From the Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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ABSTRACT |
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T cell receptor engagement activates
transcription factors important for cytokine gene regulation.
Additionally, this signaling pathway also leads to activation-induced
apoptosis in T lymphocytes that is dependent on FasL transcription and
expression. Here we demonstrate that nuclear factor Activation-induced cell death
(AICD)1 is a major mechanism
to maintain immune homeostasis. AICD occurs in mature T lymphocytes to
limit antigen-specific responses. Upon clearing antigens and/or pathogens from the host, activated T cells are deleted via the activation of apoptosis, which we and others have shown to be dependent
upon Fas ligand (FasL, CD95 ligand) expression and ligation of its
receptor, Fas (CD95) (1-5). However, despite our understanding of the
importance of FasL in controlling homeostasis, little is known about
its transcriptional regulation.
T cell receptor ligation activates a cascade of signaling events which
leads to the activation of several transcription factors that regulate
cytokine gene expression including interleukin-2 (IL-2) (6). Signaling
events post-TCR ligation and phospholipase C- Like NF-AT, nuclear factor Recently, we identified a role for NF- Cell Lines and Antibodies--
Human leukemic Jurkat cells
(ATCC) and Jurkat cells stably transfected with SV40 large T antigen
were used in this study. The T cell hybridoma A1.1 has been described
previously (2). All cells were grown in RPMI 1640 medium containing
10% fetal calf serum, 2 mM L-glutamine, and
100 units/ml each penicillin and streptomycin (complete medium).
Phorbol myristate acetate (PMA) was purchased from Sigma, and ionomycin
was purchased from Calbiochem. Mouse anti-human CD3 (OKT3) antibody and
hamster anti-mouse CD3 Induction of Apoptosis--
All experiments were performed in
96-well plates and in triplicates samples, with cells resuspended at
0.5-1 × 106/ml in complete medium. For T cell
receptor stimulation, 96-well plates were precoated with anti-CD3
antibody (2C11) in 50 mM Tris, pH 9.0. PMA and ionomycin
were added at concentrations of 50 ng/ml and 0.5 µg/ml, respectively.
Reverse Transcription (RT)-PCR for FasL Expression--
The
expression of Fas-L was determined by RT of total RNA followed by
reverse PCR analysis (RT-PCR) as described previously (16). Briefly,
cDNAs were synthesized by extension of dT primers with 200 units of
SuperScript II reverse transcriptase (Life Technologies, Inc.) in a
mixture containing 1 µg of total RNA digested by RNase-free DNase (2 units/µg of RNA) (Ambion) for 15 min at 37 °C. PCR of the cDNA
was performed in a final volume of 50 µl, containing all four dNTPs,
2 mM MgCl2, 2.5 units of AmpliTag (Life
Technologies, Inc.), and each primer at 0.2 µM using the
geneAmp 2400 PCR system (Perkin-Elmer). Amplification of
hFasL forward: TAAAACCGTTTGCTGGGGC.
hFasL reverse: CTCAGCTCCTTTTTTTCAGGGG.
Expression Vectors and Transient Transfections--
Cloning of
the 1.2-kb FasL promoter and generation of the truncated version was
described before (16). A 1.2- and a 0.9-kb fragment of the hFasL
promoter were subsequently used in the experiments described here.
Jurkat T cells containing stably transfected SV40 large T antigen were
electroporated as described previously (17). Briefly, 1.5-2 × 107 cells were washed twice with serum-free RPMI 1640 medium, resuspended in 500 µl of the same medium, and transferred to
4-mm gap electroporation cuvettes (Bio-Rad). 20-60 µg of FasL
reporter alone or with Sr Luciferase Assays--
Briefly, different hFasL promoter
constructs were transfected with or without co-expression vectors. pCMV
Electromobility Shift Assays--
DNA binding reactions were
carried out for 20 min at 4 °C in a buffer containing 50 mM Hepes, pH 7.8, 20 mM MgCl2, 0.5 mM EDTA, 20 mM spermidine, 500 µg/ml bovine
serum albumin, 10 mM dithiothreitol, 75% glycerol, and
104 counts/min labeled probe. The probes used were
double-stranded synthetic oligonucleotides (Retrogen, San Diego, CA)
consisting of the NF- Inhibition of NF-
To examine this possibility, we employed a recently described inhibitor
of NF- Characterization of the 1.2-kb FasL Promoter for TCR-mediated
Signaling Events--
Activation-induced cell death is mediated by
regulation of Fas/FasL expression. T cell receptor cross-linking and
pharmacologic agents mimicking these signals also induce FasL
expression (27). There are potential binding sites for several
transcription factors in the 1.2-kb promoter region of Fas ligand (Fig.
2A). To understand the results
obtained in Fig. 1 and to dissect the molecular mechanisms underlying T
cell receptor-mediated FasL expression, we used a 1.2-kb FasL promoter
(16) and examined whether this reporter is activated upon TCR ligation.
As shown in Fig. 2B, the 1.2-kb FasL promoter was activated
upon cross-linking of the TCR receptor with anti-CD3 antibody as well
as upon stimulation with the pharmacologic agents PMA and ionomycin.
Similar results were reported in studies using a 486-bp FasL reporter
construct (10). To identify the regulatory domains that mediate this
inducible transcription of the 1.2-kb FasL promoter, we then tested the
effect of a 0.9-kb FasL reporter (Fig. 2A) that has its 5'
enhancer truncated. In the experiment shown in Fig. 2C, we
observed that when T cells were activated with PMA and ionomycin, FasL
reporter activity of the 0.9-kb promoter was significantly reduced
versus that of the 1.2-kb promoter.
T Cell Activation Induces Binding of NF- Inhibition of NF-
We have recently identified an NF- p65 RelA Increases FasL Reporter Activity upon T Cell
Activation--
The ability of the various Rel-related proteins to
recognize DNA sequence elements through either homo- or heterodimer
formation may be a mechanism whereby selective functional regulation by this family of proteins is achieved. It has been shown that the p65
subunit of NF- AP-1 Is Not Critical for TCR-mediated FasL Promoter
Activation--
Recently, Latinis et al. (10) showed that
NF-AT is an important transcription factor required for
activation-induced FasL expression. Similarly, we identified AP-1 and
NF- TCR engagement and activation often leads to growth and
differentiation as well as apoptosis in T cells (6). Some of the proximal signaling events lead to activation of AP-1, NF- Activation-induced cell death is primarily mediated by Fas/FasL in
mature T lymphocytes (2, 3). Previously, Latinis et al. (10)
showed that NF-AT regulates activation-induced FasL promoter activity.
Similarly, we showed recently that the transcription factors AP-1 and
NF- NF- Apoptosis mediated by Fas often does not involve activation of NF- Our present study also raises some interesting questions about the
complex regulation of Fas ligand. A 0.9 kb fragment of the FasL
reporter had low activity upon stimulation (Fig. 1C), whereas a 486-bp reporter was shown to be sufficient for reporter activity (10). One possibility is that there could be repressor elements in the Fas ligand promoter that may act as regulators of this
death-promoting gene. A recent study on human FasL promoter has
suggested the existence of a repressor element between So far, NF-B (NF-
B),
which is involved in the transcriptional regulation of many cytokine
genes expressed in activated lymphocytes, also plays a role in T cell
activation-induced FasL expression. Inhibition of NF-
B activity in a
T cell hybridoma leads to decreased FasL expression and apoptosis upon
T cell receptor stimulation. We identified the NF-
B site in the FasL
promoter that contributes to such regulation. Co-expression of p65 (Rel A) with the FasL promoter enhanced its activity, and co-expression of
I
B dramatically inhibited the inducible promoter activity. In
contrast, the transcription factor AP-1 is not required for activation-induced FasL promoter activity. These results define a role
for NF-
B in mediating FasL expression during T cell activation.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
1 lead to two distinct
signals: activation of the protein kinase C pathway and release of
intracellular Ca2+ stores, subsequent Ca2+
influx, and the activation of the serine phosphatase, calcineurin (7).
These two signals can be mimicked by treatment of cells with the
pharmacological agents phorbol myristate acetate and ionomycin.
Calcineurin-mediated activation of the nuclear factor of activated T
cells (NF-AT) and its role in cytokine gene expression is well
established (7-9), and more recently it was shown that NF-AT sites in
the FasL promoter are important for T cell activation-induced FasL
expression (10).
B (NF-
B) is also involved in the
transcriptional regulation of the interleukin 2
receptor (11) and
the IL-2 genes (12). NF-
B is present in the cytoplasm as two major
precursor forms either as a RelA·p50 complex with the inhibiting
I
B or as a heterodimeric RelA·p105 complex (13). Release of the
RelA-p50 from its inhibitor, I
B, is dependent on the phosphorylation
of I
B (14), and recently, the kinases that phosphorylate I
B have
been identified (15). NF-
B is then translocated into the nucleus as
active heterodimers consisting of Rel A (p65), Rel B, and c-Rel, which
regulate gene expression.
B and another transcription
factor, AP-1, in the regulation of stress-induced FasL expression (16).
In the present study, we sought to determine whether NF-
B and AP-1
regulate FasL expression in lymphocytes activated through the T cell receptor.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(145-2C11) were purified from culture
supernatants by protein A affinity chromatography. Anti-p65(RelA)
antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
-actin
served as control for sample loading and integrity. The following
primers were designed to discriminate between the amplification of
cDNA (low size PCR products) and contaminating genomic cDNA
(high size PCR products):
-Actin forward: TGACGGGGTCACCCACACTGTGCCCATCTA.
-Actin reverse: CTAGAAGCATTTGCGGTGGACGATGGAGGG.
p65, I
B, I
B
M, or DN MEKK
expression vectors (18, 19) were added to the cells and mixed well.
Electroporation was carried out at 250 V and 960 microfarads in a
Bio-Rad Gene Pulse II.
-galactosidase was used to normalize the transfection efficiencies
in the various co-transfections. Forty h post-transfection, cells were
activated with PMA and ionomycin and incubated for another 12-18 h.
Cells were harvested, washed three times with PBS, and lysed in 100 µl of the lysis buffer. Cell debris was removed by centrifugation, and the supernatant was used in the luciferase assay using a Monolight 2010 luminometer (16).
B site from the human FasL promoter,
5'-AAGCCTGGGCAACATAGAAAGTCCCCATCTGTACAAAAA-3', and the consensus
NF-
B oligonucleotide from Santa Cruz Biotechnology, Inc. Each strand
was labeled separately with T4 polynucleotide kinase (Life
Technologies, Inc.) and [
-32P]ATP (5000 Ci/mmol), and
the strands were then slowly allowed to reanneal. The samples were
analyzed on a 4% nondenaturing acrylamide gel in 0.5% Tris-buffered
EDTA. In some experiments, nuclear extracts from Jurkat cells were made
as described (16). Binding reactions contained 5-10 µg of nuclear
extract, 32P-labeled probe (25,000 cpm), and 2 µg of
poly(dI:dC) in the binding buffer.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B Diminishes FasL Expression and T Cell
Receptor-mediated Apoptosis--
It has been suggested that NF-
B is
required for activation-induced cell death in T cells. Recently, it was
shown that antioxidants inhibit activation-induced cell death by
blocking NF-
B activation, and thereby blocking FasL expression (20).
Similarly, we have recently shown that NF-
B is required for
stress-induced FasL expression and apoptosis in T cells (16). In
contrast, previous studies by ourselves and others have suggested that
NF-
B can act as an inhibitor of apoptosis induced by TNF (21-24).
We therefore sought to determine whether inhibition of NF-
B can
inhibit activation-induced cell death in T cells.
B composed of a cell-permeable peptide carrying the nuclear
localization signal of the NF-
B p50 subunit (25). This peptide has
been shown to specifically inhibit the nuclear translocation of the
NF-
B complexes (26). We studied the effect of the NF-
B inhibitory
peptide or a control peptide in preventing activation-induced cell
death in a T cell hybridoma. The T cell hybridoma A1.1 undergoes
expression of FasL and apoptosis when cross-linked with anti-T cell
receptor antibodies (1). We observed a significant decrease in
apoptosis in the presence of the NF-
B inhibitory peptide and not the
control peptide (Fig. 1A). We
then assessed FasL expression by semi-quantitative RT-PCR in these cells and observed a corresponding inhibition in the level of FasL
mRNA (Fig. 1B). Together, these results suggest that
NF-
B may be an important mediator of activation-induced FasL
expression and subsequent cell death.
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Fig. 1.
Inhibition of NF B blocks
activation-induced cell death and FasL expression. A,
A1.1 T hybridoma cells were activated by the T cell receptor in the
presence of the
B inhibitory peptide (
) or the control peptide
(
) for 12-16 h, and cell death was assessed by propidium iodide
uptake on FACS. Apoptotic cells were also analyzed by staining with
acridine orange and ethidium bromide and examining the morphology by
fluorescent microscopy. B, RT-PCR analysis of total mRNA
isolated from A1.1 cells activated as described above in the presence
of the
B inhibitory peptide or the control peptide.
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Fig. 2.
T cell activation induces the FasL
reporter. A, schematic representation of the identified
transcription factor binding sites in the FasL promoter. B,
Jurkat T cells were transiently transfected with luciferase reporter
construct containing the 1.2-kb FasL reporter. Cells were treated with
anti-TCR antibodies (OKT3) or PMA and ionomycin (P+I), and
reporter activity was determined after approximately 16 h.
C, Jurkat T cells were transiently transfected with
luciferase reporter constructs containing 1.2 or 0.9 kb of the FasL
reporter. Cells were treated with PMA, and ionomycin and reporter
activity was determined after 16 h.
B to Its Cognate
DNA--
To further examine the possible role of NF-
B in AICD, we
studied the activation-dependent characteristics of a
NF-
B reporter construct carrying two copies of the consensus
B
motif (28) and found that NF-
B is activated upon stimulation with
PMA and ionomycin (Fig. 3A). A
mutation in the site abrogated this activity (Fig. 3A). We
have previously shown that the distal NF-
B site binds to NF-
B
proteins from nuclear extracts treated with the DNA-damaging agent,
etoposide (16). After treatment with PMA and ionomycin, we then
examined nuclear extracts for NF-
B binding activity by an
electromobility shift assay, using the distal NF-
B site from the
FasL promoter (16). As shown in Fig. 3B, T cell activation
induced NF-
B binding in an activation-dependent manner. The binding to this site was competed off by the wild type consensus NF-
B motif (GGGGACTTTCCC) (29) and by an anti-p65 antibody (30) but
not by control serum (Fig. 3B). The inhibition of complex formation by anti-p65 antibody also suggests that p65 is part of the
complex induced by PMA and ionomycin treatment.
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Fig. 3.
T cell activation induces the transcription
factor NF- B. A, Jurkat cells were transfected with
the wild type (WT)
B reporter construct containing two
copies of the consensus
B motif or a construct containing a mutation
in the NF-
B site as shown. Cells were treated with PMA, and
ionomycin and reporter activity was determined after 12-16 h.
B, an NF-
B binding site in the FasL promoter is required
for T cell receptor-mediated activation. Nuclear extracts made
from T cells activated with PMA and ionomycin (P+I)
were employed in an electromobility shift assays with an
oligonucleotide corresponding to the putative NF-
B binding sequence
in the FasL promoter (GGGGACTTTCT).
B Prevents FasL Reporter Activity--
Earlier
studies using chemical activators of cAMP signaling (31) and reactive
oxygen intermediates have suggested that NF-
B could be involved in
activation-induced cell death (20, 32). The experiments in Fig. 1
suggested that NF-
B is required for optimal activation-induced cell
death and FasL expression. We therefore investigated the possibility
that NF-
B directly regulates T cell activation-mediated FasL
transcription. Based on the dependence of NF-
B activation on the
effective degradation of I
B, we first took the approach of
inhibiting NF-
B activation using I
B or a nondegradable mutant
I
B
M (21). We therefore co-transfected Jurkat cells with the FasL
reporter construct together with either wild type I
B or the
nondegradable mutant I
B
M (21). As shown in Fig.
4A, either form of I
B
effectively blocked the basal activity as well as activation of the
FasL promoter with PMA and ionomycin.
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Fig. 4.
NF- B is required for activation-induced
FasL promoter activity. A, Jurkat cells were
transfected with the 1.2-kb FasL reporter construct plus or minus
expression constructs for I
B or I
B
M. Cells were then treated
with PMA and ionomycin (P+I), and reporter activity was
determined after 12-16 h. B, Jurkat cells were transfected
with the wild type FasL reporter construct or a construct containing
the mutation in the NF-
B site as shown. Cells were treated with PMA
and ionomycin, and reporter activity was determined after 12-16 h.
WT, wild type.
B responsive element in the FasL
promoter essential for stress-induced promoter activity (16). To
investigate the importance of this NF-
B site in activation-induced FasL promoter activation, we used a reporter construct in which this
site was mutated (16) and tested for its ability to be induced upon T
cell activation. As shown in Fig. 4B, mutating the NF-
B
site had a profound effect on T cell activation-mediated FasL
expression, further supporting the idea that NF-
B is required for
optimal activation of the FasL promoter.
B can act as a transcriptional activator (33). Co-expression of p65 (Rel A) with the FasL reporter induced
constitutive FasL reporter activity and enhanced the ability of PMA and
ionomycin to drive this promoter (Fig.
5A). The NF-
B mutation also
abolished the ability of this promoter to be induced by co-expression
of the p65 subunit of NF-
B (Fig. 5B). Transient
overexpression of p65 was sufficient to induce expression of the
endogenous Fas ligand gene in Jurkat cells as assessed by
semi-quantitative PCR (Fig. 5C). These data support a role
for NF-
B in driving the FasL promoter following T cell
activation.
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Fig. 5.
p65 Rel enhanced activation-induced FasL
promoter activity. A, Jurkat cells were transfected
with the 1.2-kb FasL reporter construct alone or together with an
expression vector for p65 Rel. Cells were treated with PMA and
ionomycin (P+I), and reporter activity was determined after
12-16 h. B, Jurkat cells were transfected with the 1.2-kb
FasL reporter construct or the 1.2-kb FasL reporter that contains the
NF- B site mutation (mut) alone or together with an
expression vector for p65 Rel. Cells were treated with PMA and
ionomycin, and reporter activity was determined after 12-16 h.
WT, wild type. C, p65 (RelA) was transiently
transfected into Jurkat cells. Forty eight h later, cells were either
left untreated or activated with PMA and ionomycin for 4 h and
harvested. Total RNA was isolated, and a semi-quantitative PCR was
performed as described under "Experimental Procedures."
B sites required for stress-induced FasL expression (16). T cell
receptor ligation activates the early genes fos and
jun that heterodimerize and bind to the AP-1 site to
regulate IL-2 and other cytokine gene transcription (34).
Phosphorylation of c-Jun by c-Jun N-terminal kinases (JNK/SAPK) is
required for AP-1-dependent promoter activity (34).
Inhibiting c-Jun phosphorylation by a DN-MEKK (K432M) also
down-regulates AP-1-dependent reporter activity (35). To study the effects of DN-MEKK on FasL reporter activity, we
cotransfected the FasL reporter with a DN-MEKK expression vector and
observed that DN-MEKK had no effect on TCR-mediated FasL reporter
activity (Fig. 6A). In
contrast and as shown earlier (16), DN-MEKK inhibited stress-induced
FasL reporter activity (Fig. 6A). We then investigated whether the distal AP-1 binding site (16) was required for TCR-mediated FasL transcription. As shown in Fig. 6B, a mutation in the
AP-1 site did not alter FasL reporter activity in response to T cell receptor signaling, thus suggesting the involvement of a different signaling mechanism. We have previously observed that this mutation abolishes the response of the promoter to DNA-damaging agents (16).
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Fig. 6.
AP-1 is not obligatory for activation-induced
FasL promoter activity. A, Jurkat cells were
transfected with hFasLPr (1.2 kb) and DN-MEKK1 (K432M). Cells were
activated with PMA and ionomycin (P+I). Reporter activity
was measured after 12-16 h. Results shown are representative of three
independent experiments. B, Jurkat cells were transfected with the
1.2-kb FasL promoter reporter construct or a construct containing the
mutation in the AP-1 site as shown. Cells were treated with PMA and
ionomycin, and reporter activity was determined after 12-16 h.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B, as well
as the NF-AT family of transcription factors (9), which are known to
activate cytokine gene transcription. A substantial amount of work in
the past several years has helped our understanding of this complex
cytokine gene regulation. There is also evidence that transcription can
play a critical role in mechanisms involved in some forms of apoptosis,
such as activation-induced cell death in T cells.
B regulate DNA damage and other stress-induced FasL expression
(16). Here, we have provided evidence that NF-
B also plays a role in
T cell activation-mediated FasL expression. We found that the
regulation of the 1.2-kb FasL reporter was dependent on NF-
B and
that inhibiting NF-
B translocation into the nucleus also inhibited
FasL expression and apoptosis in T cells.
B was originally identified as a constitutively expressed protein
that can form a complex with a 10-bp site in the immunoglobulin
light chain enhancer (36). It was soon found to also take part in T
cell activation, regulating IL-2 and IL-2
receptor genes (11). There
is also indirect evidence suggesting that NF-
B can be involved in
activation-induced cell death (20, 31). AICD has been shown to be
inhibited by antioxidants (20, 32) and inhibitors of proteasome
activity such as lactacystin (37, 38). It was recently shown that
antioxidants block NF-
B activation and that nondegradable I
B not
only blocks NF-
B activation but also cell death in T cells. Thus,
agents that induce or inhibit NF-
B activation appear to have an
impact on AICD. Inhibition of NF-
B also blocks the ability of
reactive oxygen intermediates to induce FasL expression in T cells
(20). Because reactive oxygen intermediates may be involved in T cell
signaling, we also studied the effect of inhibiting NF-
B in
H2O2-induced cell death and found that
inhibiting NF-
B blocks this mode of apoptosis (data not shown).
NF-
B has also been implicated in signaling events that prevent cell
death elicited by the cytokine tumor necrosis factor
, TNF
(39).
We and others previously showed that inhibition of NF-
B in Jurkat
and other cell types sensitizes these cells to TNF-induced apoptosis
(21). We have recently shown that the inhibition of NF-
B also
prevents DNA damage and other related stress-induced apoptosis in
Jurkat cells (16). Taken together with the results described in this
paper, NF-
B seems to serve multiple functional roles under different conditions.
B
(40). On the other hand, TRAMP (TNF-related apoptosis-mediating protein)/DR3/WSL-1 and TRAIL receptors (TNF-related apoptosis-inducing ligand) induce apoptosis and also activate NF-
B (41-44). Like FasL,
TRAIL is also induced upon lymphocyte activation, and its expression is
inhibited by cyclosporin A, suggesting a role for calcineurin and NF-AT
(45). Whether NF-
B also regulates TRAIL expression is not known. It
is possible that in T cells, some of the early signals may define the
de novo gene expression.
453-373 in
the promoter (46). This study did not, however, characterize the region
between
473 and
2365 in the promoter. Our study suggests the
possibility of a repressor element between
900 and
486 bp in this
promoter, and there could also be another between the 1.2 kb and the
2.3 kb of the enhancer. We are currently analyzing the promoter region
for any such repressor elements. It is also interesting that NF-AT
alone is sufficient to drive a 486-bp FasL promoter (10), whereas in
the context of a 1.2-kb promoter, a mutation in the NF-AT site did not
completely inhibit the promoter activity.2 In contrast, a
mutation in the NF-
B site had a profound effect on
activation-induced Fas ligand promoter activity (Fig. 4B), suggesting that NF-
B is critical for FasL expression.
B, AP-1, and NF-AT are among the transcription factors
shown to be involved in some forms of apoptosis, of which NF-
B seems
to possess quite distinct characteristics. Understanding the
characteristics of this molecule with regard to how this differential specificity is achieved and how different signaling events converge on
the same regulatory factor would also shed light on any "switch mechanism" a cell might have to regulate different cellular
functions. We studied the importance of the previously identified AP-1
site (16) in T cell activation-mediated FasL expression and found that
AP-1 was dispensable for this form of induction but was required for
induction by stress (16). Our studies indicate that the preference of
different transcription factors to regulate gene transcription varies
with the mode of cell activation. The transcription factors AP-1 and
NF-
B are involved in stress-induced FasL expression, whereas NF-AT
and NF-
B appear to co-operate in T cell receptor-mediated FasL expression.
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FOOTNOTES |
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* This work was supported by National Institutes of Health National Service Award AG 00252-01 (to S. K), Human Frontier Science Program (to L. G.), and National Institutes of Health Grants GM 52735 and CA 69831 (to D. R. G.) This is publication 255 from La Jolla Institute for Allergy and Immunology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Cellular
Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 619-678-4680; Fax: 619-558-3526; E-mail: skasibhatla{at}liai.org.
The abbreviations used are:
AICD, activation-induced cell death; IL-2, interleukin 2; TCR, T cell
receptor; NF-AT, nuclear factor of activated T cells; NF-B, nuclear
factor
B; PMA, phorbol myristate acetate; RT-PCR, reverse
transcription-polymerase chain reaction; kbp, kilobase pair(s); bp, base pair(s); hFasL, human FasL; DN-MEKK, dominant negative
mitogen-activated protein kinase kinase.
2 S. Kasibhatla, unpublished observations.
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