From the Department of Pediatric Oncology, Dana
Farber Cancer Institute, Boston, Massachusetts 02115 and the
§ Division of Hematology and Oncology, Beth Israel Deaconess
Medical Center, Boston, Massachusetts 02215
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ABSTRACT |
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T cells can undergo activation-induced cell death
(AICD) upon stimulation of the T cell receptor-CD3 complex. We found
that the extracellular signal-regulated kinase (ERK) pathway is
activated during AICD. Transient transfection of a dominant interfering mutant of mitogen-activated/extracellular signal-regulated receptor protein kinase kinase (MEK1) demonstrated that down-regulation of the
ERK pathway inhibited FasL expression during AICD, whereas activation
of the ERK pathway with a constitutively active MEK1 resulted in
increased expression of FasL. We also found that pretreatment with the
specific MEK1 inhibitor PD98059 prevented the induction of FasL
expression during AICD and inhibited AICD. However, PD98059 had no
effect on other apoptotic stimuli. We found only very weak ERK activity
during Fas-mediated apoptosis (induced by Fas cross-linking). Furthermore, preincubation with the MEK1 inhibitor did not inhibit Fas-mediated apoptosis. Finally, we also demonstrated that pretreatment with the MEK1 inhibitor could delay and decrease the expression of the
orphan nuclear steroid receptor Nur77, which has been shown to be
essential for AICD. In conclusion, this study demonstrates that the ERK
pathway is required for AICD of T cells and appears to regulate the
induction of Nur77 and FasL expression during AICD.
AICD1 was first
described in T cell hybridomas and is defined as apoptosis of
lymphocytes by any signal that results in lymphocyte activation and, in
particular, by stimulation of the TCR·CD3 complex (or B cell receptor
complex) with antigens or antibodies (1, 2). AICD is thought to play an
important role in the deletion of (a) autoreactive T cell
clones in the thymus (negative selection), (b) autoreactive
T cells in the periphery with specificity for autoantigens that are not
presented in the thymus (peripheral tolerance), and (c)
activated T cells at the termination of an immune response (1).
Several studies have shown that the expression and interaction of Fas
and FasL, which results in autocrine stimulation of the Fas death
pathway, is required for AICD of T hybridoma cells, Jurkat T leukemia
cells, and activated T cells (3-6). AICD could be inhibited with
nonstimulatory anti-Fas (CD95) antibodies or with Fas-IgFc fusion
protein. Recent studies have shown that the expression of FasL during
AICD is dependent on an intact CD3- In T cells, stimulation of the TCR·CD3 complex results in activation
of the Ras-Raf-MEK1/2-ERK1/2 pathway and, in combination with signals
through the calcium/calcineurin pathway and the Jun N-terminal kinase
pathway, leads to IL-2 production (reviewed in Ref. 13). T cell
activation through the TCR·CD3 complex can be inhibited by dominant
interfering Ras or dominant interfering Raf. ERK1/2 activity is also
required for thymic positive selection in vivo, as
demonstrated in mice expressing a dominant interfering form of Ras (14)
or a dominant interfering form of MEK1 (15).
ERK1 and -2 are proline-directed protein serine/threonine kinases that
belong to the family of MAPKs and phosphorylate Ser/Thr-Pro motifs in
various substrates (reviewed in Ref. 16). The ERK pathway can be
activated by many stimuli, including growth factors (such as epidermal
growth factor and nerve growth factor), cytokines (such as IL-2),
antigen receptor stimulation (TCR), insulin, and v-Src expression. ERK1
and -2 are directly activated through dual phosphorylation of tyrosine
and threonine residues in a conserved TEY motif by the specific protein
MAPK kinases MEK1 and -2. MEK1 and -2 can be activated by the MAPK
kinase kinase Raf. The initiation of this Raf-MEK1/2-ERK1/2 cascade
occurs through the recruitment of Raf to the plasma membrane by the
guanine nucleotide binding protein p21ras.
Nur77 is an orphan nuclear steroid receptor (17) and is essential for
AICD in T cell hybridomas and thymocytes, but not for IL-2 secretion
(18, 19). To date, no ligand for Nur77 has been identified. Rapid Nur77
mRNA expression can be found 30 min after stimulation of T cell
hybridoma cells with anti-CD3 antibody, which induces apoptotic DNA
ladders as early as 4 h poststimulation (18). In contrast, no
Nur77 expression was found during dexamethasone-induced apoptosis or
IL-2 withdrawal apoptosis of CTLL-20 cells (18, 19). A
dominant-interfering truncated Nur77 or antisense Nur77 was capable of
preventing AICD (but not IL-2 production) after TCR stimulation of T
cell hybridomas (18, 19).
In this study, we define a role for the ERK pathway in AICD and
demonstrate its requirement in the up-regulation of FasL and expression
of Nur77.
Reagents--
The MEK1 inhibitor PD98059 was obtained from New
England Biolabs (Beverly, MA) and dissolved in Me2SO at a
final concentration of 50 µM. FasIgFc fusion protein was
kindly provided by Dr. Shyr-Te Ju (Boston University School of
Medicine). Cyclosporin A was provided by Sandoz Pharmaceuticals Corp.
(East Hanover, NJ), and FK506 was received from Fujisawa USA
(Deerfield, IL). Ionomycin and 4 Cell Lines--
The murine T cell hybridoma cell line DO11.10
(22) was kindly provided by Dr. Barbara Osborne (University of
Massachusetts, Amherst, MA). The human T cell leukemia line Jurkat was
a gift of Dr. Paul Anderson (Dana Farber Cancer Institute, Boston, MA). Jurkat-16 and Jurkat-77 are subclones of the Jurkat cell line and were
kindly provided by Dr. Jannie Borst (J16; Netherlands Cancer Institute,
Amsterdam, The Netherlands) (4) and Dr. K. Smith (J77; Cornell
University, New York, NY). The murine B cell hybridoma cell line LK
35.2 (23) was obtained from Dr. Christoph Klein (Children's Hospital,
Boston). All cell lines were grown in RPMI 1640 containing 10%
heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine.
Intracytoplasmic Fluorescence Staining--
Cells (5 × 106/ml) were washed twice with PBS and incubated for 20 min
at room temperature with 4% formaldehyde. After three washes with PBS,
cells were incubated with 0.1% Triton X-100 (in PBS) for 2 min at room
temperature, washed three times with PBS, and incubated with PBS, 2%
bovine serum albumin, 0.1% azide for 10 min at 4 °C. Subsequently,
cells were incubated with anti-FasL-PE antibody for 30 min at 4 °C,
washed with PBS (with bovine serum albumin and azide), and analyzed
with a flow cytometer.
In Vitro Kinase Assay and Western Blot Analysis--
In
vitro kinase assays and Western blot analyses were performed as
described by Hibi et al. (24) with modifications. Briefly, after freezing at Apoptosis Assay--
For cross-linking experiments, cell culture
dishes were coated overnight at 4 °C with anti-CD3 or anti-TCR
(1-10 µg/ml) antibodies. Cells (106/ml) were exposed to
an apoptotic stimulus and incubated at 37 °C and 5%
CO2. Apoptotic cells were analyzed by flow cytometry after
staining with hypotonic propidium iodide (PI) solution as described
previously (25). Briefly, cells (2 × 105) were
incubated for 10 min at 4 °C in the dark with 200 µl of hypotonic
buffer containing 0.1% (w/v) sodium citrate, 0.1% Triton X-100, and
50 µg/ml propidium iodide. The cell suspension was then analyzed by
flow cytometry, and the subdiploid peak (which represents apoptotic
nuclei undergoing DNA fragmentation) was measured to determine the
percentage of apoptotic cells.
FasL Functional Activity--
FasL functional activity was
determined by the ability of FasL expressing cells to induce apoptosis
in Fas+ LK 35.2 target cells as described previously (6).
Briefly, 5 × 106 LK 35.2 cells were labeled for
1 h at 37 °C with 20 µCi 51Cr, washed three
times, and resuspended in RPMI 1640 medium with 10% fetal calf serum.
T cell hybridomas or Jurkat cells were incubated (105
cells/well) for 3 h at 37 °C in anti-CD3-coated 96-well plates before 51Cr-labeled LK 35.2 target cells (104
cells) were added. Some cells received only 51Cr-labeled
LK35.2 cells and anti-Fas antibodies (Jo2; 5 µg/ml). After an
additional 8-h incubation, 100 µl of supernatant was removed from
each well and counted in a Transient Transfection--
In transient transfection
experiments, J16 (107) cells were electroporated at 800 microfarads/250 V using a Life Technologies, Inc. electroporator. Cells
received 10 µg of control plasmid in addition to 10 µg of a FasL
promoter-luciferase reporter construct, 20 µg of experimental
construct, and 0.2 µg of pRL-TK to normalize for transfection
efficiency. Subsequently, cells were incubated for 18 h at
37 °C and stimulated with phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) plus ionomycin (2 µM) or plate-bound anti-CD3 (1 µg/ml) as described above. After 6 h, luciferase activity was measured with a luminometer according to the manufacturer's
instructions (Analytical Luminescence, San Diego, CA).
CD3·TCR-mediated AICD Involves ERK1/2 Phosphorylation and
Activation--
We performed all of our experiments in two T cell
lines that are widely used for the study of AICD in T cells: the murine T cell hybridoma DO11.10, which expresses a TCR specific for
OVA323-339, a peptide derived from chicken ovalbumin (22), and the
human T cell leukemia line Jurkat-16 (a variant of the Jurkat cell
line) (4). TCR·CD3 cross-linking leads to AICD in both cell lines, whereas Jurkat-16 cells also undergo apoptosis upon Fas cross-linking. In addition, we used in some experiments regular Jurkat cells, which
were only sensitive to Fas-mediated apoptosis, or Jurkat-77 cells,
which do not undergo AICD upon TCR·CD3 stimulation and are resistant
to Fas-mediated apoptosis.
We first determined if the ERK pathway is being activated during
TCR·CD3-mediated AICD as has been described for TCR·CD3-mediated activation, proliferation, and IL-2 production (13). Activation of ERK1
and -2 requires phosphorylation of specific tyrosine and threonine
residues, and we therefore assessed tyrosine phosphorylation of ERK1
and -2 after CD3 cross-linking in DO11.10 cells by Western blot
analysis of whole cell lysates with an antibody that specifically recognizes tyrosine-phosphorylated ERK1 and -2 (tyrosine 204). Maximal
phosphorylation of (mostly) ERK2 occurred 15 min after CD3
cross-linking, but ERK1 and -2 remained phosphorylated at later time
points (Fig. 1A) up to 6 h (data not shown). Expression of ERK1 and -2 was determined by
immunoblotting with anti-ERK antibodies, and expression levels were
unaffected by CD3 cross-linking. We also determined with an in
vitro kinase assay that the phosphorylation of ERK1 and -2 (Fig.
1A) correlated with the induction of ERK activity during
AICD (Fig. 1B). Subsequently, we analyzed ERK1 and -2 phosphorylation after CD3 cross-linking in two Jurkat subclones: J16
(sensitive to AICD and Fas-mediated apoptosis) and J77 (resistant to
AICD and Fas-mediated apoptosis). We found that the kinetics of ERK1
and -2 phosphorylation in these two cell lines was identical (Fig.
1C) and demonstrated a similar pattern as found in DO11.10 cells. All three cell lines demonstrated maximal phosphorylation at 15 min of predominantly ERK2. These data make it unlikely that the
kinetics of ERK phosphorylation can account for the differences in
susceptibility to AICD seen in these cell lines.
MEK1 Inhibitor PD98059 Inhibits ERK Phosphorylation and Activation
during AICD--
We then determined if a widely used specific
inhibitor of the ERK pathway, MEK1 inhibitor PD98059, could inhibit ERK
activation during AICD. PD98059 binds to the inactive form of MEK1 and
prevents its activation by upstream activators. This inhibitor has been shown to act as a highly selective inhibitor of the activation of MEK1
in the ERK pathway (26, 27). PD98059 inhibits activation and
phosphorylation of MEK1 by either c-Raf or MEKK1 with IC50 values of 5-10 µM but inhibits the activation of MEK2 by
c-Raf less potently with an IC50 of 50 µM. In
all experiments, cells were pretreated with 50 µM of
PD98059 for 30 min at 37 °C. We found that pretreatment with the
MEK1 inhibitor PD98059 prevented phosphorylation and activation of ERK1
and -2 completely for at least 2 h after CD3 cross-linking (Fig.
1, A and B). However, phosphorylation and
activation of ERK1 and -2 after PMA and ionomycin stimulation was only
diminished and not completely inhibited. This partial inhibition could
be due to incomplete inhibition of MEK2 by PD98059 or could be the
result of an alternative pathway for ERK phosphorylation, which does
not involve MEK1 and 2 and/or is not affected by PD98059. Similar
results were obtained with Jurkat-16 cells (data not shown).
We also determined the effect of the MEK1 inhibitor PD98059 on
the activation of MEK1 upon CD3 stimulation in J16 cells with an
antibody that specifically recognizes serine phosphorylation of MEK1
and -2 (serines 217 and 221). Maximal phosphorylation of MEK1/2
occurred after 5 min and could be partially inhibited by the MEK1
inhibitor (Fig. 1D). The incomplete inhibition of MEK1/2
phosphorylation was probably due to the less potent effect of the MEK1
inhibitor against MEK2 activation as described above.
MEK1 Activity Regulates FasL Promotor Activation during
AICD--
The up-regulation of FasL has been identified as an
essential step in TCR·CD3-mediated apoptosis in both cell lines
(DO11.10 and Jurkat-16) and, in general, during AICD (4-6). We
confirmed the requirement of the Fas/FasL interaction during AICD by
inhibiting AICD of DO11.10 T cell hybridomas with Fas-IgFc fusion
protein (data not shown). Fas-IgFc binds to FasL and prevents its
interaction with the Fas receptor (6). To evaluate the role of the ERK pathway in the regulation of FasL expression during AICD, we
co-transfected the human FasL promotor coupled to a luciferase reporter
construct along with either a dominant interfering form or a
constitutively active form of MEK1, the upstream activator of ERK, in
Jurkat-16 cells. CD3 cross-linking resulted in FasL promotor
activation, and cotransfection of a dominant-interfering form of MEK1
could inhibit this activation by almost 50% (Fig.
2A). Cotransfection of a
constitutively active MEK1 construct led to a 4-5-fold increase in
FasL promotor activation in unstimulated cells and a further 6.5-fold
increase in FasL promotor activation upon CD3 cross-linking. These
results indicate that MEK1 activity can regulate FasL expression during
AICD.
The MEK1 Inhibitor PD98059 Inhibits FasL Promotor Activation during
AICD--
We further studied the role of MEK1 (and the ERK pathway) in
FasL expression during AICD with the use of the MEK1 inhibitor PD98059.
As shown in Fig. 2B, pretreatment of Jurkat-16 cells with
the MEK1 inhibitor could inhibit FasL promotor activation upon CD3
cross-linking. This result confirms our finding with transient
transfection of MEK1 constructs that MEK1 activity is required for FasL
up-regulation during AICD.
The Inhibition of MEK1 Activity Prevents FasL Expression and
Activity after CD3·TCR Stimulation--
Subsequently, we studied the
regulation of FasL expression and activity by MEK1 during AICD. We
first determined FasL expression by flow cytometric analysis after
intracytoplasmic staining with anti-FasL antibodies. We could not
detect any FasL in unstimulated cells (<1%), whereas a 4-h incubation
with anti-CD3 resulted in 13% positive cells. Pretreatment of the
cells with Me2SO (drug vehicle as control) had no effect on
the induction of FasL expression (13% positive cells), whereas
pretreatment with the MEK1 inhibitor completely prevented expression of
FasL (<1% positive cells) (Fig. 3A).
We then determined the induction of FasL activity in a cytolytic assay
with 51Cr-labeled Fas-sensitive target cells (6, 28, 29).
This assay measures FasL activity of the effector cells (DO11.10) by their capability to lyse Fas-sensitive target cells (B cell hybridoma LK 35.2). We found that CD3 cross-linking of DO11.10 cells resulted in
the induction of FasL activity, which indicates that DO11.10 cells
up-regulate their FasL cell surface expression upon CD3·TCR activation (Fig. 3B). We confirmed the specificity of this
assay by demonstrating that preincubation of CD3-stimulated DO11.10 cells with FasIgFc could inhibit the lysis of the Fas-sensitive target
cells (data not shown). Pretreatment of the DO11.10 cells with the MEK1
inhibitor abolished the induction of FasL activity after CD3
cross-linking (Fig. 3B).
AICD Is Inhibited by the MEK1 Inhibitor PD98059--
The use
of the MEK1 inhibitor in cell cultures allowed us to test the role of
the ERK pathway in CD3-mediated apoptosis assays. Pretreatment of
DO11.10 cells with PD98059 resulted in partial inhibition and a 2-4-h
delay in the onset of AICD (Fig.
4A). Because PD98059 is only
effective for a few hours in cell cultures (see Fig. 1, A
and B), we determined if a second addition of the inhibitor would result in a stronger effect. Indeed, the inhibitory effect of
PD98059 on AICD of DO11.10 cells could be prolonged and enhanced by a
second treatment with PD98059 4 h after the initial treatment (Fig. 4B). We repeated these experiments with Jurkat-16
cells and also found inhibition of AICD after pretreatment with MEK1 inhibitor (Fig. 4C). These results suggest that inhibition
of the ERK pathway leads to a marked inhibition of AICD. The inhibitory effect of PD98059 on AICD was confirmed in similar experiments with
another murine T cell hybridoma cell line A1.1 (data not shown).
Fas-mediated Apoptosis Is Not Affected by Inhibition of MEK1
Activity--
To better evaluate the role of the ERK cascade in the
more distal Fas-mediated component of the AICD pathway, we studied
Fas-mediated apoptosis of Jurkat cells that resulted from Fas
cross-linking (4). We assessed the phosphorylation of ERK1 and -2 during Fas-mediated apoptosis of Jurkat cells by Western blot analysis of whole cell lysates with antibodies against tyrosine-phosphorylated ERK1 and -2. Only very weak, unsustained phosphorylation of ERK2 was
found 2 h after Fas cross-linking (Fig.
5A). In addition, we
determined activation of ERK1 and 2 after Fas cross-linking of Jurkat
cells by in vitro kinase assays. These results were comparable with the analysis of ERK phosphorylation by Western blotting. Very little ERK activation was seen at 2 h after Fas cross-linking, and this activation was not sustained (Fig.
5B). Subsequently, we determined the effect of the MEK1
inhibitor on Fas-mediated apoptosis of Jurkat cells and found no
inhibition (Fig. 5C). In addition, we found no inhibition by
the MEK1 inhibitor PD98059 of Fas-mediated apoptosis of LK 35.2 cells
(Fig. 3B), J16 cells (Fig. 4C), and fresh murine
thymocytes (data not shown). These results suggest that activation of
the ERK pathway is not required for Fas-mediated apoptosis but is
necessary for AICD.
The Anti-apoptotic Effect of the MEK1 Inhibitor PD98059 Is
Restricted to AICD--
To evaluate the role of the ERK pathway in
other forms of apoptosis, we pretreated DO11.10 cells with PD98059 and
induced apoptosis by several other stimuli, including corticosteroids (dexamethasone), chemotherapeutic drugs with an inhibitory effect on
topoisomerase II (adriamycin and etoposide), C2-ceramide,
and radiation. We found no inhibitory effect of the MEK1 inhibitor on
any of the forms of apoptosis tested (Fig.
6). The anti-apoptotic effect of PD98059
in T cells seems to be specific and restricted to AICD. This suggests
that activation of the ERK pathway is required for AICD but not for any
of the other forms of apoptosis tested.
The Inhibition of MEK1 Activity Prevents Nur77 Expression during
AICD--
Liu et al. (19) used the DO11.10 cell line for
the identification of an orphan steroid receptor Nur77, which is
required for AICD in T cell hybridomas and thymocytes. Nur77 was
maximally expressed 2 h after CD3·TCR cross-linking of DO11.10
cells. We confirmed CD3·TCR-stimulated expression of Nur77 within
2 h by Western blot analysis of whole cell lysates with anti-Nur77
antibodies (Fig. 7). Interestingly,
pretreatment of DO11.10 cells with the MEK1 inhibitor prevented this
rapid expression of Nur77. In further experiments, we found a 1-2-h
delay and overall decrease in Nur77 expression (data not shown). These
results suggest that the ERK pathway is required for Nur77 expression
during AICD.
In this study, we describe the requirement of signaling
through the ERK pathway for AICD of T cells. Several studies have demonstrated activation of the MAPK pathways (ERK, Jun N-terminal kinase, and p38) during apoptosis, and stimulatory or inhibitory effects of these pathways on apoptosis have been found to depend on
the cell type, activation or differentiation state, apoptotic stimulus,
and cell death pathway.
Xia et al. (31) demonstrated an important role for the MAPK
pathways in apoptosis of PC12 cells caused by withdrawal of the
survival factor nerve growth factor by transfection of dominant interfering or constitutively active mutants. In this model for neuronal apoptosis, the ERK pathway seemed to have an antiapoptotic role, whereas the Jun N-terminal kinase and p38 pathways had
proapoptotic effects.
In T cells, the ERK pathway has been linked to various cellular
processes, such as IL-2 expression, anergy, activation, and proliferation (13). Our data in AICD of T cells suggest a proapoptotic role for the ERK pathway in AICD in contrast to the antiapoptotic role
described for the ERK pathway in apoptosis after nerve growth factor withdrawal (31). Moreover, we also found that pretreatment with
MEK1 inhibitor could inhibit AICD of activated nontransformed CD4+
splenocytes (data not shown), which confirms the importance of the ERK
pathway as a proapoptotic regulator in AICD of T cells. The observation
that the ERK pathway can induce apoptosis in certain situations is in
agreement with studies by Kauffman-Zeh et al. (32), which
demonstrated a proapoptotic role for the ERK pathway in c-Myc-induced
apoptosis of fibroblasts after transfection with partial
loss-of-function Ras mutants and by Sutherland et al. (33),
who found an association between ERK2 activation and B-cell antigen
receptor-induced apoptosis in B lymphoma cells. Interestingly, we found
that pretreatment with the MEK1 inhibitor would not only inhibit AICD
but also inhibited IL-2 secretion (as measured by enzyme-linked
immunosorbent assay) after CD3 cross-linking of DO11.10 cells (data not
shown). Our data suggest a dual role for the ERK pathway in
proliferation and apoptosis in T cells, which has has also been
described for c-Myc and CDC2 kinase (1). Green and Scott (1) proposed a
two signal death/survival model to explain these results: an activation
signal (in this case TCR·CD3-mediated ERK activation) leads to
proliferation or apoptosis depending on additional signals or the
cellular context at the time of activation.
Conflicting data exist regarding a possible role of the Ras-ERK pathway
in Fas-mediated apoptosis. Goillot et al. (34) demonstrated a small and transient ERK activation during Fas-induced apoptosis of
SHEP neuroblastoma cells. Interference with the ERK pathway by ectopic
expression of dominant-interfering MEK1 resulted in a decrease in Fas
expression and inhibition of Fas-mediated apoptosis. Gulbins et
al. (35) found activation of Ras and ERK within 2-5 min of
Fas-mediated apoptosis of Jurkat cells. They could inhibit Fas-mediated
apoptosis by inhibition of Ras with a neutralizing antibody or
dominant negative N17-Ras mutant. However, several studies, including
our data, have not been able to confirm the role of the Ras-ERK pathway
in Fas-mediated apoptosis (36-38). Wilson et al. (36) found
activation of Jun N-terminal kinase, but not Ras or ERK, during
Fas-mediated apoptosis of human peripheral blood lymphocytes or Jurkat
cells. Juo et al. (37) demonstrated activation of Jun
N-terminal kinase and p38 after 1-2 h but no ERK1 activation during
Fas-induced apoptosis of Jurkat cells. Chauhan et al. (38)
found no increase in ERK1 and ERK2 activity during Fas-mediated
apoptosis of multiple myeloma cell lines. Our data also show only weak
phosphorylation and activation of ERK1/2 after 2 h of Fas
cross-linking. Moreover, Fas-mediated apoptosis of Jurkat cells (Figs.
4C and 5C), LK 35.2 cells (Fig. 3B),
and thymocytes (data not shown) was not affected by inhibition of the
ERK pathway with MEK1 inhibitor.
NUR77 is an orphan nuclear steroid receptor (17) that belongs to the
steroid/thyroid hormone receptor superfamily, which consists of
ligand-dependent transcription factors with a
characteristic centrally located and highly conserved DNA-binding
domain containing two zinc fingers. DNA binding of Nur77 was first
detected 2 h poststimulation and continued to increase until
12 h poststimulation. At present, it is not known if NUR77
contributes to the regulation of FasL expression. Expression of FasL in
the thymus is highly elevated in NUR77 full-length transgenic mice,
which have a substantial decrease in numbers of thymocytes and
peripheral T cells, and this would suggest a role for NUR77 expression
in the regulation of FasL expression. However, cross-breeding of the
NUR77 full-length transgenic mouse with the gld mouse (which
has a mutated FasL gene) only partially rescued
NUR77-induced apoptosis (17, 39). No ligand for Nur77 has been
identified, although in a yeast two-hybrid system using the Nur77 DNA
binding domain as a bait a novel cell cycle inhibitor p19 was isolated
(40). However, so far no association in vivo between Nur77
and p19 could be demonstrated. Our data suggest that the rapid
expression of Nur77 during AICD is regulated by activation of the ERK pathway.
Previous studies have shown that certain signaling molecules, including
CD3- In conclusion, our data suggest that the ERK pathway is required for
AICD of T cells and the induction of FasL and Nur77 expression during
AICD but not for Fas-induced apoptosis of T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain and Lck, ZAP-70, CD45,
calcineurin, and Ras activities (7-11). However, negative
selection in the thymus was not affected in lpr or
gld mice, which are deficient in Fas and FasL expression (12), suggesting that the Fas pathway is not an absolute requirement for thymic negative selection.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phorbol 12-myristate 13-acetate
were obtained from Calbiochem. Dexamethasone and propidium iodide were
obtained from Sigma. The antibodies used in this study include
anti-mouse CD3
(145-2C11), anti-mouse TCR (F23.1), anti-mouse Fas
(Jo2; PharMingen, San Diego, CA), anti-mouse FasL-PE (MFL3;
PharMingen), anti-human CD3 (OKT3), anti-human Fas (CH11; Upstate
Biotechnology, Inc., Lake Placid, NY), anti-phospho-MAPK (New England
Biolabs, Beverly, MA), anti-phospho-MEK1/2 (New England Biolabs), and
anti-ERK1 (C-16; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
C2-ceramide, adriamycin, and etoposide were a gift from Zhi-Min Yuan
(Dana Farber Cancer Institute, Boston, MA). The human FasL
promoter-luciferase reporter construct was generously provided by Dr.
G. Koretzky (University of Iowa, Iowa City, IA) (9). A construct
encoding a dominant interfering MEK1, in which Ser221 was
mutated to Ala, was provided by Dr. Sally Cowley (Institute of Cancer
Research, London, United Kingdom) (20), and a constitutively active
form of MEK1, in which serines 218 and 222 were mutated to Asp, was
provided by Dr. Raymond L. Erikson (Harvard University, Cambridge, MA)
(21). Both constructs were subcloned into the pEBG vector (provided by
Dr. Bruce Mayer, Harvard Medical School, Boston, MA): GST-MEK1-S221A
and GST-MEK1-S218/222D. The Renilla luciferase reporter
construct (pRL-TK) was purchased from Promega (Madison, WI).
70 °C, cell pellets were lysed for 20 min on ice
with 50 µl of lysis buffer containing 20 mM Hepes (pH
7.5), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 0.5 mM vanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin,
aprotinin, and pepstatin. The lysate was cleared by high speed
centrifugation (13,000 rpm for 10 min) and diluted at 1:3 with
equilibration buffer consisting of 20 mM Hepes (pH 7.5),
2.5 mM MgCl2, 0.1 mM EDTA, 0.05%
Triton X-100, 1 mM dithiothreitol, 0.5 mM
vanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin, aprotinin, and pepstatin. For in vitro kinase assays, the lysate was incubated for 3 h at 4 °C with anti-ERK1 antibodies (5 µl) and protein A-Sepharose
beads (30 µl of 50% slurry; Amersham Pharmacia Biotech). The
precipitates were washed twice with wash buffer containing 20 mM Hepes (pH 7.5), 0.05 M NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, and
0.1 mM vanadate; twice with high salt buffer containing 10 mM Tris (pH 7.5), 0.5 M LiCl, and 0.1 mM vanadate; twice with low salt buffer containing 10 mM Tris (pH 7.5), 0.1 M NaCl, 1 mM
EDTA, and 0.1 mM vanadate; and finally resuspended in wash buffer. The precipitate was then incubated for 20 min at 30 °C with
3 µg of myelin basic protein and 10 µCi of
[
-32P]ATP in 2× kinase buffer containing 20 mM Hepes (pH 7.5), 20 mM MgCl2,
0.03 mM ATP, 2 mM dithiothreitol, and 0.2 mM vanadate. The reaction was stopped with 4× Laemmli
sample buffer. The reaction mixtures (for in vitro kinase
assays) or lysates (for Western blot analysis) were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The membranes were then developed by
autoradiography (for in vitro kinase assays) or blocked with
5% nonfat milk powder in Tris-buffered saline, probed with primary
antibody followed by horseradish peroxidase-conjugated secondary
antibody and developed by ECL (Amersham Pharmacia Biotech) (for Western
blot analysis).
-counter to determine experimental
release. Spontaneous release was obtained from wells receiving target
cells and medium only, whereas total release was obtained from wells
receiving 1% Triton X-100. The percentage of specific lysis was
calculated with the following formula: percentage of lysis = 100 × ((experimental release
spontaneous release)/(total release
spontaneous release)).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD3 cross-linking of DO11.10 cells results in
phosphorylation and activation of ERK1/2, which can be inhibited by the
MEK1 inhibitor. A, DO11.10 T cell hybridoma cells were
preincubated with 50 µM PD98059 MEK1 inhibitor or
Me2SO (drug vehicle control) for 30 min at 37 °C and
subsequently incubated with plate-bound anti-CD3 antibody. Cells
stimulated with PMA (10 ng/ml) and ionomycin (2 µM)
(P + I) for 15 min at 37 °C and 5%
CO2 were analyzed as positive control for ERK
phosphorylation. At various intervals, cells were harvested and lysed,
and the whole cell lysates were separated by SDS-polyacrylamide gel
electrophoresis in 10% gels. ERK phosphorylation (p-ERK1
and p-ERK2) was determined by Western blotting with an
anti-phospho-ERK antibody (top panel). Subsequently, the
same nitrocellulose membrane was stripped and reprobed with antibodies
recognizing ERK1 and -2 to determine ERK1 and -2 expression at every
time interval (bottom). A representative experiment out of
seven performed is shown. B, DO11.10 T cell hybridoma cells
were treated as in A. At various intervals, cells were
harvested, and the lysates were immunoprecipitated with an anti-ERK
antibody. The immunoprecipitates were tested in an in vitro
kinase assay with myelin basic protein as substrate to determine ERK
activity. A representative experiment out of three performed is shown.
C, J16 and J77 cells were incubated with anti-CD3
antibodies, and at various intervals cells were harvested and lysed,
and whole cell lysates were separated as described in A. ERK
phosphorylation was determined by Western blotting with
anti-phospho-ERK antibody. D, J16 cells were preincubated
with 50 µM PD98059 MEK1 inhibitor or Me2SO
(drug vehicle control) for 30 min at 37 °C and subsequently
incubated with plate-bound anti-CD3 antibody. At various intervals,
cells were harvested and lysed, and whole cell lysates were separated
as described in A. MEK1/2 phosphorylation was determined by
Western blotting with anti-phospho-MEK1/2 antibody.
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Fig. 2.
. MEK1 activity is required for FasL promotor
activation during AICD of J16 cells. A, J16 cells were
transiently transfected with a FasL promotor-luciferase reporter
construct, a dominant-interfering (MEK ) or constitutively
active MEK (MEK+) construct (or pEBG vector alone
(VECTOR)), and a pRL-TK reporter construct and incubated for
18 h at 37 °C. Subsequently, cells were stimulated for 7 h
with plate-bound anti-CD3 (1 µg/ml), and luciferase activity was then
measured with a luminometer. Luciferase activity of pRL-TK was used to
correct for transfection efficiency. Experiments were performed in
triplicate, and results are expressed as mean ± S.E. A
representative experiment of three experiments performed is shown.
B, transfections were performed as above without MEK
constructs. After an 18-h incubation, cells were pretreated with MEK1
inhibitor (or Me2SO (DMSO) as control) and
subsequently activated with PMA (10 ng/ml) plus ionomycin (2 µM) or plate-bound anti-CD3 for 6 h, and luciferase
activity was measured. Experiments were performed in duplicate, and
results are expressed as mean values. A representative experiment of
three experiments performed is shown.
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Fig. 3.
The ERK pathway is required for the induction
of FasL expression and activity during AICD. A, DO11.10
T cell hybridomas were preincubated with 50 µM PD98059
MEK1 inhibitor or Me2SO (DMSO, drug vehicle as
control) for 30 min at 37 °C and subsequently incubated with
plate-bound anti-CD3 antibody. Cells were fixated with formaldehyde,
permeabilized with Triton X-100 (see "Experimental Procedures"),
and stained with PE-labeled anti-FasL antibody. Percentages of positive
cells were as follows: unstimulated cells, <1%; anti-CD3, 13%;
anti-CD3 plus Me2SO, 13%; anti-CD3 plus MEK1 inhibitor,
<1%. B, DO11.10 cells were pretreated with MEK1 inhibitor
(or Me2SO (DMSO)), stimulated for 3 h by
anti-CD3 cross-linking, and subsequently incubated for 8 h with
51Cr-labeled Fas+ LK35.2 target cells. Some
LK35.2 cells were only stimulated for 8 h with anti-Fas
antibodies. Assays were performed in triplicate. 51Cr
release, which indicates Fas-mediated lysis of target cells, was
measured in a -counter. One representative experiment from four
performed is shown.
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Fig. 4.
AICD of DO11.10 T cell hybridoma cells and
Jurkat-16 cells is decreased by pretreatment with the MEK1 inhibitor
PD98059. A, DO11.10 T cell hybridoma cells were
preincubated with 50 µM PD98059 MEK1 inhibitor
(circles) or Me2SO (squares; drug
vehicle alone as control) for 30 min at 37 °C and 5%
CO2 and subsequently incubated with (solid
symbols) or without (open symbols)
plate-bound anti-CD3. Cells were harvested at various intervals, and
the percentage of apoptotic cells was determined. A representative
experiment out of four experiments is shown. B, cells were
treated and analyzed as described for A; however, 20 µM PD98059 MEK1 inhibitor (or Me2SO as drug
vehicle control) was added a second time to the cell cultures 4 h
after incubation with (solid triangles) or without
(open triangles) plate-bound anti-CD3 antibody was begun.
C, J16 cells were preincubated with 50 µM
PD98059 MEK1 inhibitor or Me2SO (drug vehicle alone as
control, DMSO) for 30 min at 37 °C and 5% CO2 and
subsequently incubated with or without plate-bound anti-CD3 (OKT3, 10 µg/ml) or soluble anti-Fas antibodies (CH11, 0.5 µg/ml). Cells were
harvested after 12 h, and the percentage of apoptotic cells was
determined. A representative experiment out of three experiments is
shown.
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Fig. 5.
Fas-mediated apoptosis of Jurkat cells
results in weak phosphorylation and activation of ERK1/2 and is not
affected by the MEK1 inhibitor. A and B,
Jurkat cells were preincubated with 50 µM PD98059 MEK1
inhibitor or Me2SO and subsequently incubated at
106 cells/ml at 37 °C with an anti-Fas antibody. Cells
stimulated with PMA (10 ng/ml) and ionomycin (2 µM)
(P + I) for 15 min at 37 °C were analyzed as
positive control for ERK phosphorylation and activation. At various
intervals, cells were harvested, and whole cell lysates were analyzed
by Western blotting with an anti-phospho-ERK (p-ERK)
antibody (A) to evaluate ERK phosphorylation or by
immunoprecipitation with an anti-ERK antibody followed by an in
vitro kinase assay with myelin basic protein as substrate
(B) to determine ERK activity. C, Jurkat cells
were preincubated with 50 µM PD98059 MEK1 inhibitor,
Me2SO, or control medium for 30 min at 37 °C and
subsequently incubated at 106 cells/ml at 37 °C with an
anti-Fas antibody. Cells were harvested after 10 h, and the
percentage of apoptotic cells was determined by flow cytometric
analysis after staining with PI solution. A representative experiment
out of four experiments performed is shown.
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Fig. 6.
The MEK1 inhibitor has a specific
antiapoptotic effect on AICD and does not affect other forms of
apoptosis. DO11.10 T cell hybridomas were preincubated with 50 µM PD98059 MEK1 inhibitor or Me2SO and
subsequently incubated at 106 cells/ml for 22 h with
plate-bound anti-CD3 antibody, 10 µg/ml anti-Fas antibody, 5 µM C2-ceramide, 10 6 M
dexamethasone, 10 µg/ml adriamycin, 10 µg/ml etoposide or exposed
to 20-gray
-irradiation. The percentage of apoptotic cells was
determined by flow cytometric analysis after staining with PI
solution.
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Fig. 7.
The ERK pathway is required for Nur77
expression during AICD. DO11.10 T cell hybridoma cells were
preincubated with 50 µM PD98059 MEK1 inhibitor or
Me2SO for 30 min at 37 °C and subsequently incubated
with plate-bound anti-CD3 antibody. Some cells were stimulated with PMA
(10 ng/ml) and ionomycin (2 µM) (P + I) for 15 min at 37 °C and 5% CO2. At
various intervals, cells were harvested and lysed, and the whole cell
lysates were separated by SDS-polyacrylamide gel electrophoresis in
10% gels. Nur77 expression was determined by Western blot analysis
with anti-Nur77 antibodies. A representative experiment of three
experiments is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Lck, Zap-70, CD45, Ras, and calcineurin, that are involved in
TCR·CD3-mediated signaling pathways leading to proliferation and
activation are also important for AICD. We have confirmed the
requirement of activation of the calcium/calcineurin pathway for AICD,
since calcineurin inhibitors (such as cyclosporin A and FK506) can
inhibit AICD (data not shown; Ref. 29). Our data with transiently
transfected MEK1 mutants and the specific MEK1 inhibitor provide the
first evidence that the ERK pathway is required for AICD and exerts its
effect through the regulation of FasL expression and perhaps also
through the regulation of Nur77 expression. The induction of FasL
expression during AICD was shown to be the mechanism by which other
signaling molecules (CD3-
, Lck, Zap-70, CD45, Ras, and calcineurin)
regulate AICD (7-11, 28). However, the effects of these molecules on
Nur77 expression, which occurs exclusively in T cells and thymocytes during AICD, were not determined in any of these studies. NFAT binding
sites have been identified in the FasL promotor (10), which could
explain the regulation of FasL expression by the
Ca2+/calcineurin pathway. A binding site for Nur77 in the
FasL promotor has not been detected, which leaves the question of how
Nur77 regulates AICD unanswered. Also, a binding site for a
transcription factor that is regulated by the ERK pathway has not yet
been identified within the FasL promotor, although a recently
identified MEKK1-regulated response element in the FasL promotor can
bind to ATF2 and c-Jun, which are both substrates for phosphorylation
by ERK1/2 (41).
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ACKNOWLEDGEMENTS |
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We thank Drs. Sansana Sawasdikosol, Zhi-Min Yuan, and Shyr-Te Ju for reagents and helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Program Project Grant 5PO1 CA39542 (to S. J. B.) and CA09382 (to R. K.).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.
¶ Recipient of a Howard Hughes Physician Postdoctoral Fellowship. To whom correspondence should be addressed: Division of Pediatric Oncology, Dana Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-667-1930; Fax: 617-632-5144; E-mail: mvandenb{at}bidmc.harvard.edu.
Present address: Millennium Pharmaceuticals,
Cambridge, Massachusetts.
** Supported by a fellowship from The Medical Foundation.
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ABBREVIATIONS |
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The abbreviations used are: AICD, activation-induced cell death; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated/extracellular receptor protein kinase kinase; PI, propidium iodide; IL-2, interleukin-2; TCR, T cell receptor; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate.
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