(Received for publication, November 17, 1994; and in revised form, January 24, 1995)
From the
Calmodulin (CaM) antagonists chlorpromazine, trifluoperazine,
and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide HCl
inhibit Jurkat T cell activation, as monitored by measuring
interleukin-2 synthesis in cells treated by a combination of CD3
monoclonal antibody and phorbol myristate acetate. T cell activation
with CD3 monoclonal antibody is accompanied by a decreased synthesis of
phosphatidylserine due to the release of Ca from the
endoplasmic reticulum. CaM antagonists reverse the phosphatidylserine
(PtdSer) inhibition induced by CD3. This increase of PtdSer synthesis
was observed in the absence of any modification of CD3-induced
Ca
movements. Both in intact cells and in an
acellular system, the increase of PtdSer synthesis induced by CaM
antagonists was abolished in the presence of EGTA, indicating that the
base exchange enzyme system responsible for PtdSer synthesis is
regulated by CaM provided that Ca
is present. By
contrast, cyclosporin A that inhibits T cell activation through the
interaction of cyclophilin-cyclosporin A complexes with the
calmodulin-activated phosphatase, calcineurin, had no effect on PtdSer
synthesis. Calmodulin thus appears as a junction leading to at least
two independent pathways of regulation of T cell activation, one
involving the calcineurin phosphatase and the other the base exchange
enzyme system responsible for PtdSer synthesis.
T cell activation via the T cell receptor complex involves a
transduction pathway that begins with the phosphorylation on tyrosine
of phospholipase
C(1, 2, 3, 4, 5) .
This phospholipase hydrolyzes phosphatidylinositol bisphosphate into
diacylglycerol and inositol trisphosphate. This second messenger is
responsible for the release of Ca
from intracellular
stores(6, 7) . Emptying of the Ca
stores is followed by a decreased synthesis of phosphatidylserine
(PtdSer)(
)(8, 9, 10) , since the
Ca
-dependent base exchange enzyme system responsible
for the synthesis of this phospholipid is mainly located in the
endoplasmic reticulum (the major Ca
store) in T cells
as in other cell types(11, 12) . The release of
Ca
from the endoplasmic reticulum is also followed by
an influx of Ca
through the plasma
membrane(13) . The changes in the Ca
concentration both in the endoplasmic reticulum and in the
cytoplasm occurring during T cell activation modify the activity of
calmodulin. Calmodulin, a Ca
-binding protein, located
both in the cytosol and the particulate fraction of mammalian cells,
interacts with several proteins including calcineurin (14, 15, 16) and the base exchange enzyme
system(17, 18) . Cyclosporin A (CsA) inhibits T cell
activation through an interaction with cyclophilin (19, 20) that in turn interacts with calcineurin, a
phosphatase that is regulated by calmodulin. In previous reports, we
have shown that a number of inhibitors of interleukin-2 synthesis such
as K
channel blockers (21, 22, 23) , cytochrome P-450
inhibitors(24) , and diacylglycerol kinase inhibitors (25) reversed the CD3-induced inhibition of PtdSer synthesis.
Here we have examined the effects of calmodulin inhibitors on IL-2
production and PtdSer synthesis, and we have found that chlorpromazine,
trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, HCl (W7)
inhibited IL-2 production and increased PtdSer synthesis. By contrast,
the CsA-inhibiting pathway has been found to be independent of changes
in PtdSer synthesis. The CaM
Ca
complex, through
its interaction with the base exchange enzyme system, thus appears as a
target for some immunosuppressive drugs.
Figure 1:
Effect of W7 concentration on
phosphatidylserine synthesis (left panel) and interleukin-2
production (right panel) in Jurkat T cells activated with CD3
monoclonal antibody (2 µg/ml) and the phorbol ester PMA (10 ng/ml).
For IL-2 synthesis measurements, cells were incubated for 24 h as
described under ``Materials and Methods.'' For
phosphatidylserine synthesis, cells were incubated with the activators,
various concentrations of W7, and [H]serine at
time 0. Then after 2 h at 37 °C, phospholipids were extracted,
separated by thin layer chromatography (tlc), and quantified with a tlc
analyzer.
Figure 2:
Kinetics of phosphatidylserine synthesis
in control and 50 µM W7-treated Jurkat T cells.
Nonactivated cells were treated or not treated with W7 in the presence
of [H]serine for 2 h. Phospholipids were
extracted, separated by tlc, and then quantified as in Fig. 1.
Results are expressed as counts/min ± S.D. (n =
6 from two experiments with triplicate points) of
[
H]serine incorporated into
phosphatidylserine.
Figure 3:
Changes in cytosolic Ca
concentration induced by CD3 monoclonal antibody. At time 0, the cells
were incubated with 2 mM EGTA and 2 µg/ml CD3 mAb. Changes
in the cytosolic Ca
concentration due to the release
of Ca
from intracellular stores were monitored for 5
min. At 5 min, the Ca
concentration in the medium was
adjusted to 1 mM in order to study the CD3-induced
Ca
influx. Calmodulin inhibitor CPZ, TFP, or W7 (50
µM) was added at time 0. The figure is representative of
at least five independent experiments.
Figure 4: Inhibition of phosphatidylserine synthesis by 2 µg/ml CD3 mAb (panel A) and reversal of this inhibition by three anti-calmodulin drugs (panel B). In panelA, cells were either nonactivated (Control) or activated with CD3 mAb for 2 h. In panelB, cells activated with CD3 for 2 h were treated with 50 µM CPZ, TFP, or W7, and phosphatidylserine synthesis was monitored for an additional 1 h.
Figure 5:
Effect of CPZ, TFP, and W7 on
phosphatidylserine synthesis in thapsigargin-treated Jurkat cells.
Cells were incubated for 2 h in the presence of
[H]serine, 0.1 µM thapsigargin, and
50 µM of either CPZ, TFP, or W7 when
indicated.
Figure 6:
A, cells were maintained either in 1
mM EGTA (no calcium, whitebars) or in 1
mM Ca-containing medium (blackbars) in the absence or presence of 50 µM TFP, CPZ, or W7. B, 100 µl of Jurkat cell extract
(prepared as described under ``Materials and Methods'') were
used. As in A, blackbars represent
experiments done in 1 mM Ca
- containing
medium, and whitebars represent experiments done in
the presence of 1 mM EGTA. Results are expressed as percentage versus control in order to facilitate the comparison of the
two different sets of experiments. The values, ± S.D. (n = 6), correspond to three experiments with duplicate
points.
Figure 7:
Left
panel, comparison of the effect of 50 µM TFP, CPZ, or W7,
5 µM oleylamine, or 5 µM stearylamine on
phosphatidylserine synthesis in Jurkat cells maintained for 2 h in 2
mM EGTA-containing medium. [H]Serine was
added at time 0, and phosphatidylserine synthesis was measured at 2 h.
Under these experimental conditions, the Ca
concentration in the intracellular Ca
stores
was undetectable, as shown in the inset. The inset represents changes in the Ca
cytosolic
concentration elicited by 10
M ionomycin in
control cells maintained for 2 h in 1 mM Ca
-containing medium (
) and in cells
maintained in 2 mM EGTA for 2 h (
) (2 mM EGTA
was added at time 0, just before ionomycin, in order to evaluate the
Ca
content of the intracellular stores for cells
maintained in Ca
-containing medium (
)). The rightpanel represents PtdSer synthesis in the
absence or presence of either oleylamine or stearylamine (5
µM) in cells incubated in 1 mM Ca
-containing
medium.
Figure 8:
Lack
of effect of CsA on phosphatidylserine synthesis. Cells were treated
with CD3 mAb (2 µg/ml) in the absence or presence of different
concentrations of CsA and [H]serine for 2 h. At
the end of this incubation period, phospholipids were extracted,
separated by tlc, and then quantified. Results are expressed as
counts/min ± S.D. of [
H]serine
incorporated into phosphatidylserine (n = 6 from two
experiments done in triplicate).
A key phenomenon in T cell activation is the rise of
cytosolic Ca concentration. Early events leading to
this phenomenon represented by the ``tyrosine kinase
pathway'' have been extensively investigated. Yet the events that
occur in the ``postcalcium period'' are less known. One of
these events is the inhibition of PtdSer synthesis that has been
attributed to the release of Ca
from intracellular
stores(11) . The exact role of PtdSer inhibition is not known,
although changes in PtdSer synthesis seem to be implicated in T cell
signaling pathways since a number of inhibitors of IL-2 synthesis
reverse this process. Among the drugs tested in past years, potassium
channel blockers(21, 22, 23) , diacylglycerol
kinase inhibitors (25) , and cytochrome P-450 inhibitors (24) enhanced PtdSer synthesis and inhibited IL-2 production
and T cell proliferation with similar dose-response curves, indicating
that these events may be linked. We show herein that three calmodulin
antagonists increase PtdSer synthesis and inhibit IL-2 synthesis in
Jurkat T cells with similar concentration-dependent effects. The
stimulation of PtdSer synthesis by calmodulin antagonists we have
observed in Jurkat cells confirms the findings of previous works
performed in acellular systems either with human leukocyte membranes by
Niwa and Tanigushi (18) or with rat brain microsomes by
Buchanan and Kanfer(17) . CaM inhibitors have been shown to be
able to interact with K
channels(31) . Since
K
channel blockers increase PtdSer synthesis and
inhibit IL-2 synthesis, we have tested the effect of CaM inhibitors on
an acellular system in which changes in K
concentration or changes in K
channel activity
are unlikely. In this acellular system, CaM inhibitors remained able to
increase PtdSer synthesis, suggesting that CaM could be the target of
the drugs used. In order to confirm this point we have tested whether
the increase of PtdSer caused by the drugs is calcium-dependent. As
expected, CPZ, TFP, and W7 increased PtdSer synthesis in control,
CD3-activated, and thapsigargin-treated cells but were inactive in
EGTA-treated cells. We have verified that after 2 h in the presence of
2 mM EGTA the cells were depleted of Ca
as
shown in Fig. 7(inset). Furthermore, as shown in Fig. 6, the Ca
dependence of CaM antagonists
was also observed in the acellular system. On the other hand, it has
been suggested that amphiphilic cations increase PtdSer synthesis by
exposing the ethanolamine moiety of phosphtidylethanolamine (the
substrate for PtdSer synthesis) to a hydrophilic environment accessible
to the catalytic site of the base exchange
enzyme(32, 33) . This interpretation appears unlikely,
since as shown in Fig. 7the two cationic amphiphilic drugs
tested (oleylamine and stearylamine) strongly increased PtdSer
synthesis but in a calcium-independent process. Furthermore, our
previous work on K
channel blockers (22) in
which we tested five antiarrhythmic drugs belonging to the cationic
amphiphilic drug family indicated that only the potassium channel
blocker, clofilium, increased PtdSer synthesis. From this study and the
results presented herein it can be concluded that increasing PtdSer
synthesis is not a general property of cationic amphiphilic drugs.
Altogether, our results strongly support the hypothesis that CPZ,
TFP, and W7 play their role through an interaction with the
CaMCa
complex.
A second event in the
postcalcium period of T cell activation is the formation of
Ca-calmodulin complexes that in turn bind to
calcineurin A subunit(14) . This pathway is inhibited by
cyclosporin A through an interaction of cyclophilin-CsA complexes to
calcineurin (14, 15, 16) . We have tested
whether CsA modifies PtdSer synthesis, and it was found (Fig. 8)
that this immunosuppressive drug had no effect on PtdSer even at
concentrations far above the doses necessary to totally abrogate IL-2
synthesis.
CaMCa
complexes thus appear as a
junction leading to at least two independent pathways of regulation of
T cell activation, one involving the calcineurin phosphatase and the
other involving the base exchange enzyme system responsible for PtdSer
synthesis.