(Received for publication, October 6, 1995)
From the
Rho proteins have been reported to activate phospholipase D
(PLD) in in vitro preparations. To examine the role of Rho
proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho
proteins, on the regulation of PLD activity in human embryonic kidney
(HEK) cells stably expressing the m3 muscarinic acetylcholine receptor
(mAChR). Toxin B treatment of HEK cells potently and efficiently
blocked mAChR-stimulated PLD. In contrast, basal and phorbol
ester-stimulated PLD activities were not or only slightly reduced.
Cytochalasin B and Clostridium botulinum C2 toxin, mimicking
the effect of toxin B on the actin cytoskeleton but without involving
Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not
alter cell surface mAChR number and mAChR-stimulated binding of
(guanosine 5`-O-(thio)triphosphate (GTPS)) to G proteins.
In addition to mAChR-stimulated PLD, toxin B treatment also inhibited
PLD activation by the direct G protein activators,
AlF
and GTP
S, studied in intact and
permeabilized cells, respectively. Finally, C. botulinum C3
exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory
effect of toxin B on GTP
S-stimulated PLD activity. In conclusion,
the data presented indicate that toxin B potently and selectively
interferes with receptor coupling mechanisms to PLD, and furthermore
suggest an essential role for Rho proteins in receptor signaling to
PLD.
Stimulation of PLD, ()hydrolyzing the major membrane
phospholipid, phosphatidylcholine, to phosphatidic acid and choline,
has been reported in a wide range of cell types in response to many
hormones, neurotransmitters, and growth
factors(1, 2) . However, the components and mechanisms
involved in receptor signaling to PLD are only poorly defined, and may
even be distinct for different receptors and cellular systems. Besides
the receptor themselves, Ca
, protein kinase C,
tyrosine kinases, and GTP-binding proteins have been shown to be
involved in the regulation of cellular PLD
activity(1, 2) . We have recently shown that in HEK
cells, stably expressing the human m3 mAChR, various mechanisms,
including Ca
, protein kinase C, tyrosine
phosphorylation, and GTP-binding proteins, can lead to PLD
activation(3) . Coupling of mAChR to PLD in these cells is
largely independent of concomitant mAChR-mediated activation of
phospholipase C but rather involves a mechanism sensitive to tyrosine
kinase inhibitors. On the other hand, direct activation of protein
kinase C by the phorbol ester PMA leads to a strong PLD response, which
is additive to that caused by agonist-activated mAChR and directly
activated G proteins, suggesting that in this cell type PLD activity
can be stimulated by at least two distinct mechanisms(3) .
Recently, evidence has been provided that small molecular weight G proteins, notably ADP-ribosylation factors (4, 5) and members of the Rho protein family(6, 7, 8, 9, 10) , can stimulate PLD activity in in vitro preparations. The present study, therefore, was designed to examine whether Rho proteins are involved in regulation of PLD activity in HEK cells as well and in particular whether Rho proteins may play distinct roles in mAChR signaling to PLD and stimulation of PLD activity by directly activated protein kinase C. For this purpose, we used the cytotoxin B of Clostridium difficile, which enters cells by receptor-mediated endocytosis(11) . This toxin has recently been reported to monoglucosylate members of the Rho protein family, thereby inactivating these proteins and finally resulting in disorganization of the actin cytoskeleton(12, 13) . For comparison, we studied in permeabilized cells the effect of Clostridium botulinum C3 exoenzyme, which also inactivates Rho proteins, but by a distinct mechanism, namely ADP-ribosylation(14) . We report here that toxin B treatment of HEK cells potently and selectively abolishes mAChR-stimulated PLD, suggesting an essential role for Rho proteins in receptor signaling to PLD.
Pretreatment of m3 mAChR-expressing HEK cells with C.
difficile toxin B caused a time- and concentration-dependent
inhibition of mAChR-stimulated PLD activity.
[H]PtdEtOH accumulation induced by carbachol (1
mM) was inhibited by up to 90% in
[
H]oleic acid-prelabeled cells pretreated for
increasing periods of time with as low as 10 pg/ml toxin B (Fig. 1A). Half-maximal inhibition was observed after
approximately 10 h treatment with this toxin concentration (Fig. 1A). In contrast, stimulation of PLD activity by
the phorbol ester PMA (0.1 µM) was only slightly reduced
and only upon prolonged pretreatment with 10 pg/ml toxin B (Fig. 1B). When pretreated for 24 h, mAChR-stimulated
PLD activity was half-maximally inhibited with 3 pg/ml toxin B (Fig. 2A) and completely abolished upon treatment with
50 pg/ml toxin B (see Fig. 4). In contrast, PMA-stimulated PLD
activity was not altered by treatment with up to 30 pg/ml toxin B for
24 h (Fig. 2B) and was reduced by about 25% with 50
pg/ml toxin B (data not shown). Basal [
H]PtdEtOH
accumulation was not affected by toxin B treatment of HEK cells, even
when treated with a rather high toxin B concentration (1 ng/ml) (data
not shown). These data suggested that toxin B potently and selectively
interferes with the signal transduction pathway of m3 mAChR to PLD.
Figure 1:
Influence of toxin B on mAChR- and
PMA-stimulated PLD activities in HEK cells: time course studies. m3
mAChR-expressing HEK cells prelabeled with
[H]oleic acid were treated for the indicated
periods of time with 10 pg/ml C. difficile toxin B, followed
by measurement of basal PLD activity (
) and PLD activities
stimulated by 1 mM carbachol (
) (A) or 0.1
µM PMA (
) (B) as described under
``Experimental Procedures.'' Data are representative of two
similar experiments.
Figure 2:
Influence of toxin B on mAChR- and
PMA-stimulated PLD activities: concentration dependence. m3
mAChR-expressing HEK cells prelabeled with
[H]oleic acid were treated for 24 h with the
indicated concentrations of toxin B, followed by measurement of basal
PLD activity (
) and PLD activities stimulated by 1 mM carbachol (
) (A) or 0.1 µM PMA
(
) (B). Data are representative of four similar
experiments.
Figure 4:
Influence of toxin B on mAChR- and
AlF-stimulated PLD activities. In m3
mAChR-expressing HEK cells prelabeled with
[
H]oleic acid and treated for 24 h without (Control) and with 50 pg/ml toxin B, formation of
[
H]PtdEtOH was determined in the absence (Basal) and presence of carbachol (1 mM) or
AlF
(10 mM NaF plus 10
µM AlCl
). Data are representative of three
similar experiments.
As observed in other cell types(11, 12, 13) , treatment of HEK cells with toxin B potently induced rounding-up of the cells (data not shown), indicating disruption of the actin cytoskeleton. To exclude the possibility that inhibition of receptor-mediated PLD response by toxin B was merely due to its effect on the cytoskeleton, cytochalasin B and C. botulinum C2 toxin were used as controls. Both agents, by distinct mechanisms, cause depolymerization of actin, but notably without involving Rho proteins(18, 19) . As shown in Fig. 3, neither cytochalasin B (5 µg/ml, 15 min) nor C2 toxin (20 ng/ml component I plus 40 ng/ml component II, 24 h), both causing similar morphological changes (rounding-up) of HEK cells as toxin B, had any effect on mAChR-stimulated PLD activity.
Figure 3:
Lack of effect of cytochalasin B and C2
toxin on mAChR-stimulated PLD activity. m3 mAChR-expressing HEK cells
prelabeled with [H]oleic acid were treated
without (Control) and with cytochalasin B (Cyto B, 5
µg/ml, 15 min) or C2 toxin (20 ng/ml component I plus 40 ng/ml
component II, 24 h), followed by measurement of basal and carbachol (1
mM)-stimulated PLD activities. Data are representative of
three similar experiments.
To study
at which level toxin B interferes with receptor signaling to PLD,
several possibilities were considered. First, cell surface mAChR number
was determined by binding of the membrane-impermeant mAChR ligand
[H]NMS, at a receptor-saturating concentration (2
nM), to intact cells. Second, receptor coupling to G proteins
was measured as agonist-stimulated binding of
[
S]GTP
S to permeabilized adherent HEK
cells(16) . Treatment of HEK cells with toxin B (50 pg/ml, 24
h) affected neither mAChR surface number nor receptor coupling to G
proteins. In control and toxin B-treated cells,
[
H]NMS binding was 0.65 ± 0.06 and 0.78
± 0.01 pmol/dish, respectively. Basal and carbachol (1
mM)-stimulated [
S]GTP
S binding
amounted to 150 ± 5 and 258 ± 8 fmol/mg protein,
respectively, in control cells, and the corresponding numbers in toxin
B-treated cells were 138 ± 4 and 242 ± 1 fmol/mg protein,
respectively.
m3 mAChR-induced PLD activation in HEK cells is
apparently mediated by pertussis toxin-insensitive G
proteins(20) . Therefore, we studied whether toxin B treatment
also affects PLD activation caused by direct activation of
heterotrimeric G proteins. As shown in Fig. 4, similar to
carbachol-induced stimulation, stimulation of PLD activity in intact
HEK cells by AlF, a direct activator of
heterotrimeric G proteins, was completely abolished by prior toxin B
treatment (50 pg/ml, 24 h).
In permeabilized HEK cells, PLD activity
can be stimulated by the stable GTP analog GTPS, apparently by
activating small molecular weight G proteins(3, 21) ,
and by the phorbol ester PMA, which stimulation is additive to that
caused by GTP
S(3) . In control digitonin-permeabilized
cells, GTP
S (100 µM) and PMA (0.1 µM)
increased [
H]PtdEtOH accumulation by about
4-5-fold. Pretreatment of [
H]oleic
acid-prelabeled intact cells with toxin B (24 h), followed by digitonin
permeabilization and measurement of PLD activity, caused a
concentration-dependent reduction of the response to GTP
S (Fig. 5A). Half-maximal inhibition was observed at
about 5 pg/ml toxin B, and treatment with 30 pg/ml toxin B decreased
GTP
S-stimulated PtdEtOH formation by about 80%. Similar to data
obtained in intact cells, basal PLD activity and stimulation by the
phorbol ester PMA in permeabilized HEK cells were not affected by this
pretreatment with toxin B (Fig. 5B).
Figure 5:
Influence of toxin B on GTPS- and
PMA-stimulated PLD activities in permeabilized HEK cells.
[
H]Oleic acid-prelabeled HEK cells were treated
for 24 h with toxin B at the indicated concentrations. Thereafter,
formation of [
H]PtdEtOH was determined in
digitonin-permeabilized cells without (Basal,
) and with
100 µM GTP
S (
) (A) or 0.1 µM PMA (
) (B) as described under ``Experimental
Procedures.'' Data are representative of four similar
experiments.
To corroborate
the hypothesis that inactivation of Rho proteins is responsible for the
toxin B effects on PLD activity, we studied the effect of C3 exoenzyme
on GTPS-stimulated PLD activity in digitonin-permeabilized cells.
C3 exoenzyme had no effect on basal [
H]PtdEtOH
accumulation (Fig. 6). However, stimulation of PLD activity by
GTP
S was strongly reduced. [
H]PtdEtOH
accumulation induced by 10 µM GTP
S was virtually
abolished, and the stimulatory effect of 100 µM GTP
S,
the maximally effective concentration, was reduced by about 60%.
Figure 6:
Inhibition of GTPS-stimulated PLD
activity by C3 exoenzyme. In [
H]oleic
acid-prelabeled and digitonin-permeabilized HEK cells, formation of
[
H]PtdEtOH was determined in the absence (Control) and presence of 12 µg/ml C3 exoenzyme without (Basal) and with 10 µM or 100 µM GTP
S as detailed under ``Experimental Procedures.''
Data are representative of at least three similar
experiments.
Finally, we studied whether Rho proteins in HEK cells are affected
by toxin B treatment. For this, C3-catalyzed incorporation of
[P]ADP-ribose into Rho proteins was determined
in lysates of HEK cells pretreated without and with toxin B (50 pg/ml,
24 h). As reported before in other cell types, glucosylation of Rho by
toxin B prevents subsequent ADP-ribosylation by C3
exoenzyme(12, 13) . As illustrated in Fig. 7,
incorporation of [
P]ADP-ribose into Rho
proteins, using [
P]NAD as substrate and fresh C3
exoenzyme, was reduced by C3 pretreatment of permeabilized HEK cells by
about 80%. A similar, at least 80% reduction in C3-catalyzed
[
P]ADP-ribosylation of Rho proteins was also
observed in lysates of cells pretreated with toxin B, indicating that
endogenous Rho proteins in HEK cells were affected by prior toxin B
treatment.
Figure 7:
C3-catalyzed ADP-ribosylation of Rho
proteins in HEK cell lysates pretreated with toxin B or C3 exoenzyme.
C3-catalyzed [P]ADP-ribosylation of Rho proteins
was determined in lysates of control and toxin B (ToxB)-pretreated intact cells (50 pg/ml, 24 h) and for
comparison in lysates of digitonin-permeabilized HEK cells pretreated
without and with C3 exoenzyme (12 µg/ml, 60 min). Phosphorimage
data (Molecular Dynamics) of the SDS-polyacrylamide gel electrophoresis
are shown. Other labeled bands were not
detected.
In the present study, we examined the involvement of Rho proteins in stimulation of PLD activity in HEK cells stably expressing the human m3 mAChR subtype. To study the role of Rho proteins in G protein-coupled receptor signaling to PLD, an intact cellular system was necessary, since receptor stimulation of PLD activity has as yet not been reported in cell-free preparations. C. botulinum C3 exoenzyme inactivates Rho proteins; however, it does not enter intact cells or does so only poorly(14) , making it an unsuitable agent to study the role of Rho proteins in receptor coupling to PLD. Recently, C. difficile toxin B, known to enter cells by receptor-mediated endocytosis and to induce disruption of the actin cytoskeleton in a manner similar to micro-injected C3 exoenzyme, has been shown to specifically inactivate Rho proteins in intact cells, apparently by causing monoglucosylation of these proteins(12, 13) . We demonstrate here that toxin B treatment of HEK cells potently and efficiently blocks mAChR-stimulated PLD activity. For example, in HEK cells pretreated for 24 h, half-maximal inhibition of carbachol-stimulated PtdEtOH formation was observed with only 3 pg/ml, and complete inhibition upon treatment with 50 pg/ml toxin B. On the other hand, under similar treatment conditions, stimulation of PLD activity by the phorbol ester PMA, both in intact and permeabilized HEK cells, was not or only slightly reduced, and basal activity was not affected at all. These data indicate that toxin B does not modify PLD enzyme(s) itself and support the view that distinct signaling pathways leading to PLD activation exist in HEK cells. Furthermore, the differential sensitivity of PLD stimulation by the G protein-coupled mAChR and the PMA-activated protein kinase C may reflect the presence of different PLD isoenzymes in this cell type as suggested by studies in other cellular systems(9, 22) .
Control experiments with
cytochalasin B and C. botulinum C2 toxin demonstrated that the
inhibitory effect of toxin B treatment is not an event secondary to the
destruction of the actin cytoskeleton. Furthermore, toxin B treatment
had no effects on mAChR number and location and on receptor activation
of G proteins. Finally, toxin B treatment also abolished stimulation of
PLD activity by AlF, directly activating
heterotrimeric G proteins. Together, these findings indicate that the
cytotoxin interferes with the signaling pathway to PLD somewhere
downstream of the receptor-activated heterotrimeric G proteins. This
conclusion was further substantiated by the finding that toxin B
treatment also potently reduced stimulation of PLD activity by the
stable GTP analog GTP
S in permeabilized HEK cells. This inhibitory
effect on GTP
S-induced PLD stimulation was mimicked by C3
exoenzyme added to permeabilized HEK cells. We have no obvious
explanation for the reported ineffectiveness of C3 to inhibit
GTP
S-induced PLD stimulation in neutrophil and liver plasma
membranes(6, 7) , it may be due to the use of
different cellular systems. Finally, it is demonstrated that toxin B
treatment of HEK cells apparently affects endogenous Rho proteins,
thereby largely reducing C3-catalyzed incorporation of ADP-ribose.
Toxin B has been reported to monoglucosylate Rho A, Rac 1, and Cdc42,
while C3 exoenzyme ADP-ribosylates Rho A, B, and
C(13, 14) . The combined analysis with both tools
working on distinct members of the Rho protein family thus suggests
that toxin B causes its effects on receptor signaling to PLD in HEK
cells, most likely by an action on Rho A proteins.
PtdIns(4,5)P is apparently an essential co-factor for
PLD activation (23, 24) and is absolutely required for
ADP-ribosylation factor regulation of PLD
activity(4, 22) . Synthesis of PtdIns(4,5)P
in fibroblast cells has recently been reported to be stimulated
by activated Rho(25) , although more recent data suggest that
stimulation of phosphatidylinositol-4-phosphate 5-kinase by Rho A is
rather indirect(26) . In light of these data, it is tempting to
speculate that mAChR-mediated PLD activation in HEK cells involves both
ADP-ribosylation factor (21) and Rho A proteins, the former
stimulating PLD activity rather directly, while Rho A may be required
for the supply of the PLD co-factor, PtdIns(4,5)P
. As
recently reported in U937 cells, stimulation of PLD activity by protein
kinase C is apparently in part independent of PtdIns(4,5)P
as a cofactor(24) . This finding is consistent with the
data presented herein that, in comparison to mAChR-stimulated PLD,
stimulation of PLD activity by phorbol ester-activated protein kinase C
exhibited a largely reduced sensitivity to toxin B.
In conclusion, we demonstrate that C. difficile toxin B, known to inactivate Rho proteins, potently and efficiently blocks mAChR signaling to PLD in HEK cells, making an essential role of Rho proteins in this receptor action likely. On the other hand, stimulation of PLD activity by protein kinase C was markedly less sensitive to toxin B, suggesting the presence of distinct regulatory mechanisms and/or PLD isoenzymes.