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Cooperates with Calcineurin to Induce Fas Ligand Expression During Activation-Induced T Cell Death1
Divisions of
*
Cell Biology and
Cellular Immunolgy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, a Ca2+-independent PKC isoform that we have
previously isolated as a PKC enzyme selectively expressed in T cells,
plays an important role in these processes. A constitutively active
PKC mutant preferentially induced FasL expression and activated the
corresponding gene promoter; conversely, a dominant-negative PKC
mutant blocked FasL expression induced by anti-CD3 or PMA plus
ionomycin stimulation. Furthermore, PKC synergized with calcineurin
to provide a potent stimulus for FasL promoter activation. Full
activation of the promoter required its binding sites for the
transcription factors NF-AT, AP-1, and NF-
B. The biological
significance of these findings is implicated by the finding that
rottlerin, a selective PKC inhibitor, blocked FasL induction by
anti-CD3 or PMA plus ionomycin stimulation and, consequently,
protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from
activation-induced cell death. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies focused on characterization of the structure and
properties of the FasL gene promoter. This promoter possesses potential
binding sites for several transcription factors including the
"housekeeping" promoter elements, NF-AT, AP-1, and NF-B
(8). Two NF-AT-binding sites are necessary for proper FasL
induction in activated T cells (9, 10), and a role for
NF-B in FasL expression after T cell activation has been shown
(11, 12). Stress-induced FasL expression also requires the
activation of NF-B and AP-1 (8, 13). In addition, the
FasL promoter also contains a new response element (RE), designated
RE-3 (14), and a mitogen-activated protein
kinase/extracellular signal-related kinase (ERK) kinase-1 (MEKK1) RE
(13). Taken together, these findings suggest that FasL
induction is mediated by the coordinated action of several
transcription factors.
In addition to TCR ligation, the combination of phorbol ester plus Ca2+ ionophore, which mimicks the two physiologic signals required for T cell activation and IL-2 production, can also induce FasL expression (14, 15) and AICD (16). Phorbol esters mediate their pleiotropic effects mainly via the activation of protein kinase C (PKC), suggesting that some PKC isoform(s) regulates FasL expression. In contrast, phosphatase 2B or calcineurin (Cn) represents an important target of Ca2+ signals (17), and its TCR-induced enzymatic activation leads to dephosphorylation of NF-AT and its translocation to the nucleus (18), where it combines with AP-1 (19, 20) and other transcription factors to stimulate transcription of the IL-2 gene.
In the present work, we have investigated the potential involvement of
PKC in the pathway that induces FasL expression following TCR/CD3
stimulation. We focused on this particular isoform because it is
selectively expressed in T cells (21), where it
colocalizes with the TCR complex to the contact site between
Ag-specific T cells and APCs (22, 23). Moreover, PKC
selectively cooperates with Cn in the activation of JNK and induction
of the IL-2 gene (24, 25). We report that PKC plays an
important role in regulating FasL expression and AICD and, furthermore,
that it cooperates with Cn in these processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal Abs against the PKC isoenzymes and 
were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mAbs
against PKC
and PKC
were obtained from Transduction Laboratories
(Lexington, KY). The anti-CD3 mAb, OKT3, was purified from culture
supernatants of the corresponding hybridomas by protein A-Sepharose
chromatography. The anti-hemagglutinin mAb 12CA5 was obtained from
Boehringer Mannheim (Indianapolis, IN), Rottlerin and Gö6976 were
obtained from Calbiochem (La Jolla, CA). All other products were
obtained from Sigma (St. Louis, MO).
Plasmids
The cDNAs encoding human wild-type PKC and PKC, the
constitutively active mutants of human PKC (A148E) and PKC
(A25E), rat PKC (A159E), or mouse PKC (A119E), and the
dominant-negative (kinase-inactive) mutant of PKC (K409R) were
cloned in the eukaryotic expression vector pEFneo. An
hemagglutinin-tagged, constitutively active Cn A mutant (CaM-AI; Ref.
25) cDNA was cloned in pSR. The FasL reporter
constructs have been previously described (8, 12). As
control for transfection efficiencies, a ß-galactosidase (ß-gal)
expression plasmid in the pEF vector was used.
Cell culture and transfection
SV40 large T Ag (TAg)-transfected human leukemic Jurkat T cells (Jurkat-TAg), Jurkat-CE cells (which are highly sensitive to AICD), and the T cell hybridoma A1.1 were grown in RPMI 1640 medium (Life Technologies), supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, pH 7.2, 1x MEM nonessential amino acid solution (Life Technologies), and 100 U/ml of each penicillin G and streptomycin. Cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as previously described (26, 27). In each experiment, cells in each group were transfected with the same total amount of DNA by supplementing expression vector DNA with the proper amounts of the corresponding empty vector. In some experiments, the cells were pretreated with PKC inhibitors as indicated.
Luciferase and ß-gal assays
Transfected cells were harvested after 2 days, washed twice with PBS, and lysed in 100 µl of lysis buffer (100 mM KPO4, pH 7.8, 1 mM DTT, 0.5% Triton X-100) for 10 min at room temperature. The lysates were then centrifuged (15,000 x g, 5 min at 4°C). For the luciferase assay, 50 µl of the supernatants were mixed with 100 µl of assay buffer (17.5 mM glycilglycine, pH 7.8, 10 mM MgCl2, 5 mM ATP, 0.135 mM coenzyme A, 0.235 mM luciferin), and the luciferase activity was determined in a luminometer. Luciferase activity in each group was normalized to the activity of a cotransfected ß-gal reporter plasmid. For the ß-gal assay, 20 µl of the supernatants were incubated at 37°C in 150 µl of assay buffer containing 60 mM Na2HCO, 80 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 10 mM DTT, and 60 µg o-nitrophenyl ß-D-galactopyranoside until a yellow color developed. Absorbance was measured at 400 nm in a spectophotometer. The results were expressed as arbitrary luciferase units per arbitrary ß-gal units. All experiments were performed in duplicate and were repeated several times with similar results.
Induction and assessment of apoptosis
Human Jurkat-CE leukemic cells or murine A1.1 hybridoma cells were induced to undergo apoptosis by treatment with a combination of PMA (100 ng/ml) plus ionomycin (1 µg/ml), or with anti-human (OKT3) or anti-mouse (2C11) CD3 mAbs (28). For TCR stimulation, 96-well plates were precoated with optimal Ab concentrations in 50 mM Tris-HCl, pH 9.0. Apoptosis of Jurkat-CE cells was assessed by staining the cells using the annexin-V-FLUOS staining kit (Boehringer Mannheim) and analyzing them on a FACScan analyzer following the manufacturer’s instructions. In A1.1 cells, AICD was determined by propidium iodide staining (28).
In vitro PKC kinase assay
Sixty nanograms of purified PKC (29) and PKC
(Panvera, Madison, WI) proteins were incubated for 5 min at 30°C with
gentle shaking in a reaction mixture containing 10 µg of the
synthetic peptide substrate RFARKGSLRQKNVY homologous to
the pseudosubstrate sequence of PKC, 100 ng
L--phosphatidyl-L-serine,
20 ng diacylglycerol, 10 µCi [
-32P]ATP, 20
mM HEPES, pH 7.5, 10 mM MgCl2, and 0.1 mM ATP. In
additon, the PKC and PKC kinase reactions contained 0.1 mM
CaCl2 or 0.1 mM EGTA, respectively
(30). Samples were placed on ice to stop the reaction,
collected on phophocellulose units (Pierce, Rockford, IL), and
processed following the instructions of the manufacturer. The amount of
radioactivity incorporated into the synthetic peptide substrate was
determined in a scintillation counter.
FasL expression analysis by RT-PCR
In experiments performed with transfected cells, 20 x 106 Jurkat-TAg cells were transfected with 5 µg of the appropriate plasmid. After 2 days, total RNA was extracted and subjected to RT-PCR analysis of FasL and actin expression as previously described (8). In experiments performed with Jurkat-CE cells, 10 x 106 cells were incubated for 30 min at room temperature with 30 µM of rottlerin and then stimulated for 4 h with PMA (100 ng/ml) plus ionomycin (1 µg/ml) or with anti-human (OKT3) CD3 mAb (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays an important role in FasL expression
To determine whether a particular PKC isoform is preferentially
involved in FasL expression, we cotransfected Jurkat-TAg cells with a
plasmid carrying the FasL promoter cloned upstream of a luciferase
reporter gene (8) plus constitutively active mutants of
several PKC isoforms representing Ca2+-dependent
(), -independent (, ), or atypical () PKC isoforms. These
mutants have been previously characterized (30, 31, 32). The
constitutively active PKC mutant (-A/E) induced a 
5-fold
activation of the FasL reporter, whereas similar mutants of the other
PKC isoforms caused a much weaker stimulation that did not exceed
2-fold (for PKC-A/E) (Fig. 1
A). Under the same
conditions, and in agreement with a recent report (25),
the various PKC isoforms tested were capable of stimulating the kinase
activity of an ERK2 reporter plasmid to a similar degree (data not
shown), indicating that they were functional in the transfected cells.
The mean ± SEM of eight experiments was 103 ± 25-fold and
332 ± 53-fold induction for pEF- or PKC-A/E-transfected cells,
respectively. Wild-type PKC (or PKC) was unable to induce FasL
promoter activation, suggesting that PKC activation is required for
FasL promoter stimulation. Immunoblotting with the corresponding
PKC-specific Abs confirmed the proper overexpression of the transfected
PKC enzymes in all groups (Fig. 1B). RT-PCR analysis showed
that transient overexpression of PKC-A/E, but not PKC-A/E, also
induced expression of the endogenous FasL (Fig. 1C,
top), although both PKC isoforms were similarly
overexpressed (Fig. 1C, bottom), ruling out the
possibility that the differential induction of FasL mRNA reflects lower
expression of PKC.
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is necessary for FasL promoter activation
induced by an anti-CD3 Ab or the combination of PMA plus ionomycin.
First, we determined the effect of selectively inhibiting cellular
activity of PKC on FasL promoter induction. Because no specific
PKC inhibitor was described to date, we addressed the possibility
that rottlerin, which was previously found to inhibit the activity of
PKC
(33) also inhibits PKC. This was prompted by the
high degree of similarity between PKC and PKC (21).
The effect of rottlerin was tested on the in vitro enzymatic activity
of purified PKC or PKC, the latter being a representative of the
Ca2+-dependent PKC subfamily. As a control, the
effect of another compound, Gö6976, which is a selective
inhibitor of Ca2+-dependent PKC enzymes
(34), was also determined. Fig. 2A shows that, as expected,
Gö6976 blocked the enzymatic activity of PKC, but had no
effect on the activity of PKC (Fig. 2A, left).
Conversely, rottlerin inhibited the activity of PKC by nearly 50%,
but had only a marginal effect on PKC (Fig. 2A,
right). Importantly, the various Jurkat cell sublines used
(see below) express PKC but not PKC; conversely, and as a
specificity control, COS cells expressed PKC but not PKC
(Fig. 2B).
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, we
examined whether this drug can inhibit activation of the FasL promoter
in intact T cells induced by transiently overexpressed PKC or
activating stimuli. Jurkat-TAg cells were transfected with
constitutively active (A/E) PKC, and the luciferase activity of a
cotransfected FasL reporter was analyzed in rottlerin-pretreated vs
untreated cells. As an additional control, some cells were pretreated
with Gö6976. In unstimulated cells, rottlerin reduced FasL
promoter activity by 90% in empty vector-transfected cells, and
completely blocked the PKC-A/E-induced activity (Fig. 2
C,
left). In that regard, the effect of rottlerin was very
similar to that of the PKC-K/R mutant (see below). In contrast,
Gö6976 failed to inhibit FasL promoter activity and, in fact,
appeared to augment it. In other experiments, we found that rottlerin
also blocked PKC-, but not ionomycin-induced NF-AT activation (data
no shown). When the cells were stimulated with PMA plus ionomycin,
rottlerin again blocked FasL promoter activation in empty vector- or
PKC-A/E-transfected cells, whereas Gö6976 was markedly less
effective (Fig. 2C, right). Similar results were
obtained when Jurkat-TAg cells were stimulated with an anti-CD3
Ab (data no shown).
In a second approach, Jurkat-TAg cells were cotransfected with
dominant-negative (K/R) PKC mutant and the FasL reporter plasmid,
and luciferase activity was determined in resting or stimulated cells.
Dominant-negative PKC profoundly inhibited the basal (Fig. 2C, left) as well as PMA plus ionomycin-induced
(Fig. 2C, right) or anti-CD3-induced (data
not shown) FasL promoter activity. The level of inhibition reached
80–95%. Taken together, these findings strongly suggest that PKC
is required for FasL activation.
PKC cooperates with Cn to induce FasL expression
Recent studies demonstrated that PKC cooperates with Cn to
activate JNK, NF-AT, and the IL-2 promoter in T cells (24, 25). Therefore, the cooperative induction of FasL by the
combination of PMA and Ca2+ ionophore led us to
determine whether Cn represents a potential Ca2+
target in this system as well. Jurkat-TAg cells were transfected with
various combinations of constitutively active (A/E) mutants of PKC,
PKC, and/or Cn (CaM-AI), and the luciferase activity of a
cotransfected FasL reporter was determined (Fig. 3). Transient overexpression of PKC or
CaM-AI, but not PKC, led to a limited activation of the FasL
promoter. However, when PKC and CaM-AI were combined, a
synergistic activation of the promoter was observed (Fig. 3A). The mean values ± SEM of six independent
experiments were 103 ± 26-fold and 2,740 ± 507-fold
induction for the empty vector- or PKC plus CaM-AI-transfected
groups, respectively. PKC also cooperated with CaM-AI to stimulate
the promoter’s activity, but this effect was 4.5-fold lower than that
induced by PKC plus CaM-AI. Both PKC and PKC, as well as
CaM-AI, were properly overexpressed in the cells (Fig. 3B).
This result indicates that there is considerable selectivity to the
cooperation of PKC with CaM-AI in the activation of the FasL
promoter.
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To investigate in more detail the transcription factor(s) involved
in FasL expression, cells were transfected with constitutively active
PKC and/or Cn mutants together with various FasL reporter
constructs. The latter consisted of the wild-type promoter or mutations
in its distal NF-AT-binding site (
NF-AT), or in the binding sites
for AP-1 (AP-1) or NF-B (NF-B), respectively. These binding
sites were previously found to be involved in FasL expression
(8). The results (Fig. 4A) show that maximal FasL
promoter activation by PKC plus CaM-AI in unstimulated T cells
required all three binding sites, because mutation of any binding site
resulted in at least 68% decrease in promoter activity. Mutation of
the NF-AT-binding site had the most deleterious effect. This was also
evident when the suboptimal induction of the FasL promoter by either
PKC or CaM-AI alone was assessed. Thus, mutating the NF-AT-binding
site completely blocked FasL promoter activity induced by CaM-AI, in
agreement with previous reports (9, 10) and also had a
more severe effect on the PKC-mediated promoter activity when
compared with mutations in the AP-1- or NF-B-binding sites. This is
consistent with our finding that PKC can stimulate the activity of
an NF-AT reporter derived from the IL-2 promoter in T cells (data not
shown).
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B). The
finding that this stimulation did not increase the activity of the FasL
promoter above the level observed in unstimulated cells (compare Fig. 4, B and A) strongly suggests that PKC and Cn
are the predominant, if not exclusive, targets for the action of PMA
and ionomycin, respectively, on FasL. It is noteworthy that the
combination of constitutively active PKC and Cn was by far more
effective than PMA plus ionomycin stimulation in stimulating the
activity of the FasL promoter (Fig. 4, A vs B).
This may reflect the possibility that the less selective stimulation by
PMA and ionomycin induces signals that negatively regulate FasL
expression, e.g., the down-regulation and degradation of PKC by PMA or
the induction of negative regulatory effectors such as
Ca2+/calmodulin-dependent protein kinase II
(35) by ionomycin. This explanation is consistent with our
finding that constitutively active PKC is much more potent than PMA
in inducing an NF-B reporter activity in T cells (N. Coudronniere
and A. Altman, manuscript in preparation). Rottlerin inhibits AICD
FasL expression leads to apoptosis in cells that also express Fas,
a process termed AICD (2, 3, 4, 5, 6). Therefore, to determine
whether PKC plays a physiologically relevant role in regulating
AICD, we investigated whether the inhibition of cellular PKC
activity by rottlerin can reduce AICD. First, we determined that
rottlerin abolishes FasL expression induced by an anti-CD3 mAb or
PMA plus ionomycin in Jurkat CE cells (Fig. 5A). As a control, the level
of actin mRNA was not affected by this pretreatment, suggesting that
rottlerin specifically inhibits FasL expression and, moreover, that
this inhibition is not due to drug toxicity.
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15% of the cells underwent apoptosis
compared with only 3% spontaneous apoptosis in unstimulated and
vehicle-treated cells, or 6–7% apoptosis in unstimulated cells
pretreated with rottlerin or Gö6976 (Fig. 5B).
Rottlerin inhibited AICD by 50% in both anti-CD3- or PMA plus
ionomycin-stimulated cells. In contrast, Gö6976 was completely
ineffective in protecting the T cells from AICD. Under the same
conditions, rottlerin was ineffective in protecting cells from
apoptosis induced by an anti-Fas mAb, indicating that it mediates
AICD protection by inhibiting a step preceding the binding of FasL to
its receptor, Fas, and supporting the notion that it inhibits FasL
expression. Furthermore, the failure to inhibit anti-Fas-induced
apoptosis indicates, again, that rottlerin is not toxic to the cells in
these 6 h cultures. However, due to the toxicity of rottlerin for
Jurkat T cells under prolonged culture conditions (20 h), we could
not evaluate its effects on AICD in longer-term cultures where AICD
reached a level of 50%.
Because of the relatively low level of AICD in this system, we decided
to use the mouse T cell hybridoma A1.1. These cells were characterized
in AICD studies, and undergo extensive apoptosis when stimulated with
an anti-CD3 Ab for a relatively short time period (2).
Pretreatment with rottlerin concentrations of up to 30 µM reduced
cell survival (i.e., increased cell death) by only 10%, i.e., from
a basal level of 90% to 80% (Fig. 5C). When
stimulated for 6 h with an anti-CD3 Ab, the majority of the
cells (65%) underwent apoptosis. However, pretreatment with
rottlerin protected the cells from AICD in a dose-dependent manner,
with 50% protection achieved by 2–3 µM rottlerin. Complete
protection was observed when the cells were pretreated with 10–30 µM
rottlerin (Fig. 5C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Cn are important
components of the TCR-induced pathway that leads to FasL expression.
Previous studies demonstrated that the combination of phorbol ester and
Ca2+ ionophore can replace and mimic the
physiological FasL-inducing signal provided by TCR ligation (14, 15) and induce AICD (16). Furthermore, these and
other studies have linked Cn, and its principal cellular target, NF-AT,
to this pathway, demonstrating that Cn represents the target for the
Ca2+ signal (9, 10, 28). However,
the identity of the target for the other inducing signal provided by
phorbol ester has remained elusive. Because phorbol esters mediate
their pleiotropic cellular effects predominantly via the activation of
conventional (cPKC: , ß, and ) or novel (nPKC: , , 
,
, and µ) PKC enzymes, we sought to determine whether a particular
phorbol ester-responsive PKC isoform is selectively involved in the
positive regulation of FasL expression. For this analysis, we chose
four T cell-expressed isoforms that represent the three PKC
subfamilies, i.e., PKC (cPKC), PKC and PKC (nPKC), and PKC,
a member of the atypical PKC subfamily (aPKC).
Our findings demonstrate that PKC activation preferentially plays an
important, albeit perhaps not exclusive, role in up-regulation of FasL
expression following TCR ligation. This conclusion is based on the
following results. First, constitutively active PKC caused a marked
activation of the FasL promoter, whereas other PKC isoforms were much
less effective or ineffective (Fig. 1). Second, PKC activity is
necessary for FasL expression because transient overexpression of
wild-type PKC in resting cells failed to induce the FasL promoter
(Fig. 1A). Third, dominant-negative PKC effectively
inhibited FasL promoter activation induced by PMA plus ionomycin
treatment (Fig. 2C) or TCR/CD3 ligation (data not shown).
Finally, rottlerin, a compound that inhibits the activity of PKC (as
well as PKC), but not Ca2+-dependent PKCs such
as PKC (Ref. 33 ; Fig. 2A), blocked PKC-
or PMA plus ionomycin-induced FasL promoter activation (Figs. 2C and 5A), and protected two types of T cells
from AICD (Fig. 5, B and C). The absence of
detectable PKC in Jurkat T cells (Fig. 2B) strongly
suggests that PKC is the predominant target for this inhibitory
effect of rottlerin.
Although these results demonstrate that PKC is a critical
rate-limiting factor in the induction of FasL expression, they do not
rule out the possibility that other T cell-expressed PKC isoforms also
play a role in FasL expression, either as components of a
PKC-mediated pathway or as parts of PKC-independent contributory
pathways. This may account for the weak activation of the FasL promoter
by PKC either alone (Fig. 1A) or in combination with Cn
(Fig. 3A) and for the partial inhibition of
PKC-A/E-induced FasL promoter activity by Gö6976 in PMA plus
ionomycin-stimulated cells (Fig. 2C, right). In
this regard, PKC was recently found to up-regulate the expression of
PKC (36), an isoform that is closely related to PKC
(21). This raises the possibility of a cross-talk between
PKC and PKC.
The second novel finding emerging from the current study is that PKC
synergizes with Cn to activate the FasL promoter. This result, as well
as the selective role of PKC in FasL induction, are in general
agreement with recent findings by Villunger et al. (32).
The synergy between PKC and Cn is consistent with the findings that,
first, phorbol ester and Ca2+ ionophore also
synergize to induce FasL expression (14, 15) and, second,
cyclosporin A or FK506 (3, 28, 37) and rottlerin (Fig. 2B), which selectively interfere with the activity of Cn or
PKC, respectively, can each block TCR- or PMA plus ionomycin-induced
FasL expression in different T cell lines. The findings that inhibition
of either Cn or PKC is sufficient to inhibit FasL expression
suggests that the combination of these two signaling elements is
required for optimal induction.
The concentration of rottlerin required for half-maximal protection
from AICD (2–3 µM; Fig. 5C) was 100-fold lower than
that which caused a 50% inhibition of the in vitro PKC kinase
activity (300 µM; Fig. 2A). One possible explanation is
that rottlerin is taken up by the cells in a highly efficient manner
and, thus, accumulates intracellularly to a higher concentration than
in the culture medium. A similar situation has been observed with
another PKC inhibitor, bisindolylmaleimide, in an analysis of apoptosis
in cerebellar granule cells (38).
Several recent studies demonstrated a unique role for PKC in
intracellular signaling pathways induced by TCR engagement. For
example, PKC specifically translocates to the contact site between T
cells and APCs during Ag-specific interactions (22, 23)
and, together with the TCR complex, forms the central core of the newly
described supramolecular activation cluster (23). Second,
PKC specifically activates the JNK/AP-1 pathway (24, 25, 30) and, furthermore, similar to our finding herein (Fig. 3), it
cooperates with Cn to activate JNK and the IL-2 promoter in T cells
(25). Finally, it was very recently suggested that PKC
may be involved in the negative selection of thymocytes
(39). The current study demonstrates that FasL and the
physiological process of AICD represent novel targets of activated
PKC in T cells.
A dual role for PKC in signaling pathways that lead to either T cell
proliferation or apoptosis is not surprising. Proliferative and
apoptotic signals can coexist and mediate crosstalk in the same pathway
(40), with the final result depending on how and when
these signals are integrated. For example, several studies demonstrated
that induction of apoptosis by the JNK pathway is enabled or enhanced
when the activation of the anti-apoptotic MEK/ERK pathway is
blocked (41, 42). Similarly, c-Myc (43, 44)
or Ras (40, 45, 46, 47) are known to generate signals that can
lead to either proliferation or apoptosis, depending on the input of
additional signals provided in stimulus- and cell type-specific
manners.
The upstream and downstream signaling elements that are linked to
PKC in the TCR-initiated signaling pathway leading to FasL
expression and AICD are not well understood, but several relevant clues
emerge from recent studies. Thus, it has recently been shown that the
recruitment of the Zap-70 tyrosine kinase to the TCR-associated
phospho- chain following TCR engagement (48) is
necessary for FasL expression, but not for IL-2 production in
apoptosis-defective cells (49, 50), suggesting a
bifurcation of TCR signals at this level. The related kinase, Syk, can
induce JNK activation (51), suggesting that the effects of
Zap-70 and/or Syk on FasL expression and T cell apoptosis may be
mediated by JNK. In contrast, PKC functions as a selective JNK
activator in T cells (24, 25, 52). Therefore, Zap-70/Syk
and PKC could be linked in a physiological pathway leading to
FasL expression. One possibility is that following their activation,
these tyrosine kinases phosphorylate and activate PLC1, which leads
to diacylglycerol production and subsequent activation of PKC enzymes,
including PKC.
PKC and PKC were found to be involved in the induction of
apoptosis in different cell types (38, 53, 54, 55, 56, 57), but the
molecular basis for their action is poorly understood. These data do
not necessarily mean that PKC and are proapoptotic but, rather,
may reflect the possibility that these enzymes are involved in a signal
transduction pathway, such as the induction of FasL expression
demonstrated here, that can lead to apoptosis under certain
circumstances.
In summary, we have shown that PKC activation is necessary for FasL
expression following TCR engagement or PMA plus ionomycin treatment.
PKC cooperates with Cn to induce FasL expression, and this
cooperation appears to depend on the integrity of the different
transcription factor-binding sites in the FasL promoter. Nonetheless,
the presence of functional PKC is necessary for AICD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Drs. Amnon Altman or Martin Villalba, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address:
3 Current address: Cytovia, San Diego, CA 92121.
4 Current address: Institut National de la Santé et de la Recherche Médicale Unité 80, Hopital E. Herriot, 69437 Lyon Cedex 03, France.
5 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; RE, response element; PKC, protein kinase C; Cn, calcineurin; JNK, c-Jun N-terminal kinase; ß-gal, ß-galactosidase; ERK, extracellular signal-related kinase; MEKK, mitogen-activated protein kinase/ERK kinase; TAg, SV40 large TAg.
Received for publication July 13, 1999. Accepted for publication September 13, 1999.
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  A. G. Granja, E. G. Sanchez, P. Sabina, M. Fresno, and Y. Revilla African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-{theta}-Mediated p300 Transactivation J. Virol., January 15, 2009; 83(2): 969 - 980. [Abstract] [Full Text] [PDF]
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 H. Kao, J. Lin, D. R. Littman, A. S. Shaw, and P. M. Allen Regulated Movement of CD4 In and Out of the Immunological Synapse J. Immunol., December 15, 2008; 181(12): 8248 - 8257. [Abstract] [Full Text] [PDF]
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J. Garaude, R. Farras, G. Bossis, S. Charni, M. Piechaczyk, R. A. Hipskind, and M. Villalba SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes J. Immunol., May 1, 2008; 180(9): 5983 - 5990. [Abstract] [Full Text] [PDF]
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 N. A. Shahabi, K. McAllen, and B. M. Sharp Stromal cell-derived factor 1-{alpha} (SDF)-induced human T cell chemotaxis becomes phosphoinositide 3-kinase (PI3K)-independent: role of PKC-{theta} J. Leukoc. Biol., March 1, 2008; 83(3): 663 - 671. [Abstract] [Full Text] [PDF]
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A. G. Granja, N. D. Perkins, and Y. Revilla A238L Inhibits NF-ATc2, NF-{kappa}B, and c-Jun Activation through a Novel Mechanism Involving Protein Kinase C-{theta}-Mediated Up-Regulation of the Amino-Terminal Transactivation Domain of p300 J. Immunol., February 15, 2008; 180(4): 2429 - 2442. [Abstract] [Full Text] [PDF]
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 G. Despouy, M. Joiner, E. Le Toriellec, R. Weil, and M. H. Stern The TCL1 oncoprotein inhibits activation-induced cell death by impairing PKC{theta} and ERK pathways Blood, December 15, 2007; 110(13): 4406 - 4416. [Abstract] [Full Text] [PDF]
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 J. Kanchanapoo, M. Ao, R. Prasad, C. Moore, C. Kay, P. Piyachaturawat, and M. C. Rao Role of protein kinase C-{delta} in the age-dependent secretagogue action of bile acids in mammalian colon Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1851 - C1861. [Abstract] [Full Text] [PDF]
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 J. A. Jones, R. E. Stroud, B. S. Kaplan, A. M. Leone, J. E. Bavaria, J. H. Gorman III, R. C. Gorman, and J. S. Ikonomidis Differential Protein Kinase C Isoform Abundance in Ascending Aortic Aneurysms From Patients With Bicuspid Versus Tricuspid Aortic Valves Circulation, September 11, 2007; 116(11_suppl): I-144 - I-149. [Abstract] [Full Text] [PDF]
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K. Podar, M. S. Raab, J. Zhang, D. McMillin, I. Breitkreutz, Y.-T. Tai, B. K. Lin, N. Munshi, T. Hideshima, D. Chauhan, et al. Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl) Blood, February 15, 2007; 109(4): 1669 - 1677. [Abstract] [Full Text] [PDF]
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M. J. Grybko, A. T. Pores-Fernando, G. A. Wurth, and A. Zweifach Protein kinase C activity is required for cytotoxic T cell lytic granule exocytosis, but the {theta} isoform does not play a preferential role J. Leukoc. Biol., February 1, 2007; 81(2): 509 - 519. [Abstract] [Full Text] [PDF]
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S. Manicassamy and Z. Sun The Critical Role of Protein Kinase C-{theta} in Fas/Fas Ligand-Mediated Apoptosis J. Immunol., January 1, 2007; 178(1): 312 - 319. [Abstract] [Full Text] [PDF]
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J. Garaude, S. Cherni, S. Kaminski, E. Delepine, C. Chable-Bessia, M. Benkirane, J. Borges, A. Pandiella, M. A. Iniguez, M. Fresno, et al. ERK5 Activates NF-{kappa}B in Leukemic T Cells and Is Essential for Their Growth In Vivo J. Immunol., December 1, 2006; 177(11): 7607 - 7617. [Abstract] [Full Text] [PDF]
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M. D. Godeny and P. P. Sayeski ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKC{zeta}-controlled ERK1/2 activity within the nucleus Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1297 - C1307. [Abstract] [Full Text] [PDF]
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 F. Renault-Mihara, F. Beuvon, X. Iturrioz, B. Canton, S. De Bouard, N. Leonard, S. Mouhamad, A. Sharif, J. W. Ramos, M.-P. Junier, et al. Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase C{delta}-dependent Mechanism Mol. Biol. Cell, December 1, 2006; 17(12): 5141 - 5152. [Abstract] [Full Text] [PDF]
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K. Hayashi and A. Altman Filamin A Is Required for T Cell Activation Mediated by Protein Kinase C-{theta} J. Immunol., August 1, 2006; 177(3): 1721 - 1728. [Abstract] [Full Text] [PDF]
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 M. Oda, S. Ikari, T. Matsuno, Y. Morimune, M. Nagahama, and J. Sakurai Signal Transduction Mechanism Involved in Clostridium perfringens Alpha-Toxin-Induced Superoxide Anion Generation in Rabbit Neutrophils. Infect. Immun., May 1, 2006; 74(5): 2876 - 2886. [Abstract] [Full Text] [PDF]
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 M. Ding, C. Huang, Y. Lu, L. Bowman, V. Castranova, and V. Vallyathan Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L291 - L297. [Abstract] [Full Text] [PDF]
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P. Contini, M. Ghio, A. Merlo, A. Poggi, F. Indiveri, and F. Puppo Apoptosis of Antigen-Specific T Lymphocytes upon the Engagement of CD8 by Soluble HLA Class I Molecules Is Fas Ligand/Fas Mediated: Evidence for the Involvement of p56lck, Calcium Calmodulin Kinase II, and Calcium-Independent Protein Kinase C Signaling Pathways and for NF-{kappa}B and NF-AT Nuclear Translocation J. Immunol., December 1, 2005; 175(11): 7244 - 7254. [Abstract] [Full Text] [PDF]
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R. M. Marshall, D. Salerno, J. Garriga, and X. Grana Cyclin T1 Expression Is Regulated by Multiple Signaling Pathways and Mechanisms during Activation of Human Peripheral Blood Lymphocytes J. Immunol., November 15, 2005; 175(10): 6402 - 6411. [Abstract] [Full Text] [PDF]
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 J.-A. Park, F. He, L. D. Martin, Y. Li, B. N. Chorley, and K. B. Adler Human Neutrophil Elastase Induces Hypersecretion of Mucin from Well-Differentiated Human Bronchial Epithelial Cells in Vitro via a Protein Kinase C{delta}-Mediated Mechanism Am. J. Pathol., September 1, 2005; 167(3): 651 - 661. [Abstract] [Full Text] [PDF]
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K. Gulow, M. Kaminski, K. Darvas, D. Suss, M. Li-Weber, and P. H. Krammer HIV-1 Trans-Activator of Transcription Substitutes for Oxidative Signaling in Activation-Induced T Cell Death J. Immunol., May 1, 2005; 174(9): 5249 - 5260. [Abstract] [Full Text] [PDF]
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M. Cippitelli, C. Fionda, D. Di Bona, M. Piccoli, L. Frati, and A. Santoni Hyperthermia Enhances CD95-Ligand Gene Expression in T Lymphocytes J. Immunol., January 1, 2005; 174(1): 223 - 232. [Abstract] [Full Text] [PDF]
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 Y. Aiba, M. Oh-hora, S. Kiyonaka, Y. Kimura, A. Hijikata, Y. Mori, and T. Kurosaki Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation PNAS, November 23, 2004; 101(47): 16612 - 16617. [Abstract] [Full Text] [PDF]
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 N. N. Berg-Brown, M. A. Gronski, R. G. Jones, A. R. Elford, E. K. Deenick, B. Odermatt, D. R. Littman, and P. S. Ohashi PKC{theta} Signals Activation versus Tolerance In Vivo J. Exp. Med., March 15, 2004; 199(6): 743 - 752. [Abstract] [Full Text] [PDF]
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 J. Pardo, M. Buferne, M.-J. Martinez-Lorenzo, J. Naval, A.-M. Schmitt-Verhulst, C. Boyer, and A. Anel Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression Int. Immunol., December 1, 2003; 15(12): 1441 - 1450. [Abstract] [Full Text] [PDF]
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 M. Ishaq, G. DeGray, and V. Natarajan Protein Kinase C{theta} Modulates Nuclear Receptor-Corepressor Interaction during T Cell Activation J. Biol. Chem., October 10, 2003; 278(41): 39296 - 39302. [Abstract] [Full Text] [PDF]
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 S. A. Trushin, K. N. Pennington, E. M. Carmona, S. Asin, D. N. Savoy, D. D. Billadeau, and C. V. Paya Protein Kinase C{alpha} (PKC{alpha}) Acts Upstream of PKC{theta} To Activate I{kappa}B Kinase and NF-{kappa}B in T Lymphocytes Mol. Cell. Biol., October 1, 2003; 23(19): 7068 - 7081. [Abstract] [Full Text] [PDF]
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S. A. Crist, T. S. Griffith, and T. L. Ratliff Structure/Function Analysis of the Murine CD95L Promoter Reveals the Identification of a Novel Transcriptional Repressor and Functional CD28 Response Element J. Biol. Chem., September 19, 2003; 278(38): 35950 - 35958. [Abstract] [Full Text] [PDF]
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 C. E. Runyan, H. W. Schnaper, and A.-C. Poncelet Smad3 and PKC{delta} mediate TGF-{beta}1-induced collagen I expression in human mesangial cells Am J Physiol Renal Physiol, September 1, 2003; 285(3): F413 - F422. [Abstract] [Full Text] [PDF]
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M. B. Hock and M. A. Brown Nuclear Factor of Activated T Cells 2 Transactivation in Mast Cells: A NOVEL ISOFORM-SPECIFIC TRANSACTIVATION DOMAIN CONFERS UNIQUE Fc{epsilon}RI RESPONSIVENESS J. Biol. Chem., July 11, 2003; 278(29): 26695 - 26703. [Abstract] [Full Text] [PDF]
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E. J. Nelson, K. Hellevuo, M. Yoshimura, and B. Tabakoff Ethanol-induced Phosphorylation and Potentiation of the Activity of Type 7 Adenylyl Cyclase. INVOLVEMENT OF PROTEIN KINASE C delta J. Biol. Chem., February 7, 2003; 278(7): 4552 - 4560. [Abstract] [Full Text] [PDF]
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M. A. del Pozo, M. A. Schwartz, J. Hu, W. B. Kiosses, A. Altman, and M. Villalba Guanine Exchange-Dependent and -Independent Effects of Vav1 on Integrin-Induced T Cell Spreading J. Immunol., January 1, 2003; 170(1): 41 - 47. [Abstract] [Full Text] [PDF]
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S. Mukherjee, P. K. Maiti, and D. Nandi Role of CD80, CD86, and CTLA4 on mouse CD4+ T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin J. Leukoc. Biol., November 1, 2002; 72(5): 921 - 931. [Abstract] [Full Text] [PDF]
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Y.-J. Chwae, M. J. Chang, S. M. Park, H. Yoon, H.-J. Park, S. J. Kim, and J. Kim Molecular Mechanism of the Activation-Induced Cell Death Inhibition Mediated by a p70 Inhibitory Killer Cell Ig-Like Receptor in Jurkat T Cells J. Immunol., October 1, 2002; 169(7): 3726 - 3735. [Abstract] [Full Text] [PDF]
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M. Ishaq, M. Fan, K. Wigmore, A. Gaddam, and V. Natarajan Regulation of Retinoid X Receptor Responsive Element-Dependent Transcription in T Lymphocytes by Ser/Thr Phosphatases: Functional Divergence of Protein Kinase C (PKC){theta} and PKC{alpha} in Mediating Calcineurin-Induced Transactivation J. Immunol., July 15, 2002; 169(2): 732 - 738. [Abstract] [Full Text] [PDF]
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 M. Villalba, K. Bi, J. Hu, Y. Altman, P. Bushway, E. Reits, J. Neefjes, G. Baier, R. T. Abraham, and A. Altman Translocation of PKC{theta} in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C J. Cell Biol., April 15, 2002; 157(2): 253 - 263. [Abstract] [Full Text] [PDF]
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A. T. Miller and L. J. Berg Defective Fas Ligand Expression and Activation-Induced Cell Death in the Absence of IL-2-Inducible T Cell Kinase J. Immunol., March 1, 2002; 168(5): 2163 - 2172. [Abstract] [Full Text] [PDF]
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A. R. Moise, J. R. Grant, T. Z. Vitalis, and W. A. Jefferies Adenovirus E3-6.7K Maintains Calcium Homeostasis and Prevents Apoptosis and Arachidonic Acid Release J. Virol., February 15, 2002; 76(4): 1578 - 1587. [Abstract] [Full Text] [PDF]
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 P. SIGNORELLI, C. LUBERTO, and Y. A. HANNUN Ceramide inhibition of NF-{kappa}B activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response FASEB J, November 1, 2001; 15(13): 2401 - 2414. [Abstract] [Full Text] [PDF]
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D. Krappmann, A. Patke, V. Heissmeyer, and C. Scheidereit B-Cell Receptor- and Phorbol Ester-Induced NF-{kappa}B and c-Jun N-Terminal Kinase Activation in B Cells Requires Novel Protein Kinase C's Mol. Cell. Biol., October 1, 2001; 21(19): 6640 - 6650. [Abstract] [Full Text] [PDF]
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 S.-C. Hsieh, K.-H. Sun, C.-Y. Tsai, Y.-Y. Tsai, S.-T. Tsai, D.-F. Huang, S.-H. Han, H.-S. Yu, and C.-L. Yu Monoclonal anti-double stranded DNA antibody is a leucocyte-binding protein to up-regulate interleukin-8 gene expression and elicit apoptosis of normal human polymorphonuclear neutrophils Rheumatology, August 1, 2001; 40(8): 851 - 858. [Abstract] [Full Text] [PDF]
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M. Villalba, P. Bushway, and A. Altman Protein Kinase C-{{theta}} Mediates a Selective T Cell Survival Signal Via Phosphorylation of BAD J. Immunol., May 15, 2001; 166(10): 5955 - 5963. [Abstract] [Full Text] [PDF]
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R. Wang, L. Zhang, X. Zhang, J. Moreno, X. Luo, M. Tondravi, and Y. Shi Differential Regulation of the Expression of CD95 Ligand, Receptor Activator of Nuclear Factor-{{kappa}}B Ligand (RANKL), TNF-Related Apoptosis-Inducing Ligand (TRAIL), and TNF-{{alpha}} During T Cell Activation J. Immunol., February 1, 2001; 166(3): 1983 - 1990. [Abstract] [Full Text] [PDF]
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 F. Rusnak and P. Mertz Calcineurin: Form and Function Physiol Rev, October 1, 2000; 80(4): 1483 - 1521. [Abstract] [Full Text] [PDF]
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C. E. Ahnadi, P. Giguère, S. Gravel, D. Gagné, A.-C. Goulet, T. Fülöp Jr, M. D. Payet, and G. Dupuis Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibition of CD3- but not Ti-dependent antibody-triggered Ca2+ signaling J. Leukoc. Biol., August 1, 2000; 68(2): 293 - 300. [Abstract] [Full Text]
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B. Bauer, N. Krumbock, F. Fresser, F. Hochholdinger, M. Spitaler, A. Simm, F. Uberall, B. Schraven, and G. Baier Complex Formation and Cooperation of Protein Kinase Ctheta and Akt1/Protein Kinase Balpha in the NF-kappa B Transactivation Cascade in Jurkat T Cells J. Biol. Chem., August 17, 2001; 276(34): 31627 - 31634. [Abstract] [Full Text] [PDF]
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N. Coudronniere, M. Villalba, N. Englund, and A. Altman NF-kappa B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-theta PNAS, March 28, 2000; 97(7): 3394 - 3399. [Abstract] [Full Text] [PDF]
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M. Villalba, K. Bi, J. Hu, Y. Altman, P. Bushway, E. Reits, J. Neefjes, G. Baier, R. T. Abraham, and A. Altman Translocation of PKC{theta} in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C J. Cell Biol., April 15, 2002; 157(2): 253 - 263. [Abstract] [Full Text] [PDF]
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