimulated PLD Activity by LT Treatment also
Involves a Modification at the Cytosol Level in HL-60 Cells--
The
possibility that LT treatment could have an effect on a factor present
in the cytosol was also investigated. For this purpose, cytosolic
fractions from control and LT-treated cells were prepared, and their
respective ability to stimulate PLD activity in the presence of GTP
S
was tested in control HL-60 cells that were depleted of cytosol by a
pre-permeabilization step. As reported in Fig.
10A, cytosol (60 µg of
proteins/assay) extracted from LT-treated cells did not stimulate
GTP
S-dependent PLD activity in control cells to the same
extent as cytosol extracted from untreated cells. This result indicates
that a cytosolic factor involved in PLD activity is either missing or
decreased after LT treatment.

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Fig. 10.
Effect of a cytosol from LT-treated cells on
PLD activity in cytosol-depleted HL-60 cells. HL-60 cells were
treated or not (control) with 100 ng/ml LT. After an overnight
incubation, cells were washed and then lysed by hypotonic shock
followed by freeze-thawing as reported under "Experimental
Procedures." Unbroken cells and nuclei were eliminated by a low speed
centrifugation. Supernatants were then centrifuged for 20 min at
300,000 × g to pellet particulate fractions. The same
amount of cytosolic protein (60 µg) from LT-treated and control cells
was then used to reconstitute PLD activity in untreated HL-60 cells
pre-permeabilized for 15 min and depleted of their cytosol as in Fig.
4. A, basal level of PLD activity (in the absence of
GTP S) (open bar); B, control level (in the
presence of 25 µM GTP S) (filled bar);
C, GTP S-stimulated PLD activity in the presence of
cytosol extracted from untreated cells (striped bar);
D, GTP S-stimulated PLD activity in the presence of
cytosol extracted from LT-treated cells (100 ng/ml) (dotted
bar). In the experiment shown, the total radioactivity in cells
was 116,060 ± 4,989 dpm. This experiment was repeated three times
with similar results.
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Several proteins involved in PLD regulation are known to be cytosolic
including phosphatidylinositol transfer protein (PITP) reported to be
essential for maintaining the level of PtdInsP2 (32) and
ARF and PKC
, potent activators of PLD. Thus, the level of these
proteins was measured in the cytosol recovered from untreated and
LT-treated cells. As reported in Fig.
11, A and B, no
modification in the level of PITP and ARF was observed after LT
treatment. In contrast, cytosolic PKC
was decreased by about 40% in
LT-treated cells (Fig. 11C). Quantification of the bands by
scan densitometer or PhosphorImager showed that the amount of PITP and
ARF differed by around 6% (decrease for PITP and increase for ARF)
between control and LT-treated cells, and PKC was decreased by 61% in LT-treated cells by comparison to untreated cells.

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Fig. 11.
Effect of LT treatment on cytosol content in
PITP, ARF, and PKC in HL-60 cells.
Cytosols prepared from untreated (control) and LT treated cells were
electrophoresed, and after transfer onto nitrocellulose membrane, their
respective content in PITP, ARF, and PKC was analyzed using specific
antibodies as reported under "Experimental Procedures." This
experiment was repeated twice with similar results.
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 |
DISCUSSION |
In the present work, we have investigated different effects of
lethal toxin (LT) purified from C. sordelli (strain IP-82) in HL-60 cells. Toxins purified from different Clostridia
origins have been demonstrated to inactivate small G-proteins of the
Rho family, and target profiles of their intracellular activity are specific for each of them. It has been reported that, in HeLa cells, LT
enters cells by endocytosis and, after toxin entry, cells round up as a
consequence of F-actin reorganization since disruption of actin stress
fibers and formation of filopodia occur. Possessing a
glucosyltransferase activity, LT monoglucosylates and inactivates Ras
by monoglucosylation on threonine 35 in HeLa cells (16). LT was also
shown to glucosylate Ras, Rac, Rap, and Ral in vitro (16,
18). Under their GTP-bound form, all these small G-proteins except Rap
have been reported to participate in PLD activation (5, 6, 33).
In the present study, we observed that LT treatment provokes, in HL-60
cells, glucosylation of small G-proteins, among which are Rac and Ras,
modification of the actin cytoskeleton, as in HeLa cells (16), and a
decrease in PLD activity stimulated by GTP
S in permeabilized cells
and by PMA in intact cells. In this cell line, GTP
S is known to
provoke a major activation of PLD increasing basal activity by a factor
8 to 10 in the presence of optimal concentrations of Mg-ATP and
Ca2+ (34). In all our attempts to measure PLD activity
using artificial vesicles with various phospholipid compositions as the
enzyme substrate, addition of sodium oleate to vesicles provoked a
major decrease in phosphatidylcholine breakdown in this cell
line.2 These results indicate
that HL-60 cells contain little or no oleate-activated PLD. These cells
have been shown to express high levels of mRNA for PLD1,
particularly, and PLD2 (35), both isoforms requiring
PtdIns(4,5)P2 as a cofactor for activity (36, 37). The
former human isoform can be directly activated in vitro by ARF, Rho protein family, and PKC
(33), and the latter was recently reported to be stimulated by ARF also (38). A direct interaction between the carboxyl terminus of PLD and activated RhoA using the
two-hybrid technique has been reported by Sung et al. (39). So far, no direct interaction between PLD and ARF was reported; this
could be due to a pre-required binding of ARF and/or PLD to
PtdIns(4,5)P2 (40, 41) for interaction. In HL-60 cells, PLD
activity was shown to be calcium-dependent (34). In these promyelocytic cells, LT inhibits PLD activity, whichever its pathway of
stimulation, and hence its effect is partly different to that observed
in an epithelial cell line, HEK. Indeed, in a recent work, Schmidt
et al. (42) found that LT was able to decrease exclusively
the PKC pathway of PLD activation in HEK-293 cells stably expressing m3
muscarinic receptors by inactivating Ral proteins. In HL-60 cells, Ral
proteins also appeared to be the major target for LT effect on PLD
activity. Indeed, LT extracted from strain 9048 of C. sordellii, which does not inactivate Ral proteins in
vitro, was not able to provoke modification in PLD activity in
these cells.
In HL-60 cells, LT (from strain IP-82) was found to generate major
changes in cytoskeleton organization, membrane phospholipid composition, and cytosolic PKC
level. These profound modifications could be related to the concomitant alteration in PLD activity stimulated via different signaling pathways.
LT treatment induced an almost complete disorganization of cortical
actin cytoskeleton in HL-60 cells. In HeLa cells, cytoskeletal modifications induced by this toxin were reported to be different from
those provoked by toxin B from C. difficile. Such
differences can be due to their different major targets in
vivo, Ras and Rac for LT and Rho proteins and RhoA, Rac, and Cdc42
for toxin B. All these small G-proteins have been reported to exert a
control on the actin cytoskeleton through different pathways (43, 44). In HL-60 cells, which grow in suspension, a similar modification was
observed with both toxins, a major decrease in polymerized actin. LT
could modify the actin cytoskeleton in inactivating Ras and/or Rac.
Several studies suggest an implication of the cytoskeleton in PLD
regulation. Small G-proteins of the Rho subfamily, well established to
be involved in the regulation of actin polymerization, are also
activators of PLD (33, 45), and RhoA was reported to interact directly
with PLD1 (39). The actin-binding protein, fodrin, was found to inhibit
PLD activity (8), and gelsolin was shown to interact with PLD and
activate the enzyme (7).
From the present study, the major decrease in actin polymerization
observed after LT treatment in HL-60 cells is likely to be related to
Ras/PtdIns 3-kinase/Rac pathway(s), but changes at the cytoskeleton
level do not appear to be directly responsible for PLD inhibition. In
HL-60 cells, we observed that LT treatment, giving a depolymerization
in cortical actin, was accompanied by a profound decrease in PtdIns
3-kinase activity, a direct target for Ras (46). Indeed, control of
actin cytoskeleton by Ras has been related to PtdIns 3-kinase activity,
and Rac was shown to function downstream of Ras/PtdIns 3-kinase (43).
As it has been recently reported that gelsolin dissociation from actin
filament is under the control of Rac (47), Rac inactivation by LT could also specifically depolymerize actin by blocking actin/gelsolin interaction, preventing actin uncapping and blocking nucleating activity at the barbed end of F-actin. Thus, inactivation of both Ras
and Rac by LT are likely to be responsible for cortical actin disorganization in HL-60 cells. However, a direct relationship between
PLD activation and the actin cytoskeleton organization can be ruled out
as neither PtdIns 3-kinase inhibition nor drugs such as cytochalasin D,
which caps the barbed end of actin filaments, and iota toxin, which
ADP-ribosylates the actin monomers (31), have an effect on PLD activity
in HL-60 cells. A similar observation has been made by Schmidt et
al. (13) in HEK cells. In these cells, cytochalasin B and toxin C2
from Clostridium botulinum both disorganize the actin
cytoskeleton but do not inhibit GTP
S-stimulated PLD activity as C3
toxin does, which inactivates Rho proteins.
Treatment with LT (strain IP-82) was found to lead to a net decrease in
polyphosphoinositides in HL-60 cells. Such changes in the levels of
negatively charged phospholipids could explain both the decrease in
polymerized actin and the inability for any PLD-activating small
G-protein to stimulate fully PLD activity in LT-treated cells. Proteins
participating in the cytoskeleton or in the regulation of actin
polymerization including profilin, gelsolin, and fodrin bind
PtdIns(4,5)P2 and PtdIns(3,4,5)P3 with high
affinity (48). Therefore, a decrease in these minor
polyphosphoinositides would lead to changes in actin organization. As
PLD activity in HL-60 cells appears to be mostly due to PLD isoforms
requiring PtdInsP2 as cofactors, the important decrease in
polyphosphoinositides observed after LT treatment can explain the lack
of PLD activation by small G-proteins such as ARF and RhoA that are not
modified by LT. The function of ARF is related to its interaction with PtdIns(4,5)P2 (40) and has also been shown to be the small
G-protein responsible for PLD activation involving RalA (3). This
latter small G-protein was shown not to activate directly PLD but to recruit ARF to membranes which, thus, would stimulate PLD activity (4).
Thus, the inability to restore PLD activity with
PtdIns(4,5)P2 in treated HL-60 cells could explain why RalA
and ARF together could not stimulate PLD activity in LT-treated HL-60 cells.
In HL-60 cells, GTP
S- and PMA-stimulated PLD activities were
decreased to a similar extent and with similar doses of LT. This toxin
was shown to inhibit only the PMA pathway in HEK cells (42). The
discrepancies observed in the effects of LT are likely to be due to the
difference in cell types. In HL-60 cells, the effect of LT was similar
to that obtained with another bacterial toxin, toxin B from C. difficile, in this cell line; GTP
S- and PMA-stimulated PLD
activities were both inhibited, and addition of
PtdIns(4,5)P2 was not able to modify PLD activity
significantly (49). In contrast, in HEK cells, this latter toxin only
inhibits receptor and G-protein-stimulated PLD activity (13), and
PtdIns(4,5)P2 was able to restore membrane-located PLD
activity (14). HL-60 cells and HEK cells are likely to possess
different contents in PLD isoforms and/or different PLD regulators
participating in each pathway of activation. HL-60 cells were found to
have a more marked GTP
S-stimulated PLD activity than HEK cells and
do not possess a PLD activity measurable in the presence of sodium
oleate or sodium cholate as HEK cells do (13, 14, 50). Moreover, although inactivation of Ral proteins appears to be sufficient for LT
to inhibit PLD activity, it is likely that other small G-proteins
inactivated by the toxin exert additional effects which might have
stronger consequences in HL-60 cells than in HEK cells. Indeed, in
these latter cell lines, LT was found to inhibit specifically PMA-stimulated PLD activity in the absence of modification in c-PKC
cellular levels but not a pathway involving G-proteins. In these cells,
the same effect was observed with toxin B-1470 from C. difficile that does not glucosylate Ras. In HL-60 cells, LT
treatment was also found to decrease the amount of PKC
in cell
cytosol. Such a cytosol from LT-treated cells was found to be less
efficient to stimulate PLD activity in control cells. The exact
mechanism of action for PKC and Ca2+ in PLD activation is
not clear. It has been reported that the regulatory domain of PKC
in
the presence of Ca2+ or PMA is sufficient to stimulate PLD
activity (50). However, it has also been reported that PKC activity
could also play a role by phosphorylating a phospholipase D-related
component in the plasma membrane (51). In HL-60 cells, previous studies
have shown that both Ca2+ and PMA can stimulate PLD in
synergy with GTP
S (34). Thus, the regulatory domain of a c-PKC in
the presence of Ca2+ could be necessary as a starter for
other activators such as ARF or Rho proteins to stimulate PLD activity
and the net decrease in PKC
cytosolic content could be responsible,
at least in part, for a diminution in the synergistic effect between
small G-protein, PKC and/or Ca2+. The decrease in cytosolic
PKC
level could be a consequence of Ras inactivation as this small
G-protein and PKC interaction have been reported to be required in
activation signaling pathways (52-56). In glucosylating and
inactivating Ras and other small G-proteins downstream of it, LT could
inactivate PKC and in HL-60 cells rapidly lead to its
"down-regulation."
In conclusion, we demonstrate that LT from strain IP-82 of C. sordellii, which inactivates several small G-proteins, inhibits GTP
S- and PMA-stimulated PLD activity similarly. Using LT extracted from strain 9048 of C. sordellii which has no effect on Ral
proteins, the responsibility for PLD inhibition is essentially due to
Ral although recombinant Ral could not reconstitute PLD activation after LT treatment. Major changes in the level of
polyphosphoinositides, acting as PLD cofactors, and in the cytosolic
level of PKC
, an important PLD activator, are also observed and
could be related to the high sensitivity of HL-60 cells to inactivation
by LT not only of Ral proteins but also Rac and Ras.
 |
ACKNOWLEDGEMENTS |
We thank Dr. F. Russo-Marie (Inserm U332) for
stimulating discussions and Isabelle Bouchaert for excellent technical
assistance in confocal studies. We also thank K. W. A. Wirtz
and C. van Tiel who kindly performed immunoanalysis and quantification
of cytosolic PITP in HL-60 cells.
 |
FOOTNOTES |
*
This work was supported by Grant 6931 from the Association
de Recherche sur le Cancer (ARC) and by "la Fondation pour la
Recherche Médicale" (FRM).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.
**
To whom correspondence should be addressed.
2
V. Montalescot and B. Geny, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
PLD, phospholipase D;
ARF, ADP-ribosylation factor;
DTT, dithiothreitol;
GTP
S, guanosine 5'-O-(3-thiotriphosphate);
HPLC, high
pressure liquid chromatography;
LT, lethal toxin from C. sordelli;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid);
PITP, phosphatidylinositol transfer protein;
PtdEt, phosphatidylethanol;
PtdIns3-K, phosphoinositide 3-kinase;
PtdIns 4-K, phosphatidylinositol
4-kinase;
PtdIns(4)P 5-K, phosphatidylinositol 4-phosphate 5-kinase;
PtdIns, phosphatidylinositol;
PtdInsP, phosphatidylinositol
monophosphate;
PtdIns(3)P, phosphatidylinositol 3-phosphate;
PtdIns(4)P, phosphatidylinositol 4-phosphate;
PtdInsP2, phosphatidylinositol bisphosphate;
PtdIns(4, 5)P2, phosphatidylinositol
4,5-bisphosphate;
PtdIns(3, 4)P2, phosphatidylinositol
3,4-bisphosphate;
PtdIns(3, 4,5)P3, phosphatidylinositol
3,4,5-trisphosphate;
PKC, protein kinase C;
PtdCho, phosphatidylcholine;
PAGE, polyacrylamide gel electrophoresis;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline;
HEK, bovine
serum albumin.
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